University of Nevada, Reno
Coils, Loops, and Fingers: Functional Motifs in Hantavirus Proteins
A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Cellular and Molecular Biology
by
Daniel M. Boudreaux
Dr. Stephen C. St Jeor / Dissertation Advisor
December 2009
THE GRADUATE SCHOOL
We recommend that the dissertation prepared under our supervision by
DANIEL M. BOUDREAUX
entitled
Coils, Loops, And Fingers: Functional Motifs In Hantavirus Proteins
be accepted in partial fulfillment of the requirements for the degree of
DOCTOR OF PHILOSOPHY
Stephen C. St Jeor, Ph.D., Advisor
Gregory S. Pari, Ph.D., Committee Member
David P. AuCoin, Ph.D., Committee Member
William H. Welch, Ph.D., Committee Member
Ellen J. Baker, Ph.D., Graduate School Representative
Marsha H. Read, Ph. D., Associate Dean, Graduate School
December, 2009
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ABSTRACT
New world hantaviruses are a threat as an emerging pathogen with the potential to cause
outbreaks in rural regions remote from medical facilities. Medical authorities emphasize the need
for preventative education programs and the development of antiviral drug treatments based on an
understanding of the viral replication process. The virus is composed of three negative sense
RNA genomic segments and four proteins; the RNA-dependent RNA polymerase (RdRP),
glycoprotein Gn, glycoprotein Gc, and the nucleocapsid protein (Npro). This dissertation
presents the structures of three motifs within hantavirus proteins as part of an effort to identify
potential targets for antiviral drugs. 1) The NMR structure of the N-terminal region of Npro is
described here to form a helical coiled-coil motif. Three acidic amino acids in this structure
significantly contribute to the ability of Npro to self associate, an important process in viral
replication. 2) The middle region of Npro is proposed at a low resolution to contain a helix-loop-
helix motif which interacts with the cytoskeletal filament, vimentin. The Npro-vimentin interaction contributes to the proper trafficking of Npro through the cytoplasm. 3) The glycoprotein Gn contains an unusually long cytoplasmic tail with a conserved dual CCHC sequence. NMR resolution revealed that this sequence forms a zinc finger motif. The potential for this zinc finger to bind viral RNA was predicted by molecular modeling and molecular dynamic simulations. This interaction is thought to coordinate the packaging of each of the three viral genome segments. The presentation of these three structures contributes a better understanding of the physical properties of proteins involved in viral infections.
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TABLE OF CONTENTS
Abstract……………………………………………………………………………………………i
Table of Contents…………………………………………………………..………………….….ii
List of Tables………………………………………………………………………………….….iii
List of Figures………………………………………………………………………...………….iii
Introduction…………………………………………………………………………...…………..1
Prologue: Identification of Hantavirus Nucleocapsid Binding partners suggests essential interactions in the assembly process……………...………………………………………..…..19
Chapter 1: NMR structure of the N-terminal coiled coil domain of the Andes hantavirus nucleocapsid protein...... 29
Abstract…………………………………………………………………………………..29
Introduction…………………………………………………………………………...….30
Methods…………………………………………………………………………………..31
Results……………………………………………………………………………………35
Discussion………………………………………………………………………………..43
Chapter 1 Addendum: The N-Terminal Coiled Coil Domain of the Andes Nucleocapsid Protein is associated with Trimerization………………………………………………………48
Introduction………………………………………………………………………………48
Materials and Methods…………………………………………………………………...49
Results…………………………………………………………………………………....50
Discussion……………………………………………………………………………..…51
Chapter 2: The Middle Region of Hantavirus nucleocapsid binds Vimentin………….……54
Introduction………………………………………………………………………………54
Methods………………………………………………………………………………..…57
Results…………………………………………………………………………..………..62
Discussion……………………………………………………………………………..…74
Chapter 3: The Hantavirus Glycoprotein G1 Tail Contains Dual CCHC-type Classical Zinc Fingers...... 79
Abstract…………………………………………………………………………………..79
Introduction………………………………………………………………………………80 iii
Materials and Methods…………………………………………………………….……..81
Results……………………………………………………………………………………86
Discussion………………………………………………………………………………..94
Chapter 3 Addendum: The Zinc Finger Motif of Andes Hantavirus Gn has the potential to bind to the viral RNA panhandle………………………………………………………………98
Introduction……………………………………………………………………………....98
Materials and Methods………………………………………………………..………...100
Results…………………………………………………………………………………..103
Discussion………………………………………………………………………………111
Conclusion………………………………………………………………………………….…..114
References………………………………………………………………………………………119
Appendix I Supplementary Figures for Chapter 1…………………………………………..146
Appendix II Supplementary Figures for Chapter 3………………………………………....150
LIST OF TABLES
Introduction Table 1. Distribution, Vectors, and Diseases of Hantavirus Genotype Representatives………………………………………………………………………………….....5
Prologue Table 1. Proteins identified as potential binding partners for SNV-Npro using co- immunoprecipitation and MALDI-TOF peptide analysis………………………………..…….....23
Chapter 1 Table 1. Melting temperatures (Tm) and ellipticity ratio at 222 and 208 nm of N1-74……………………………………………………………………………...………………..41
Chapter 2 Table 1. Interaction energies of wild type and mutant SNV-N-Mid docked to VimH- 1A…………………………………………………………………………...………………...…..74
Chapter 3 Table 1. Restraints and structural statistics for 20 NMR structures…………...……..89
Appendix I Table 1. Structural statistics for 20 NMR structures of Andes virus N1-74 coiled coil domain………………………………………………………………………………………...…148
LIST OF FIGURES
Introduction Figure 1. The Life Cycle of Hantaviruses…………………………………...……12
Introduction Figure 2. Properties of Hantavirus Proteins………………………………………16 iv
Prologue Figure 1. Outline for the Interactions of Hantavirus proteins described in this dissertation ………………………………………………………………………………………28
Chapter 1 Figure 1. Assigned 1H – 15N HSQC spectrum of Andes virus N1-74 domain ……..…36
Chapter 1 Figure 2. Heptad repeats of conserved hydrophobic residues form the interface of the helix alpha1 and alpha2 that stabilize the coiled coil domain ………………………...... …39
Chapter 1 Figure 3. CD spectra of wild type N1-74 (WT) and point mutants (K26E, (R22F)...…40
Chapter 1 Figure 4 Immunocytochemistry of full-length n protein with point mutations in the N1-74 coiled coil domain…………………………………………………………………...………43
Chapter 1 Addendum Figure 1.Granular versus Globular distribution of N in transfected Cos-7 cells …………………………………………………………………………………………...….51
Chapter 1 Addendum Figure 2. Cross linking of ANDV N proteins individually expressed….52
Chapter 2 Figure 1. Structure and Assembly of Intermediate filaments…………..…...…….…55
Chapter 2 Figure 2. Immunocytochemistry of Hantavirus genotypes…………………...…….. 63
Chapter 2 Figure 3. Co-immunoprecipitation of Hantavirus Genotypes and SNV-N- Mid………………………………………………………………………………………………..65
Chapter 2 Figure 4. Model for the SNV-Npro middle region …………………...…………..…66
Chapter 2 Figure 5. Primary Sequence of SNV-N-Mid…………………………………...……67
Chapter 2 Figure 6.Co-immunoprecipitation of Vimentin with Mutant Nucleocapsids.…….…68
Chapter 2 Figure 7. Helix-Loop-Helix motif………………………………………...…………69
Chapter 2 Figure 8. Primary sequence of the Vimentin Head…………………………..………71
Chapter 2 Figure 9. Model for VimH-1A……..……………………………………………..….72
Chapter 2 Figure 10. Docking of the SNV-Npro Middle Region to VimH-1A……………...…73
Chapter 3 Figure 1. The G1 tail of Hantaviruses, Nairoviruses, and Orthobunyaviruses (genera of Bunyaviridae) contains a cysteine/histidine-rich region with two CCHC arrays………..…….85
Chapter 3 Figure 2. CD spectroscopy and titration with EDTA and ZnSO4 for recombinant Andes virus G1 tail CCHC-region………………………………………………………………..87
Chapter 3 Figure 3. The Andes virus G1 tail zinc finger domain 9residues 543-599) shows a well dispersed two-dimensional 1H – 15N HSQC spectrum……………………………………...90
Chapter 3 Figure 4. The NMR structure of the Andes virus G1 tail zinc-binding domain reveals two classical beta beta alpha fold zinc fingers that are joined together…………..………………93
Chapter 3 Addendum Figure 1. ChIP assay of pAD-ANDV-M transfected cells……………104
Chapter 3 Addendum Figure 2. Primary sequence of Gn-CT-Arms protein……….…...……105 v
Chapter 3 Addendum Figure 3. Addition of N- and C-terminal arms to ANDV-Gn-ZF is predicted to form a dsRNA binding motif………………………………………………………106
Chapter 3 Addendum Figure 4. Molecular Docking of Gn-ZF-Arms to dsRNA………….…108
Chapter 3 Addendum Figure 5. The ANDV-S viral panhandle……………………...………109
Chapter 3 Addendum Figure 6. Interaction energies and percentage of pretone residues within 3A of the RNA for 11 docking attempts of Gn-ZF-Arms with the ANDV-S-panhandles …..…110
Chapter 3 Addendum Figure 7. The Gn-ZF-Arms protein initially docked with the ZF region on top of the second bulge (g18) ………………………………………………...…………...…111
Appendix I Supplementary Figures for Chapter 1 Figure 1. Sequence alignment of hantavirus nucleocapsid N1-74 coiled coil domain with the conserved hydrophobic and polar heptads highlighted……………...………………………………………………………………146
Appendix I Supplementary Figures for Chapter 1 Figure 2. Secondary C, H, C’, and C chemical shifts…………………………………………………………………………………..147
Appendix I Supplementary Figures for Chapter 1 Figure 3. Immunocytochemistry of full length N protein with mutations in the N1-74 coiled coil domain……………………...…....…149
Appendix II Supplementary Figures for Chapter 3 Figure 1. The proper folding of the hantavirus G1 tail zinc finger domain depends on Zn2+ binding………………………...…..…150
Appendix II Supplementary Figures for Chapter 3 Figure 2. Secondary C, H, C’, C chemical shifts of the Andes virus G1 zinc finger domain……………………………………………..…151
Appendix II Supplementary Figures for Chapter 3 Figure 3. The tautomeric states of the Zn2+ coordinating histidines (His590 and His 564)………………. … ……..…………………151
Appendix II Supplementary Figures for Chapter 3 Figure 4. Effect of point mutation in the Zn2+ coordinating cysteine and histidine residues………………………………………...……152
Appendix II Supplementary Figures for Chapter 3 Figure 5. Effect of point mutation in histidine and cysteine residues that do not coordinate Zn2+ ion……………………………..…153
Appendix II Supplementary Figures for Chapter 3 Figure 6 The structures (top panel) and zinc binding topology (bottom panel) of zinc fingers………………………...…………………154
Appendix II Supplementary Figures for Chapter 3 Figure 7 The hantaviral zinc finger domain does not bind RNA…………………………..…………………………….……………155
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INTRODUCTION
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HISTORY
Hantaviruses are considered emerging infectious diseases because they have caused two major epidemics and new serotypes are continually discovered in non-endemic regions (1).
Hantavirus outbreaks have been reported on the World Health Organization’s Epidemic and
Pandemic Alert and Response Program twice, in 1997 and 2000 (2). As global trade, viral evolution, and host-human interactions increase the likelihood of outbreaks will also increase.
In 1951 approximately 3000 United Nations troops stationed near the Hantaan river during the Korean War were debilitated by illness caused by an unknown disease. Patients displayed fever, headaches, acute kidney failure and hemorrhages (3). Many patients were debilitated for several weeks. This severely hampered the success of their mission. Until 1973 the cause of this outbreak was unknown (4). A virus found in rodents near this site was correlated with the infections in 1951 and thereby named the Hantaan (HTNV) serotype of hantaviruses (5). Further evidence for hantaviruses being the causative agent of these fevers during the Korean War was presented in 1990 when sera from these patients was tested by ELISA using HTNV antigens (3). In this study, 240 out of 245 patients who displayed hemmorhagic fever like symptoms were positive for IgM antibodies against hantaan virus. This prompted further investigation of hantaviruses in Asia and Europe. To date, hantaviruses have a relatively low incidence rate with a fatalaty rate of 5 to 10% in Asia (6). Incidence rate was noted to be related to the growth rate of wheat crops as a food source for the primary reservoir of hantaviruses, small rodents (7). The dogma for incidence rate is defined as: increased crop 2
production increases rodent population which increases human-to-rodent interaction, thereby increasing hantavirus disease incidence.
Before 1993 it was thought that hantaviruses were confined to Europe and Asia. In the
Four Corners region of the United States, members of the Navajo tribe were admitted to the hospital with symptoms like an influenza infection such as fever, muscle aches, coughs, and headaches. Within 24 hours their symptoms drastically changed to life-threatening conditions.
Approximately 56 people contracted hantavirus infections and 7 people died within a period of about 2 weeks (8). Case studies commonly involved previous exposure with rodent infested
buildings in isolated areas. A correlation was made between increased rainfall in the spring
which increased vegetation for rodents in the area. The outbreak was eventually reduced as
health care officials enforced preventive measures to separate humans from infected rodents.
Characterization of the virus’s genetic compostion classified it as a new type of hantavirus (9).
This virus was diplomatically named “Sin Nombre” (SNV) virus as to not offend the local
inhabitants and tourist industry of the four corner’s region. Though this was the first reported
hantavirus infection, local Native Americans stated that very similar diseases have occurred as far back as the 1940s (10). A milder outbreak occurred in 1998 as a result of El Nino conditions increasing rainfall in the US (11). Since the 1993 outbreak, SNV has caused over 450 cases of disease with a fatality rate of 35% in North America (12, 13). Additional genotypes have been isolated in North America, they include; Prospect Hill (PHV), New York (NY-1), and Black
Creek Canal (BCCV).
To date hantaviruses have been identified throughout the world. Findings of hantaviruses in North America spurred investigation in South America (New World Hantaviruses).
Interestingly, hantavirus incidence rates are higher in South America than North America. In
South America the dominant serotype is Andes (ANDV), followed by Laguna Negra (LNV),
Lechiguanas (LECV), Maciel (MACV), and Pergamino (PGMV) (14, 15, 16). ANDV has a 3
slightly higher incidence rate than SNV (12). Both have case fatality rates of ~35%. ANDV was the first and only genotype reported to have the ability to transmit from human to human (17). In general hantavirus infections in Europe and Asia occur more often with a lower case fatality. The serotypes of Puumala (PUUV) and Dobrava (DOBV) have both caused mild outbreaks in Europe
(18, 19, 20, 21). Further identification of Asian genotypes discovered the Seoul (SEOV),
Topografav (TOPV), and Thattapalayam (TPMV) hantaviruses. Recently hantaviruses have been found in rodents of Australia and Africa (22, 23). The medical impact of hantaviruses in these continents is under investigation.
EPIDIMIOLOGY
Studies of hantavirus distributions reveal a common link between rodent population and hantavirus serotypes. Viral genotypes correlate with the niche of their rodent host (Table 1).
Rodent populations can have an incidence rate of up to 50% (24). Measurement of incidence rates by the presence of both IgG and IgM antibodies suggests that rodents infected within a period of two months still produce antibodies against the virus (25). Despite being infected, producing a humoral immune response and carrying the virus for most of their lifetime, rodents do not demonstrate any symptoms caused by the Old World viruses (26, 27). Mild disease-like symptoms have been observed in deer mice as a result of SNV infection (28). This suggests a long term co-evolution between virus and host (12). The virus is transmitted between rodents during contact between each other by saliva (24). Inhalation of dried feces and urine from infected rodents is the most common mode of rodent to human transmission. With the exception of two cases of ANDV infection, human to human transmission is uncommon (29, 30).
The symbiotic relationship between hantaviruses and rodents suggest that the virus has coevolved with its host. The rare passing of the virus into non-natural hosts such as humans, results in disease. Genetic changes could alter this normal relationship to either cause disease in 4
rodents, increase disease in humans, or increase the likelihood for person-to-person transmission.
Two properties of hantaviruses can promote changes in the genetic composition of the virus. (I)
The viral genome is segmented into three individual negative stranded RNAs (31). Each of the segments is required for viral replication. Genetic shift can occur when a rodent is co-infected with two different genotypes. The segments can reassort and assemble producing new genotypes made of segments from each of the genotypes. Viral reassortment has been detected in naturally occuring co-infections between PUUV and DOBV and between SNV and PHV-like serotypes
(32, 33, 34). Reassortment has also been induced in the laboratory between SNV and ANDV (35,
36, 2). (II) The virus replicates its genome through the use of a proofreading and error correcting deficient RNA-dependent RNA polymerase. Typically RNA viruses that use this enzyme have an error rate of 10–3 to 10–4 nucleotide substitutions/site/year (37). Hantaviruses have been predicted to have an error rate of 10–2 to 10–7 nucleotide substitutions/site/year (38, 39, 40). A
rate of mutation that high would cause a gradual genetic shift in the phenotypic characteristics of
the virus. To date no evidence for changes in the disease transmission or cause of an outbreak
has been directly linked to shift or drift. The potential for genetic changes that result in a highly
communicable and pathogenic virus poses a significant medical threat.
DISEASES
The genetic differences between hantavirus genotypes is responsible for the pathology and severity of the disease (Table 1). New World Hantaviruses (SNV, ANDV, LNV) cause
hantavirus cardiopulmonary syndrome (HCPS) (41). PHV is exceptional because it is not known
to cause disease in humans. Old World Hantaviruses (HTNV, SEOV, DOBV) cause hemmorhagic fever with renal syndrome (HFRS) (42) . In addition to HFRS, PUUV occasionally causes nephropathia epidemica (NE) (43). The severity and pathology of the disease is multi-factorial. The observation that fatality rates correlate with specific serotypes does not specifically implicate the virus as the direct cause for its effects. Other factors such as; 5
immunological health of individual populations in the geographical region of the viral genotype,
specific HLA types of individuals, length of time between diagnosis and treatment, prior
exposure or pre-existing immunity, and amount of exposure, should be considered when
interpreting the effects on humans caused by the virus (12, 44, 45, 46).
Geography Genotype Vector Disease Case Reference Fatality N. America SNV Peromyscus maniculatus (deer HCPS ~35% (47) mouse) PHV Microtus pennsylvanicus unknown unknown (48, 49) (meadow vole) S. America ANDV Oligoryzomys longicaudatus HCPS ~35% (14, 50,51) (long-tailed pygmy rice rat) LNV Calomys laucha (vesper HCPS + ~35% (52) mouse) renal variant Asia HTNV Apodemus agrarius (striped HFRS 1-15% (53, 54) field mouse) SEOV Rattus norvegicus (norway rat HFRS 1-15% (55, 56, 57) and brown rat) Europe PUUV Clethrionomys glareolus HFRS + mild >1% (42, 58, 59) (bank vole) NE DOBV Apodemus flavicollis (yellow- HFRS 1-15% (22, 60) necked mouse) TABLE 1. Distribution, Vectors, and Diseases of Hantavirus Genotype Representatives.
The severity of HFRS can range from severe (DOBV and HTNV), to moderate (SEOV), or mild (PUUV) (61). The onset of the disease appears much like influenza with fevers, headaches, swelling and fatigue. It progresses within a few days to hemorrhaging on the face and conjunctiva in the eyes and mucous membranes. Dysfunction of the kidneys is marked initially by albumin in the urine and then a decrease in urine. Patients display nausea, vomiting, low platelet counts and rashes when the disease worsens. 15% of the patients go into shock at the latest stage of the disease. Those patients that recover are diuretic for several weeks (61, 62).
NE is the milder form of HFRS. Generally the symptoms are similar but much less pronounced. 6
NE is characterized by stomach and backpains. Those infected with PUUV are rarely
hospitalized (63).
While the predominant organ affected in HFRS is the kidney, HCPS is most harmful to
the lungs and heart. Fever, headache, fatigue, and muscle pain begin about 2 weeks after
infection (64). As the disease progresses vomiting and nausea may occur. At the time of full disease onset blood platelets will rapidly decrease and the cardiopulmonary system will elevate
causing head rushes, rapid breathing and high blood pressure (65). Vascular leakage around the
lungs causes pulmonary edema (66). As a result of an immunological response to damage in the
lungs, myocardial lesions are formed (67). In the most severe cases cardiovascular shock can
occur. Often the shock is so severe it is difficult to recover the patient (68). Those patients that
recover require several weeks of hospitalization before the cardiovascular system is repaired (68).
PATHOGENESIS
The majority of hantavirus antigens are primarily found in endothelial cells of the lungs
for New World hantaviruses and the kidneys of Old World hantaviruses. Antigen is also found
in high concentration in macrophages and monocytes and lower concentrations in the peripheral
organs (28). The common pathology of patients of HCPS or HFRS is increased capillary
permeability in the infected tissues (69, 70). Little is known to explain the tropism of viruses
which cause HFRS to the kidneys. It is speculated that initial infection occurs in macrophages of
the lungs from inhaled virus which then travel to the kidney and initiates the renal disease (71).
Despite being permissive to hantavirus infection the endothelial cell line VeroE6 (African green spider monkey kidney) does not exhibit cytopathic effect. Because the virus itself is not responsible for cellular damage it has been concluded that the vascular permeability is a result of cellular immunological responses to the infection. In fact, CD8+ T cells, macrophages and monocytes are found densely associated with infected endothelial cells in patients (65, 66).
Presumably, the action of these immune cells on endothelial cells would result in vascular 7
leakage (72). The recruitment of immune cells to the region is induced by many processes.
Pathogenic hantaviruses induce interferon type I responses to upregulate interferon inducible genes such as MxA (73, 74). They also induce the expression and secretion of chemokines and cytokines such as RANTES from the infected cells (75). Cytokines TNF -α, IL6, and IL10 have been found at elevated levels in the serum of HFRS patients (76) . In addition to the cellular effects caused by immune cells on infected endothelial tissues, some reports indicate that hantavirus infected cells may undergo apoptosis (77, 78, 79). Together, these cellular responses are thought to result in vascular leakage.
Studies on pathogenesis have mostly been performed using autopsies of patients who did not survive the disease. Because pathogenesis is rare in the natural rodent host of the virus there is not a good model system (28, 80, 81). Therefore, a model system was developed using Syrian hamsters to study pathogenesis (82). Syrian hamsters are injected with a mild dose of ANDV and monitored for disease. About 24 days post injection, fatality rates are 93%. Pathogenically this model mimics human infections with regard to length of incubation, pathological effects like vascular leakage and histopathological damage as well as distribution of antigen in tissues (82).
In addition to being a useful model for pathogenesis, it has also been used to study the efficacy of vaccines and antiviral treatments.
TREATMENT
The CDC recommends the best defense against seasonal transmissions and potential outbreaks of hantaviruses, is prevention (83). Prevention includes; avoidance, sanitation, engineering controls, and chemical repellents to separate humans from rodent hosts. In the event that the virus is transmitted to humans early detection is critical. Detection is complicated because the virus may incubate for up to 3-4 weeks after exposure and initial symptoms are similar to the seasonal flu. Patients admitted to the hospital with flu-like symptoms which 8
develop into severe disease are often diagnosed by the presence of viral RNA in their serum using
RT-PCR (84). Additional confirmatory tests involve hemagglutination assays, immunoflourescence assays, and ELISAs which use IgM type antibodies (85, 86, 87, 88). Upon conformational diagnosis of hantavirus infections the best treatment is supportive therapy in intensive care wards to control bodily fluid levels, reduce fevers, and control of blood pressure, and in severe HCPS infections dialysis is performed (89, 90).
HCPS and HFRS pose two different types of medical challenges for future management of hantaviruses. Because of the higher fatality rates and lower incidence rates of the HCPS caused by most New World viruses it is suggested that early detection methods and development antiviral drug treatment are the best medicine. Conversely the Old World hantaviruses with much higher incidence rates warrants the development of vaccines and prophylaxis. To date there have been several advances to develop these tools.
VACCINES
Several approaches to develop a successful vaccine for both new and Old World
hantavirus have been attempted. Hantavax, a formalin inactivated viral vaccine, was developed
and licensed for use in Korea in 1988 to protect against SEOV (91) . The established delivery of
this vaccine, two doses within a two month period, did not elicit high enough neutralizing
antibodies to reduce virus replication (92). This protocol was not considered for use on New
World viruses, partially because of the safety regulations concerning the production of the live
virus for attenuation (93). Other vaccine strategies are aimed at safely delivering either genes
that will express hantavirus proteins or injecting purified hantavirus proteins safely, while
combining two or more serotypes to cover potential infections in different geographic regions,
and the ability to elicit both neutralizing antibodies and cytotoxic T-lymphocyte production (94).
Viral vector vaccines are attractive for their high level expression of hantavirus proteins and 9
relative safety. Vector systems used include vesicular stomatitis virus, vaccinia, cytomegalovirus, and adenovirus (95, 96, 97, 98, 99). Gene gun delivery of plasmids expressing hantavirus genes has also been very successful at eliciting an immune response and some immunological memory (100, 101, 102). Recombinant hantavirus proteins have been delivered to mice through fusion with hepatitis B core particles and with HSP70 (103, 104). Each of these systems either elicited neutralizing antibodies or elevated cytotoxic T-lymphocyte production in small rodents and in some cases provided immunological memory. While there is currently no
US FDA approved vaccine for hantavirus these strategies show promise and should continue to be investigated as potential vaccine candidates.
ANTIVIRAL DRUG THERAPY
In addition to clinical supportive therapy for infected patients a few drugs and immune components are currently under consideration as potential treatments for hantavirus infections.
The biggest challenge to antiviral therapy is that most treatments are not effective when the disease is most severe. In many cases early diagnosis of the disease and treatment with antivirals have reduced the mortality rate within infected patients. Ribavirin is an antiviral commonly used to treat severe influenza infections. It is a nucleoside analog which is thought to function by increasing the mutation rates of the virus during replication thereby decreasing the virulence of the virus. Ribivirin was administered intravenously at the onset of detecting hantavirus proteins in the serum of HFRS patients. This treatment resulted in a sevenfold reduction in the severity of the disease (105). Currently, intravenous Ribivirin treatment is considered for a investigational new drug protocol for treating HFRS (106). Unfortunately, Ribivirin is not as effective in treating HCPS. Treatment of patients with hantavirus proteins in their serum and displaying cardiopulmonary syndrome with Ribivirin did not result in any clinical benefit as compared to those patients receiving placebo (107, 108). 10
Several alternatives to Ribivirin have been investigated for use against HFRS. The
Ribivirin analog, 1-β-d-ribofuranosyl-3-ethynyl-[1,2,4]triazole (ETAR), causes greater
mutational defects and decreases viral reduction as measured by plaque reduction assays of
HTNV and ANDV than Ribivirin (109, 110). N-(1)-3-fluorophenyl-inosine is another nucleoside analog which decreases viral titers twice as effectively as Ribivirin in HTNV infected cells (111). The use of Ribivirin in combination with Lactoferrin, another common antiviral, had a synergistic effect on reducing plaque formation in HTNV infected cells (112). Currently, these potential antiviral treatments have either not been tried against New World hantaviruses or have not shown any significant level of virulence reduction.
Antivirals currently being developed for the treatment of HCPS include immunotherapy and short peptide inhibitors. Immunotherapy is most effective in treating infections of multiple serotypes using one formulation of polyclonal antibodies (113, 114). Two studies showed that serum from SNV or ANDV infected patients was effective in animal models to passively protect against a challenge for their respective homologous serotype (115, 116). Immunotherapy has also been effective during the most severe stage of the disease (117, 118). Sera from rhesus macaques producing antibodies to ANDV antigens were administered to hamsters 5 days after they were treated with a lethal dose of ANDV (115). Fifteen out of sixteen hamsters survived the infection with no clinical support other than the sera (115). This suggests that immunotherapy could offer hope as a post-exposure prophylaxis.
Serendipitously, it was discovered that a single-chain variable fragment (scFv) antibody against the cellular receptors of endothelial cells blocks attachment and subsequent infection of these cells (119). Hjelle et al. have targeted the attachment phase in the viral life cycle in their design of a potential antiviral drug (120). From a screening of multiple cyclic peptides of nine or more amino acids 8 peptides reduced the infection of cells by 50% (120). These peptides have 11
similarity to hantavirus protein sequences. Conversely, peptides were made that complemented these hantaviruses in order to block the viral protein responsible for attachment. The use of these peptides reduced viral infection by up to 78% (121, 122). The cyclic peptide, [CQATTARNC] was most effective (122). This strategy has not been employed in animal models thus far.
The majority of these proposed treatments are still in the developmental stage. Many of the treatments have not been tested for their efficacy on New World hantaviruses. To date there is no FDA approved treatment for HCPS. In the event of a large outbreak, medical facilities would become overburdened from attempting to provide traditional supportive therapies. There is a need for more potential antiviral treatments. The basis for the design of these antivirals is a thorough understanding of the molecular processes in the life cycle of the virus. Two examples of potential antiviral drugs which were developed from a knowledge of viral molecular biology are; Ribivirin affects the ability of the genome to replicate based on the mechanism of the polymerase and the cyclic peptide [CQATTARNC] exploits virus to cell attachment and prevents infection in cells, not in in vivo models. To design alternative antivirals a better understanding of the molecular interactions occurring within the virus is needed.
REPLICATION AND ASSEMBLY
The genus of Hantaviruses is classified into the Bunyaviridae family (123). Other genuses of the Bunyaviridae include the Orthobunyaviruses which are represented by the
Bunyamwera virus, phleboviruses represented by Rift Valley Fever Virus (RVFV) , Tosporivirus, represented by Tomato Spotted Wilt virus, and the Nairoviruses represented by Crimean Congo hemorrhagic fever virus (CCHFV). Bunyaviruses are arthropod-borne with the exception of hantaviruses which are rodent-borne. Another exception is that Tosporiviruses infect plants while the other four genuses infect mammals (124). It is uncommon that these viruses cause disease in humans. Outbreaks of many of these viruses can impact livestock and agricultural production 12
(125, 126). Interestingly, CCHFV viruses are considered a potential biowarfare agent and have been on the CDC select agents category A list for many years (127). Many of its genetic components are similar to hantaviruses.
FIGURE 1. The Life Cycle of Hantaviruses. Virions attach to integrins on the cell and enter through endocytosis (top left). Dissociation of the viral RNA and proteins initiates replication of an intermediate positive-strand RNA used for either replication of (-) sense viral RNA or translation of the four viral proteins; Nucleocapsid (Npro, N), Glycoproteins (Gn) (Gc), and the RNA-dependent RNA polymerase (RdRP). Assembly occurs in the Golgi where it is speculated that either RNA or Npro interacts with Gn in the Golgi membrane. The virion egresses from the cell (top right).
The commonality of the bunyaviridae family is its genetic composition (128). Genomes in this family are composed of 3 negative sense RNA segments. These negative sense RNAs are converted into positive sense mRNAs by the viral RNA-dependant RNA-polymerase (RdRP).
Each of the mRNAs encodes a single protein such that the smallest RNA (S) codes for the nucleocapsid (Npro), the largest RNA (L) codes for the RdRP, and the medium sized (M) codes 13
for the glycoprotein precursor (GPC) (128, 129, 130). This precursor is cleaved by cellular
proteases to produce the N-terminal (Gn) and C-terminal (Gc) glycoproteins (131). The 3 vRNA segments, Npro, and RdRP form the ribonucleoprotein complex (RNP) which is assumed to interact with the two glycoproteins and the Golgi membrane and possibly the cytoplasmic membrane to form the virion (132, 133, 134).
The viral life cycle begins when the virion attaches to the cell (Fig. 1) (135). The viral glycoproteins primarily attach to integrins on the surface of epithelial cells (136). Interestingly, pathogenic and non-pathogenic virions bind to different integrin variants. It was shown that the
NY-1 serotype binds alphaV beta3 and PHV binds to alpha1beta3 integrins (137, 138). The virus fuses with the cell and enters the cell in clathrin containing, low pH, endosome vesicles (139,
140, 141). In acidic endosomes, the viral components dissociate from the viral membrane and begin replication in the cytoplasm (142). As the RNP disassembles the Npro protein directs the vRNA into the RdRP for synthesis of (+) mRNA (143). The RdRP captures cellular RNA templates to prime the synthesis of mRNA from vRNA (144, 145). mRNA is then chaperoned to the ribosomes by the N protein (146). The nucleocapsid protein mimics the activity of the cellular eukaryotic Initiation Factor 4F (eIF4F) by binding the 5’end of the viral mRNA, binding the 43S ribosome, and unwinding any secondary structure in the mRNA to initiate translation
(146). Expression of the GPC, which is subsequently cleaved into Gn and Gc, initiates the viral assembly process as these proteins traffic to the Golgi (147, 148, 149). It is speculated that upon reaching a threshold level of viral protein expression the RdRP switches to viral RNA replication
(150). The RdRP produces cRNA as a template for the replication of (-) RNA. It is this step in the viral replication process that is affected by Ribivirin treatment because the incorporation of nucleoside analogues into the RdRP disrupts the replication process, resulting in an increased mutational rate (110). In untreated cells, each of the segments are encapsidated by the Npro protein within the first 2 hours of infection (151). The mechanism by which vRNA segments, 14
Npro, and RdRP are directed to the Golgi membrane to interact with Gn and Gc and assemble the
virions is unkown (150). The appropriate use of the cytoskeletal components; actin, tubulin, and
vimentin as well as endoplasmic reticulum Golgi intermediate compartments (ERGIC) are likely
to play a significant role in directing the N protein to the Golgi (152, 153). Though some reports
suggest assembly of the virion at the cell membrane, it is generally thought that the viral
components assemble together in the Golgi and the formed virion egresses out of the cell (133,
154). It takes approximately 24 hours for the first release of progeny virions after the initial infection of cells in culture (151) .
VIRAL COMPONENTS
Hantavirus replication is dependent on the ability of the virus to enter the cell, replicate the genome, assemble the virion and avoid the immune response of the cell. Many studies have been conducted to define the functions of motifs in the viral proteins and RNA. Genetic studies involve aligning the known sequences of the many serotypes and assigning functions to conserved motifs. These functions are tested by modifying the proteins expressed either individually or together in cell culture. A reverse genetics system was designed to test the replication of hantavirus-like (-) vRNA “minigenomes” (155). This system’s main benefit is that it can test the requirement of specific features of the viral components to replicate the genome.
To date there is no system which can be used to modify the genetics of a live hantavirus in order to study its phenotypic characteristics. Despite the inability to study the effect of genetic manipulation on the infective virus, the functions of many sequence motifs in hantaviral proteins have been identified as having a potential role in the viral life cycle.
The function of the RdRP is to transcribe the negative sense RNA genome into mRNA for protein translation and to use this positive sense RNA as a template to replicate the genome into negative sense RNA segments (145). This protein is found in small quantities in the virion 15
prior to infection and in the perinuclear region of the cytoplasm during infection (156, 157). The
protein is an approximately 250kDa and 2153 amino acids. To date no work has been performed
to determine the structure. The protein has been divided into five conserved regions A, B, C, D,
and E (158, 159). The relevancy of these motifs to the function of the RdRP is currently
unknown. Based on the similarity of hantavirus RdRP sequences to the sequences of
Bunyamwera RdRP it is speculated that a group of aspartates in motifs A and C are required to
dissociate any secondary structure in the RNA in preparation for transcription (160, 161). Other
than what can be inferred from other known viral polymerases the structural motifs of hantavirus
RdRP are open for discovery.
The two glycoproteins Gc and Gn are cleaved from a glycoprotein precursor in the ER
(131). Both of these proteins make up the spikes on the surface of the virion (133, 134). During
infection the proteins are found either associated with the ER or in the Golgi where they are
retained. The major role of both these proteins is to attach the virion to integrins on the cell
surface, fuse the viral membrane with the cell membrane, and initiate the formation of the virion
in the Golgi complex (162, 163). Both proteins are classified as type I membrane proteins
because the large N-terminal portion is exposed extracellularly (162). This extracellular region,
often called the ectodomain, contains N-linked glycosylation sites which are responsible for
appropriate trafficking of the protein, and cellular attachment (135, 164). Gc contains a
hydrophobic region in the N-terminal portion which has been identified as the fusion peptide to drive the entry of the virus into the cell (140). The ectodomains of both glycoproteins provide surface epitopes that induce neutralizing antibodies in infected humans (113, 165). On the C- terminal side of the transmembrane domain of both glycoproteins there is a cytoplasmic tail which is located internal to the virion. Gc contains a short (8-10 amino acid) tail of positively
charged residues. Gn has an unusually long cytoplasmic tail with some notable features.
16
FIGURE 2. Properties of Hantavirus Proteins. A. Translation of the 4 viral proteins occurs from three negative sense viral RNA segments (S, M, L). B. Mapping of the domains in viral proteins includes regions important to viral infection.
The cytoplasmic tail of Gn is approximately 135 amino acids long. This is significantly longer than most viral transmembrane proteins (166). Because it has been shown to regulate several cellular responses, it is implicated in the pathogenesis of the virus (73). The tail contains two immunoreceptor tyrosine activation motifs (ITAM) which alter the phosphorylation- 17
dependent immune cell signaling in response to viral infection (167). It contains both tyrosines
and a hydrophobic region identified as a degron motif that directs the proteosomal degradation of
Gn thereby disrupting the regular cell protein degradation system (168, 169). This process is
thought to contribute to viral immune evasion (169). The cytoplasmic tail also inhibits RIG-1 and TBK-1 directed interferon responses which are normally upregulated as a response to infection by RNA viruses (170). The binding of TRAF3 by the tail is associated with disruption of interferon responses (171). The specific residues for this interaction have not been identified as of yet.
The nucleocapsid is the most abundant protein in the virion and in infected cells.
Though it has many functions involved in both cellular regulation and viral assembly, its main function is to bind the viral RNA and protect it from cellular RNAses (150). The protein is found inside the virion and in a cytoplasmic region around the nucleus which is associated with the
ERGIC in infected cells (133, 152, 154). The protein exists in monomer, dimer, and trimer form
(172). Domains required for oligomerization have been mapped to alpha helical structures in both the N and C terminal ends (173, 174). The RNA-binding domain has been mapped to the conserved central region (175). The formation of RNP complexes is linked to both oligomerization and RNA binding processes as a major step in virion assembly (150). The nucleocapsid also binds several cellular proteins with the intent of regulating the cellular immune response to the infection (150). A conserved region in the middle of the nucleocapsid was mapped and found to bind the small ubiquitin-like modifier SUMO-1 (176). This binding is thought to localize the protein to the cytoplasmic region (150). Other cellular binding partners of the nucleocapsid which have not been mapped or whose effect on viral pathogenesis has been elucidated include; death domain associated protein (DAXX), myxovirus resistance protein A
(MxA), and actin (74, 153, 177). Because Npro is highly abundant in infected cells it is likely 18
individual poplulations of Npro can be involved in regulating the different cellular processes and
assembling the virus simultaneously.
Each of the three viral RNA genomic segments shares a common stem-loop structure
(178). The large loop is single stranded and contains the open reading frame (178). The stem is formed by slightly imperfect base pairing between the first and last 16-25 bases of the genome
(178). During replication the two ends hybridize together forming a panhandle. The panhandle region is the minimal length of RNA to be recognized by the nucleocapsid trimer (179, 180). The pattern of base-pairing followed by unmatched bases creating bulges in the panhandle region directs the interaction between the RNA and the nucleocapsid (181). These interactions are specific for each of the genotypes (182). It stands to reason that the specificity for viral RNA packaging by the nucleocapsid of certain serotypes would determine the ability of reassortants between different serotypes. The panhandles are part of the non-coding regions found on both ends of the RNA (179). Though the panhandle regions contain the necessary RNA secondary structure for N-recognition, they do not contain poly-adenylation sites to enhance the translation
of the open reading frames (128). Upon replication of the (-) RNA to mRNA the binding of the
nucleocapsid replaces the polyA binding proteins and promotes translation of the RNA in the
cellular ribosomes (146). To date there has been no significant structural motifs found in the
single stranded RNA loop region. Its purpose appears to be the site for the ORF. Further
research may identify that the loop region contains secondary structure.
19
______
PROLOGUE:
Identification of Hantavirus Nucleocapsid Binding partners suggests essential interactions in the assembly process
______
INTRODUCTION
The hantavirus nucleocapsid (Npro) is the most abundant protein in the virion and the
most highly expressed protein in infected cells. Additionally, the recombinant expression of the
nucleocapsid protein is robust. There are also multiple antibodies commercially available to
detect the protein. This protein is considered to play multiple roles during the infective process
by binding many cellular partners and coordinating the viral assembly process through
interactions with the viral genome and viral proteins (150). The majority of studies to determine
the function of this protein based on its binding partners have used yeast two-hybrid assays which
overexpress cellular proteins that are synthetically fused to the appropriate protein domain for the
assay. In these assays the nucleocapsid is expressed alone without other viral proteins to
influence its activity. In this study we identified several cellular proteins which co- immunoprecipitated (Co-IP) with the nucleocapsid in Sin Nombre genotype (SNV) infected cells.
Several proteins were identified as potential binding partners to Npro. The overall focus of this dissertation will be to relate binding properties to structural motifs within hantavirus proteins.
20
METHODS
Co-Immunoprecipitation Assay
VeroE6 cells were grown overnight in T175s at 37 °C and 5% CO2 in Dulbecco's
modified Eagle's medium containing 10% fetal bovine serum. Cells were infected with SNV at
0.1 multiplicity of infection and incubated for 7 days. One flask of cells was not infected as a
negative control. At time of harvest, the cells were washed 2 times with ice cold phosphate
buffered saline (PBS; pH 7.4.) Cells were lysed in 5 ml lysis buffer (50 mM Tris-HCl [pH 7.4],
150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% Tween 20, 10 µl protease inhibitor
cocktail/ml). Cells were removed from the flask by scraping and passed through a 22-gauge
needle three times to dissociate cellular material. Cellular debris was removed by centrifugation
at 4,000 x g for 10 min. The supernatant was transferred to a 1.5ml tube. 5ul of Normal mouse immunoglobulin G (5ug/ml) was added to the supernatant, and the mixture was rotated for 30 min
at 4°C. Lysates were treated with monoclonal anti HN-2 antibody (Santa Cruz) and rotated at 4°C
for 2 hours. 50 µl/ml of protein G plus agarose beads was added and rotated at 4°C overnight.
The complexes were spun at 6,000 rpm for 3 minutes. The precipitate was washed and spun with ice-cold PBS 5 times. The protein complexes were removed from the beads by the addition of 2X
Laemmli sample buffer (Bio-Rad) containing 20% 2-mercaptoethanol and then heated to 95°C for
5 min.
Samples were acetone precipitated overnight and resuspended in 100ul of 2X Laemmli sample buffer. 10ul and 40ul of samples were loaded on either a 7.5% or a 12% SDS-PAGE gel.
Gels were coomassie stained and visualized on a BioRad VersaDoc Imager. Intensity of IgG heavy and light chains was used as a frame of reference to compare the intensities of the uninfected and infected cell immunoprecipitates. Those bands which had a significantly higher intensity in the infected cell lane were cut and processed for tryptic digestion. 21
MALDI-TOF identification of proteins
Bands are digested using InvestigatorTM ProprepTM (Genomic Solutions, Ann
Arbor;MI), using a previously described protocol (Rosenfeld, J., et al. Analytical Biochemistry
(1992) 203. pp173-179) with some modifications. Samples are washed twice with 25mM ammonium Bicarbonate (ABC) and 100% acetonitrile, reduced and alkylated using 10mM DTT and 100mM Iodoacetamide and incubated with 75ng Trypsin in 25mM ABC for 6 hrs at 37 °C.
Samples are prepared and spotted onto a MALDI (Matrix Assisted Laser Desorption
Ionization) target with ZipTipu-C18 (Millipore, Billerica; MA). Samples are aspirated and dispensed 3 times and eluted with 70% ACN, 0.2% formic acid and overlaid with 0.5μl (5mg/ml)
MALDI matrix (α-Cyano-4-hydroxycinnamic acid) and 10mM ammonium phosphate.
All mass spectrometric data was collected using an ABI 4700 MALDI TOF/TOF
(Applied Biosystems, Foster City; CA). The data was acquired in reflector mode from a mass range of 700 – 4000 Daltons and 1250 laser shots were averaged for each mass spectrum. Each sample was internally calibrated on trypsin’s autolysis peaks. The eight most intense ions from the MS analysis, which were not on the exclusion list, were subjected to MS/MS. For MS/MS analysis the mass range was 70 to precursor ion with a precursor window of -1 to 3 Daltons with an average 5000 laser shots for each spectrum. The data were stored in an Oracle database.
The data was extracted from the Oracle database and a peak list was created by GPS
Explorer software (Applied Biosystems, Foster City; CA) from the raw data generated from the
ABI 4700. This peak list was based on signal to noise filtering and an exclusion list and included de-isotoping. The resulting file was then searched by Mascot (Matrix Science, Boston; MA). A tolerance of 20 ppm was used if the sample was internally calibrated and 200 ppm tolerance if the default calibration was applied. Database search parameters include 1 missed cleavage, oxidation of methionines and carbamidomethylation of cysteines. 22
RESULTS AND DISCUSSION
In order to identify both cellular and viral binding partners of the Sin Nombre virus nucleocapsid (SNV-Npro) for the ultimate purpose of identifying binding domains as potential antiviral drug targets, we immunoprecipitated viral and cellular proteins bound to the nucleocapsid in SNV infected cells. Bands visualized on the coomassie gel were compared between the non-infected and infected samples. Bands unique to the infected sample were analyzed using MALDI-TOF analysis. We found 20 unique bands that were immunoprecipitated with the nucleocapsid. Reported here are proteins which have ion scores (CI%) of at least 98%, have molecular weights that match their mobility on the SDS-PAGE gel, and have peptide matching to genuses of either rhesus, macaca, or homo (Table 1) . The majority of these proteins are unreported binding partners for nucleocapsid. Those proteins previously identified by yeast two hybrid screening to be bound to nucleocapsids of Old World hantavirus Hantaan ( HTNV),
Seoul (SEOV), Tula (TULV), and Puumala (PUUV) were not detected by this assay on SNV
Npro (142, 183, 184). Identification of bands in this assay is dependent on their visual intensity on a coomassie gel as a property of the total number of molecules bound to the nucleocapsid in the pulldown. It can’t be ruled out that proteins such as DAXX, SUMO-1, and UBC-9 may in fact also bind the New World hantavirus of SNV though their concentration is below the detectable level of this assay. Instead of finding the expected partners for Npro, many novel nucleocapsid binding proteins were identified. Though there are no current reports describing their interaction with Npro, the significance of these findings to hantaviral infections warrants some consideration because many of these cellular proteins have been reported to either bind directly to viral proteins in other families or play significant roles in viral infections in general.
Alteration of cellular metabolism by virus infected cells is considered one of the main factors towards pathogenesis in hepatitis infected liver cells, rotavirus infected enterocytes, and 23
Band Protein Annotation Protein Molecular Protein # of # Accession Weight Score Pep- Number (kD) (CI%) tides
1 tubulin 5-beta [Homo sapiens] CAA25318 49598.9 100 7
2 ACTB protein, Actin [Homo sapiens] AAH12854 40194.1 100 15
3 glutathione S-transferase P [Macaca mulatta] NP_001036141 23423 100 13
4 peptidyl-prolyl cis-trans isomerase B precursor P23284 22728 98.8 6 (PPIase) [Homo Sapians]
5 ribosomal protein S15a [Homo sapiens] CAA59127 14844 100 5
6 heat shock 10kDa protein 1 (chaperonin 10), isoform EAW70154 9257.9 99.9 3
7 heat shock 70kDa protein 8 isoform 1 [Homo sapiens] NP_006588 70854.2 100 13
8 heat shock 90kDa protein 1, beta isoform 5 [Macaca XP_001098925 72800.8 100 16 mulatta]
9 phosphoglycerate mutase 1 isoform 1 [Macaca mulatta] XP_001082686 25864.3 100 10
10 H3 histone, family 3A [Homo sapiens] CAH73371 14043.8 99.9 4
11 granulin, isoform CRA_b [Homo sapiens] EAW51601 46724.6 99.7 3
12 heat shock 60kDa protein 1 (chaperonin) [Pongo abelii] NP_001127086 60959.3 100 13
13 nucleocapsid protein [Sin Nombre virus] AAG03030 48194.9 100 19
14 keratin 1 isoform 1 [Macaca mulatta XP_001097988 61838.3 100 11
15 vimentin [Macaca fascicularis] Q4R4X4 53733 100 58
16 lactate dehydrogenase A isoform 2 [Macaca mulatta] XP_001084264 36893 99.8 19
17 histone H1.4 (H1 VAR.2) (H1e) [Macaca mulatta] XP_001086400 21882 99.9 14
18 histone H1.2 (H1d) [Macaca mulatta] XP_001084417 21594 99.8 10
19 lamin A/C [Macaca fascicularis] BAD51965 65098 100 24
20 myosin VI [Macaca mulatta] NP_001098006 146340 100 26
Table 1. Proteins identified as potential binding partners for SNV-Npro using co- immunoprecipitation and MALDI-TOF peptide analysis. 24
HIV infected lymphocytes (185, 186, 187). It is thought that as part of a viral immune evasion
strategy the virus would act to moderate cellular metabolic processes to maintain normal cell
function during the infection. One mechanism for this may be to bind and sequester proteins
which are often over-expressed as a response to the infection. In the co-IP experiment we
identified nucleocapsid binding to three metabolic related proteins; phosphoglycerate mutase 1,
glutathione S-transferase P, and lactate dehydrogenase A. These enzymes are involved in the processes of glycolysis, lipid detoxification, and oxygen-dependent lactic acid production (188).
These processes could all be potentially increased during viral infections. More specifically
lactate dehydrogenase levels are highly elevated in tissues of the upper respiratory tract of HIV
patients (189). Although no current research suggests that hantaviruses altar cellular processes
and the possibility that the nucleocapsid may act to reduce this alteration through its binding
actions is mere speculation, these possibilities do suggest further research in this area may
provide insight into hantavirus pathogenesis.
Heat shock proteins (HSPs) were first identified as proteins expressed in abundance in
cells stressed by heat or other stresses such as viral infection (190). HSPs are considered part of
the innate immune response because HSP70 is part of the antigen presentation process (191).
They are also important for chaperoning proteins in the ER to support proper folding (192).
Several viral proteins bind to HSP90, primarily while being presented on the cells to MHCs
(191). The relationship between HSPs and viral proteins contributes to the pathogenesis of the
virus. Structural proteins from rotavirus, japanese encephalitis virus, and simian virus 40 bind to
HSP70 on the cell surface to attach and enter into cells (193, 194, 195). HSP60 is bound by the
HIV integrase, hepatitis B X protein, and hepatitis C core protein to influence apoptotic pathways
(196, 197, 198). SNV nucleocapsid was found to bind to HSP90, HSP70, HSP60, HSP10, and
cyclophilin B (a protein up regulated during heat shock) in this co-immunoprecipitation
experiment. Interestingly, HSP90 inhibitors were reported to reduce the infectivity of multiple 25
negative stranded viruses including la crosse virus, a member of the bunyaviridae Family (199).
It was shown that HSP90 stabilizes the RdRP of these viruses to promote replication (199).
Because Npro and RdRP are cooperatively involved in replication of the hantavirus genome, it could be speculated that the binding of Npro to HSP90 stabilizes the RdRP and therefore promotes the replication of the process. Further experiments are needed to further investigate the roles of HSPs in hantavirus infections.
Several nucleus- and ribosomal- associated proteins were identified as binding partners for the nucleocapsid. They include; histone H3, histone H1.2, histone H1.4, granulin, lamin A/C, and S15a protein. Presently there is no evidence to suggest that the nucleocapsid would enter the nucleus to be exposed to histones (200, 201). Hantaviruses also do not possess the property of latency which is partially caused by the interaction of herpesviruses with histones (202, 203).
The majority of hantavirus nucleocapsids are distributed in a perinuclear ring around the nucleus
(154). This positions Npro to be in proximity with the nuclear membrane where it could possibly interact with nuclear lamin A/C. Nucleocapsids direct viral mRNA to the ribosomes to initiate translation (146, 204). A possible interaction with Ribosomal S15a protein could occur during the loading of RNA into the ribosome. The binding to histones is a peculiar finding that requires further experimentation.
The nucleocapsid was harvested from bands migrating at ~55, ~110, and ~165. This confirms previous findings that this protein exists as a monomer, dimer, and trimer (172, 205,
206). It’s notable that even under low detergent conditions the self-association of Npro remains intact. The formation of trimers is critical in the process of RNA binding and packaging of the genome (207). The region responsible for oligomerization of Npro has been mapped to both the
N and C terminal regions of Npro (174, 176, 208). These studies were performed for the Old
World viruses of HTNV SEOV, TULV, and PUUV. The N-terminal region was predicted to 26
form two helixes in a coiled coil type structure (205, 209). To further relate the predicted
structure of this region with self-association work presented in Chapter 1 will describe the
structure of the ANDV Npro N-terminal domain and relate its cellular localization to the
process of oligomerization.
During infection viral proteins must interact with multiple components of the
cytoskeleton to traffic throughout the cell and reach the viral assembly site (210). Direct binding
between viral proteins and cytoskeletal components has been broadly reported (211, 212, 213).
Members of the cytoskeleton which could be potential binding partners for the nucleocapsid are;
actin, beta tubulin, keratin, myosin VI, and vimentin. Though keratin is usually considered a
contaminant in MALDI-TOF analysis, this protein will be considered as a potential binding partner keratin is abundant in VeroE6 cells (214). In the Bunyaviridae family the relationship between the cytoskeleton and the virus has been investigated. Crimean Congo Hemmorhagic
Fever virus (CCHFV), Rift Valley Fever virus (RVFV) and Black Creek Canal virus (BCCV) require actin to functionally traffic in the cells suggesting an interaction between viral proteins and actin (154, 215, 216). Disruption of microtubule associated transport by dynamitin reduces the infection of HTNV and CCHFV (217, 218). Most interestingly vimentin was found to be rearranged into cages which surround the Npro protein in HTNV infected cells (201). This phenomenon was not observed for SEOV, BCCV, or ANDV infected cells (201). Chapter 2 of this dissertation will confirm the binding of SNV NPro to vimentin and predict the mechanism of vimentin rearrangement by HTNV and not SNV with a proposed model for the structure of SNV Npro middle region. Because this region also contains the RNA binding domain the significance of vimentin and RNA binding during the assembly process will be discussed (175). 27
It is predicted that both Gn and RdRP would associate with Npro during viral replication
(150, 219). Gn is expected to bind to Npro as part of the assembly process which would link the
membrane proteins to the ribonucleoprotein complex in order to package the genome into the
budding virion. Because both Npro and RdRP are required for viral RNA synthesis it was
suggested that these two proteins would interact (155, 220). Our co-IP experiment did not
identify either Gn or RdRP as a binding partner for Npro. It is possible that the interaction
between RdRP and Npro may be transient and may require co-association with RNA. The conditions of the coIP would disrupt any RNA interactions.
Gn was expected to bind Npro and to have a gel mobility similar to vimentin. Gn was not found as one of the potential binding partners of Npro. Many viruses are known to have interactions between the cytoplasmic tail of their transmembrane containing glycoprotein and their core, capsid, nucleocapsid proteins. Sindbis virus protein E2 binds its nucleocapsid through the aromatic residues in its cytoplasmic tail (221, 222). The cytoplasmic tail of Gp41 and the matrix protein of HIV have been shown to interact (223). The neuraminidase and haemaglutinin glycoproteins of influenza do not have long cytoplasmic tails, however they do associate with the matrix protein M1 (224) . The region near the zinc finger motif in influenza M1 is implicated in the binding to ribonucleoproteins and viral RNA which is free from capsidation (225, 226).
More specifically, Tomato Spotted Wilt Virus, Bunyamwera virus, and Uukanemei virus, members of the Bunyaviridae family, possess interactions between the Gn tail and Npro (227,
228, 229). Each cytoplasmic tail of the Bunyavirus family has conserved cysteines and histidines which are hypothesized to form a zinc finger and have properties similar to the zinc finger in influenza M1. Chapter 3 will demonstrate that this region of the ANDV Gn protein does in fact form a zinc finger motif which potentially could bind viral RNA. The binding of viral RNA could act in place of interactions between Gn and Npro. It will be proposed that the 28
packaging mechanism may occur by Gn binding to RNA which is bound to Npro and together the
RNP complex is incorporated into the membrane for budding of the virion.
The three chapters in this dissertation relate the structure of the hantavirus nucleocapsid
and Gn cytoplasmic tail with its protein binding partners. The bindings of Npro to Npro, Npro to
vimentin, and Gn-tail to RNA are events that occur during the assembly of the virus (Fig. 2). The
long-term goal of this work is to define mechanisms that are essential to infection which could
potentially be exploited as antiviral drug targets.
FIGURE 1 Outline for the Interactions of Hantavirus proteins described in this dissertation.
29
______
CHAPTER 1
NMR Structure of the N-terminal Coiled Coil Domain of the Andes Hantavirus Nucleocapsid Protein
Yu Wang1, Daniel M. Boudreaux1, D. Fernando Estrada, Chet W. Egan, Stephen C. St. Jeor, and Roberto N. De Guzman2
J. Biol. Chem., Vol. 283, Issue 42, 28297-28304, October 17, 2008
1 These authors contributed equally to this work.
______
ABSTRACT
The hantaviruses are emerging infectious viruses that in humans can cause a cardiopulmonary syndrome or a hemorrhagic fever with renal syndrome. The nucleocapsid
(Npro) is the most abundant viral protein, and during viral assembly, the N protein forms trimers
and packages the viral RNA genome. Here, we report the NMR structure of the N-terminal
domain (residues 1–74, called N1–74) of the Andes hantavirus N protein. N1–74 forms two long
helices ( 1 and 2) that intertwine into a coiled coil domain. The conserved hydrophobic residues
at the helix 1- 2 interface stabilize the coiled coil; however, there are many conserved surface residues whose function is not known. Site-directed mutagenesis, CD spectroscopy, and immunocytochemistry reveal that a point mutation in the conserved basic surface formed by Arg22
or Lys26 lead to antibody recognition based on the subcellular localization of the N protein. Thus,
Arg22 and Lys26 are likely involved in a conformational change or molecular recognition when the
N protein is trafficked from the cytoplasm to the Golgi, the site of viral assembly and maturation.
30
INTRODUCTION
Hantaviruses can cause two emerging infectious diseases known as the
hantaviruscardiopulmonary syndrome (HCPS) and the hantavirus hemorrhagic fever with renal syndrome (230). Annually, there are over 150,000 cases of hantaviral infections reported world
wide (231). Rodents are the primary reservoir of hantaviruses, and humans are normally infected
by inhalation of aerosol contaminated with the excreta of infected rodents. The first reported cases
of HCPS in North America (232) was caused by a novel hantaviral species (233) (9), the Sin
Nombre virus, and had an initial mortality rate of 78%. HCPS has since been reported throughout
the United States with a current mortality rate of 35% when correctly diagnosed (234). The major cause of HCPS in South America is the Andes virus, and person-to-person transmission of the
Andes virus was reported in Argentina and Chile (235). Hantaviruses are known to invade and
replicate primarily in endothelial cells, including the endothelium of vascular tissues lining the
heart (236, 237,238).
The genome of hantaviruses consists of three negative-stranded RNAs, which encode the
nucleocapsid (Npro) protein, two integral membrane glycoproteins (G1 and G2), and an RNA- dependent RNA polymerase (L protein). The N protein is highly immunogenic (239, 240) and
elicits a strong immune response, which confers protection in mice (241, 242, 243). It is highly conserved and is the most abundant viral protein, and it plays important roles in viral
encapsidation, RNA packaging, and host-pathogen interaction (150) . The N protein binds to viral
proteins (150), host proteins (142, 153, 183, 184, 200, 244) and viral RNA (180, 181, 182, 207,
246). The self-association of the N protein into trimers was shown by gradient fractionation and chemical cross-linking (172). Deletion mapping identified that regions at the N and C termini are
important in Npro-Npro interaction (172, 208, 247), and a model of trimerization was proposed 31
based on the head-to-head and tail-to-tail association of the N-terminal and C-terminal domains, respectively (174, 247).
The N-terminal region in the Sin Nombre virus N protein (residues 3–73) (209) and the
Tula virus (residues 1–77) (205) were predicted to form coiled coil domains. Recently, the structure of the N-terminal coiled coil domain (residues 1–75 and 1–93) of the Sin Nombre virus was determined by crystallography (248). The highly conserved hydrophobic residues stabilize the structure of the coiled coil; however, there are highly conserved polar residues that appear to
have no function in stabilizing the coiled coil domain. Here, we report the solution structure of the
N-terminal 1–74 residues of the Andes virus N protein, which also forms a coiled coil domain.
Further, we identified that the coiled coil contains distinct regions of positively and negatively
charged surfaces involving conserved polar residues. We hypothesize that these regions are also
important in N protein function. We used site-directed mutagenesis to alter the surface of the N protein and assayed for the subcellular localization of the N protein by immunocytochemistry. We used CD spectroscopy to confirm that mutations did not alter the coiled coil structure of the N1–74
(residues 1–74 of the N protein) domain. However, immunocytochemistry showed that despite the
N protein being present throughout the cytoplasm, a monoclonal antibody only recognized the
Arg22 and Lys26 mutants when nucleocapsids are associated with the Golgi, the site of viral
assembly and maturation. We propose that the conserved surface residues Arg22 and Lys26 are important in the proper conformation or molecular recognition of the N protein.
METHODS
Protein Expression and Purification of N1–74
The N1–74 domain of the Andes virus (strain 23) nucleocapsid protein was subcloned into
pET151 (Invitrogen), which appends a 33-residue His6 tag and a TEV (tobacco etch virus) 32
protease cleavage site at the N terminus. Isotopically (15N,13C) labeled protein was overexpressed
in Escherichia coli BL21(DE3) (DNAY) grown in 1 liter of M9 minimal medium with
15 13 [ N]ammonium chloride and [ C]glucose. The cells were grown at 37 °C to A600 0.8, induced with 1 mM isopropyl-β-D-thiogalactopyranoside, and incubated overnight (16 h) in a 15 °C
shaker. The cells were harvested by centrifugation, resuspended in 30 ml of binding buffer (20
mM Tris-HCl, pH 8.0, 500 mM NaCl, 5 mM imidazole), and lysed by sonication. The cells were
centrifuged at 22,500 x g for 15 min, and the supernatant was loaded on a Ni2+ affinity column
(Sigma), washed with 35 ml of binding buffer, and eluted with elution buffer (500 mM NaCl, 20 mM Tris-HCl, pH 8.0, 250 mM imidazole). The purified His-tagged N1–74 was dialyzed into buffer (10 mM sodium phosphate, pH 6.9, 10 mM NaCl) and used for NMR structure
determination. Typical NMR samples contained 1–1.4 mM N1–74. For CD spectroscopy, the His
tag was cleaved by adding 0.08 mM TEV protease into purified His-tagged N1–74 and dialyzing the mixture in an 8000 molecular mass cut-off dialysis tubing in buffer (50 mM Tris-HCl, pH 8.0,
0.5 mM EDTA, 1 mM dithiothreitol) for 16 h at room temperature.
Mutagenesis of N1–74
Site-specific mutations in the N1–74 domain were introduced by PCR using the Stratagene
QuikChange kit in two plasmids: (i) pET151-N1–74, used to overexpress recombinant His-tagged
N1–74 in E. coli, and (ii) pcDNA3.1-AND-N, used to express full-length Andes virus N protein in a
mammalian cell line for immunocytochemistry (see below). The mutations were confirmed by
DNA sequencing.
NMR Spectroscopy
NMR data were acquired at 25 °C using a Bruker Avance 800 MHz spectrometer
equipped with a cryoprobe, processed with NMRPipe (249), and analyzed with NMR-View (250). 33
Backbone assignments were obtained from two-dimensional 1H-15N HSQC (251) and three-
dimensional HNCA (252), CBCA(CO)NH (252), HNCACB (253), and HNCO (254). Secondary structures were identified from the C , Cβ, C', and H chemical shifts (255). Side chain
assignments were obtained from two-dimensional 1H-13C HMQC (256), three-dimensional
13 HBHA(CO)NH (257), and three-dimensional C-edited HMQC-NOESY (tmix = 120 ms) (258).
Nuclear Overhauser effect (NOE) cross-peaks were identified from three-dimensional 15N-edited
13 NOESY-HSQC (tmix = 120 ms) (259) and three-dimensional C-edited HMQC-NOESY (tmix =
120 ms) (258). Hydrogen-deuterium exchange was performed by lyophilizing a 600-µl 15N-
labeled NMR sample and resuspending in 600 µl of 50% D2O, 50% H2O, followed by acquisition of six consecutive 20-min two-dimensional 1H-15N HSQC spectra. Peak volumes were analyzed to identify residues with slower hydrogen-deuterium exchange rates.
Structure Calculation
NOE distance restraints were classified into upper bounds of 2.7, 3.5, 4.5, and 5.5 Å and
lower bound of 1.8 Å based on peak volumes. Backbone dihedral angles in the -helical regions
were restrained to (-60 ± 20°) and (-40 ± 20°). Hydrogen bonding distance restraints were used for -helical residues that showed slow hydrogen-deuterium exchange rates. Initial
structures were generated by torsion angle dynamics in CYANA (260), followed by molecular
dynamics and simulated annealing in AMBER7 (261), first in vacuo and then with the generalized
Born potential to account for the effect of solvent during structure calculation. CYANA and
AMBER structure calculation protocols have been described elsewhere (262). Iterative cycles of
AMBER calculations followed by refinement of NMR-derived restraints were performed until the
structures converged with low restraint violations and good statistics in the Ramachandran plot. A
family of 20 lowest energy structures was analyzed using PRO-CHECK (263), and graphics were 34
generated using Pymol. The surface electrostatic potentials were calculated using APBS (264) and visualized in Pymol.
CD Spectroscopy
N1–74 samples for CD spectroscopy contained 5–10 µM protein in buffer (25 µM Tris-
HCl, pH 8, 3 µM EDTA, and 5 µM dithiothreitol). CD spectra were collected on a Jasco J-815 spectropolarimeter in triplicate. Wavelength scans were collected at 25 °C at a scanning rate of 50
nm/min. Thermal denaturation scans at 222 nm were acquired with a temperature ramp rate of 1
°C/min to a final temperature of 80 °C, followed by cooling at 1 °C/min to 25 °C. The melting
temperature (Tm) was determined from calculating the first derivative of thermal denaturation
plots using the Jasco CD software.
Immunocytochemistry
Immunocytochemistry was performed as reported (265). Briefly, Cos-7 cells (ATCC; no.
CRL-1651) were grown overnight in 24-well plates with coverslips at 37 °C and 5% CO2 in
Dulbecco's modified Eagle's medium containing 10% fetal bovine serum. The cells at 80% confluence were transfected using Lipofectamine 2000 (Invitrogen) with 0.8 µg of pcDNA3.1-
ANDV-N plasmid, which expresses full-length wild type or mutated N protein. At 48 h after transfection, the cells were washed with ice-cold phosphate-buffered saline and fixed at room temperature with methanol:acetone (3:1) for 10 min. The cells were incubated in 10 mM glycine for 30 min and permeabilized in phosphate-buffered saline with 0.1% Triton X-100 for 30 min.
Permeabilized cells were incubated with antibodies for 60 min at room temperature and washed for 5 min three times with 0.3% Tween in phosphate-buffered saline after each incubation. Goat serum (10%) was used as a blocking agent. Primary antibodies were of two sets: (i) rabbit
polyclonal anti-hantavirus nucleocapsid (1:1000) (Immunology Consultants Laboratory; no. 35
RSNV-55) and mouse monoclonal anti-hantavirus-nucleocapsid (1:1000) (Abcam; no. AB34757)
or (ii) rabbit anti-Golgi matrix protein GM130 (1:200) (Calbiochem; no. CB1008) and mouse- anti-hantavirus nucleocapsid (1:1000). Secondary antibodies used were Alexa-Fluor-488 (1:1000)
(Invitrogen; no. A11008 [GenBank] ) and Alexa-Fluor-594 (1:1000) (Invitrogen; no. A11005).
Lastly, the cells were stained with 4',6-diamidino-2-phenylindole (Bio-Genex; no. CS2010-06),
mounted on slides, and visualized at 60X on an Olympus FV1000 confocal microscope. The images were cropped and adjusted using Adobe Photoshop CS2.
RESULTS
NMR Structure Determination of N1–74
The His-tagged N1–74 expressed well in soluble form in E. coli and yielded an excellent
two-dimensional 1H-15N HSQC spectrum that showed distinct and well dispersed peaks (Fig. 1A).
Nearly complete backbone assignments were obtained from three-dimensional HNCA,
CBCA(CO)NH, HNCACB, and 15N-edited NOESY-HSQC. The histidine residues of the His tag were overlapped and could not be assigned unambiguously. The C , H , Cβ, and C' secondary
chemical shifts (supplemental Fig. S2) showed that the first 33 residues, which were part of the
His tag, were in random coil orientation, and the native N1–74 sequence contained two -helices
(255). Side chain assignments were completed using two-dimensional 1H-13C HMQC, three- dimensional HBHA(CO)NH, and three-dimensional 13C-edited HMQC-NOESY. Manual analysis of three-dimensional 15N- and 13C-edited NOESY spectra identified 1432 unambiguous
interproton distance restraints. The NOE restraints together with 73 and 62 dihedral angle restraints and 38 hydrogen bond restraints (supplemental Table S1) were used in structure
calculation and refinement in CYANA and AMBER. The 20 low energy NMR structures of N1–74 converged into a family of structures (Fig. 1B) with low restraint violations and good
Ramachandran plot statistics (supplemental Table S1). 36
FIGURE 1. A, assigned 1H-15N HSQC spectrum of Andes virus N1–74 domain. The 33 N- terminal residues (shown with asterisks) are part of the His tag introduced by pET151. The boxes show expansions of the crowded regions. B, superposition of 20 low energy NMR structures of the Andes virus N1–74 coiled coil domain. N1–74 forms two -helices, Met1–Val34 and Val39–Leu74. The anti-nucleocapsid monoclonal antibody used in immunocytochemistry below recognizes an epitope somewhere between residues 1 and 45.
The N1–74 Coiled Coil Domain
1–74 1 34 39 74 N forms two well defined -helices ( 1, Met –Val ; 2, Val –Leu ) that are connected by an ordered acidic loop (Asp35-Pro36-Asp37-Asp38) (Fig. 1B). The two helices are
intertwined into a coiled coil, and the helix 1- 2 interface is lined with hydrophobic amino acids
4 11 18 25 32 positioned in every seventh residue on helix 1 (Leu , Ile , Leu , Leu , and Val ) and helix 2
(Leu44, Val51, Leu58, and Leu65) (Fig. 2A). This heptad repeat of hydrophobic residues is a
hallmark of coiled coils and is highly conserved among hantaviruses (205). Together with Pro36, the heptad repeats of leucines, isoleucines, and valines form the hydrophobic core that stabilize the structure of the coiled coil (Fig. 3A). On the same face of the hydrophobic heptad, there is 37
8 15 another seven-residue repeat, in this case, composed of polar residues on helix 1 (Gln , Glu ,
Arg22, and Glu29) (Fig. 2B), which are invariant among the hantaviruses (supplemental Fig. S1).
Helix 2 also contains a polar heptad, however, with more residue variability at positions 41
(Lys), 48 (Arg/Gln/Glu), 55 (Glu/Gln), and 62 (Lys/Arg). These polar residues form two
15 62 22 55 8 conserved salt bridges between helix 1- 2 (Glu –Lys and Arg –Glu ) (Fig. 2B). Gln and
Lys41 are surface-exposed and do not form any salt bridges; however, they are invariant among the hantaviruses, suggesting some unknown function.
In addition to the conserved heptad repeats mentioned above, there are other highly
conserved residues whose side chains are pointed toward the helix 1- 2 interface. These residues
are nonpolar (Leu7 and Leu54), aromatic (His14), polar (Gln17, Asn40, and Thr43), or charged (Glu6,
15 29 41 47 57 Glu , Glu , Lys , Arg , and Lys ), and their side chains are pointed toward the helix 1- 2
interface (Fig. 2C). The polar and charged residues in this group do not participate in any salt
bridge or hydrogen bonding contacts; however, their polar moieties are pointed toward the surface of the coiled coil, whereas the aliphatic portion of their side chains are involved in hydrophobic
interaction that contribute to the stabilization of the hydrophobic core. The methyl groups of two
21 28 invariant alanines, Ala and Ala , in helix 1 (Fig. 2C) are oriented toward the helix 1- 2
interface but do not contact any other residues on helix 2, indicating that small side chains are required in those positions.
Conserved Surface Residues
A striking feature of the N1–74 coiled coil is the presence of large numbers of highly
conserved residues whose side chains are pointed away from the coiled coil. These residues are nonpolar (Ala66 and Val19), polar (Gln8 and Gln23), basic (Lys24, Lys26, Arg63, and Lys73), and
acidic (Asp27, Glu33, Asp35, Asp37, Asp38, Glu60, and Asp67) (Fig. 2, B and C). These polar residues 38
are identical (Gln8, Gln23, Lys24, Glu33, Asp35, Asp37, Arg63, and Lys73) or semi-identical (basic residues in position 26 and acidic residues in positions 27, 38, and 60) among hantaviruses
(supplemental Fig. S1). Further, many residues in this group are clustered together on the surface.
The first cluster (Gln23, Lys24, and Lys26 together with Arg47 and Arg22 discussed in the preceding paragraph) forms a basic surface (Fig. 2D), and the second cluster (Asp27, Glu33, Asp35, Asp37, and
Asp38) forms an acidic surface (Fig. 2D). We mutagenized many residues in this group (see below) to test the hypothesis that these residues are important in molecular recognition rather than
in stabilizing the coiled coil structure.
Electrostatic Surface of N1–74
The N1–74 domain is acidic (theoretical pI of 5.8), and the surface electrostatic potential map of N1–74 shows distinct regions of negatively charged (red) and positively charged (blue) surfaces (Fig. 2D). The tip of the coiled coil, where the loop connecting the two helices are located, is negatively charged (Fig. 2D) because of clustering of conserved acidic residues (Asp27,
Glu29, Glu33, Asp35, Asp37, and Asp38) and polar residues (Asn40 and Thr43). Although the N1–74 domain is acidic, there are conserved basic residues (Arg22, Lys24, Lys26 and Arg47) that form a
positively charged surface just below the negatively charged tip (Fig. 2D). Point mutations in this positively charged surface have a dramatic effect on the antibody recognition of the N protein in vivo (see below).
In addition, there is a smaller negatively charged surface formed by Glu9 and Glu6 (Fig.
2D). Residue 9 could be acidic (Glu or Asp) or basic (Arg or Lys). Residue 9 is acidic among
American hantaviruses (which cause the cardiopulmonary syndrome) and Old World hantaviruses that are nonpathogenic or cause a milder form of hemorrhagic fever with renal syndrome. Residue 39
9 is basic among Old World hantaviruses that causes the severe form of hemorrhagic fever with
renal syndrome.
FIGURE 2. A, heptad repeats of conserved hydrophobic residues form the interface of the helix 1 and 2 that stabilize the coiled coil domain. B, there is also a heptad repeat of polar residues, some of which (Arg22–Glu55 and Glu15–Lys62) form salt bridges that contribute in stabilizing the coiled coil. C, there are many highly conserved residues that point away from the coiled coil and thus are not involved in stabilizing the coiled coil. D, electrostatic surface potential map of N1–74. The orientation of the left panel is identical to that in A and is rotated 180° from the right panel, which is identical in the orientation of C. Conserved surface residues forming the acidic (red) and basic (blue) surfaces are indicated. Point mutations of Arg22 and Lys26 had a dramatic effect on the antibody recognition of the N protein in vivo. 40
FIGURE 3. A, CD spectra of N1–74 wild type (WT) and point mutants (K26E and R22F) showing the characteristic -helical dips at 208 and 222 nm. All other N1–74 point mutants (listed in Table 1) showed similar -helical CD spectra. B, CD thermal denaturation curves, monitored at 222 nm, of wild type N1–74 and two point mutants, K26E and R22F. The rest of the point mutants showed similar thermal denaturation curves. The ellipticity scales on the y axes are shown on left (wild type and R22F) and right (K26E).
Circular Dichroism Spectroscopy of N1–74
Point mutations were introduced in the basic (Arg22, Lys24, Lys26, and Arg47) and acidic
(Glu33 and Asp38) surfaces. In addition, we mutated Gln23, which is near the basic region, and
Pro36, which is near the acidic region. These residues are surface-exposed (Fig. 2D) and are nearly
invariant among hantaviruses (supplemental Fig. S1). CD spectroscopy was used to assess the folding and stability of N1–74 mutants. Wild type and point mutants showed nearly identical CD 41
spectra (Fig. 3A), indicating that the -helical structure of N1–74 was preserved. In addition, the
ratio of ellipticity at 222 and 208 nm can be used to characterize -helices. A 222/ 208 ratio of
1.0 indicates -helices with extensive interhelical contacts as in coiled coils and helical bundles,
whereas a 222/ 208 ratio of 0.8 indicates extended -helices with little interhelical contacts
1–74 (266) (267) (268). All N constructs have a 222/ 208 ratio higher than 0.9 (Table 1), suggesting
that all mutants have the intact coiled coil structure. Further insight was provided by acquiring the
CD melting temperatures (Fig. 3B and Table 1). Compared with wild type N1–74, the majority of
mutants showed lower Tm, with D38R having the lowest value, whereas two mutants (K24A and
R47A) showed higher Tm (Table 1). Nevertheless, all mutations were within ±5°Cof wild type Tm
(Table 1), indicating that the mutations did not drastically alter the thermal stability of N1–74.
Thus, the point mutations maintained the structural integrity of the N1–74 coiled coil.
Immunocytochemistry of N Protein
Hantaviruses are believed to mature intracellularly; specifically, in the Golgi complex
(219). During infection, the N protein was shown to localize cytoplasmically in the endoplasmic 42
reticulum-Golgi intermediate compartment, presumably as they traffic from the endoplasmic
reticulum to the Golgi (200). In addition, immunofluorescence of Cos-7 cells transfected with the
N protein alone showed a granular pattern of staining in the perinuclear region (174) (205), suggesting colocalization with the Golgi. To test our hypothesis that the conserved surface residues of N1–74 are important in molecular interaction, we introduced point mutations designed
to keep the N1–74 coiled coil domain intact while altering only specific surface residues and
transfected full-length N protein in mammalian cells to observe the subcellular localization of the
N protein. We used two types of anti-nucleocapsid antibodies, rabbit polyclonal and mouse monoclonal antibodies. The polyclonal antibody detected that wild type Npro and mutants (Arg22,
Gln23, Lys24, and Lys26) were located throughout the cytoplasm (Fig. 4A). The monoclonal
antibody also detected wild type Npro and the Gln23 and Lys24 mutants throughout the cytoplasm in a similar pattern of staining as the polyclonal antibody (Fig. 4A). However, the monoclonal antibody showed a dramatic difference between the recognition of wild type Npro and the Arg22 and Lys26 mutants (Fig. 4A). Using the monoclonal antibody, Arg22 and Lys26 mutants were
observed in a compact location lateral to the nucleus (Fig. 4A). To further define the subcellular localization of these Npro mutants, a Golgi-specific antibody (targeting the Golgi matrix protein
GM130) was used (Fig. 4B). The Arg22 and Lys26 mutants were only detected by the monoclonal antibody when the N protein colocalized with the Golgi (Fig. 4B); however, these mutants were also present throughout the cytoplasm as shown by the polyclonal antibody (Fig. 4A). Thus, for the Arg22 and Lys26 mutants, the monoclonal antibody was able to distinguish between two
populations of the N protein based on its subcellular localization in the cytoplasm or in the Golgi,
the site of viral assembly and maturation (219). Other mutants (Glu33, Asp35, Pro36, Asp37, Asp38,
and Arg47) did not show this localization-dependent antibody recognition (supplemental Fig. S3).
43
FIGURE 4. Immunocytochemistry of full-length N protein with point mutations in the N1–74 coiled coil domain. Cos-7 cells were transfected with a plasmid expressing Andes virus N protein. Two days after transfection, the cells were fixed for immunofluorescence microscopy and double labeled with monoclonal (red) and polyclonal (green) anti-nucleocapsid antibodies (A) and monoclonal anti-nucleocapsid antibody (red) and anti-Golgi antibody (green) (B). The cell nuclei were stained blue using 4',6-diamidino-2-phenylindole. Point mutations in Arg22 and Lys26 showed a dramatic difference in the monoclonal antibody recognition of Golgi-associated N protein, suggesting that the conformation or molecular interaction (or both) of the N protein is different when it is in the cytoplasm or when it is associated with the Golgi., wild type (WT).
DISCUSSION
The NMR structure of the Andes virus N1–74 domain is similar to the recent crystal
structure of the Sin Nombre virus nucleocapsid protein N-terminal coiled coil (N1–75) (248). The C 44
root mean square deviation between the two structures is 1.3 Å. The crystal structure determination of the N protein addressed the issue of the trimerization of the N protein (172, 174,
209, 247, 248) because earlier models suggested the trimerization of the nucleocapsid N-terminal domain. A proposed model of N protein trimerization involves, first, the association of three N- terminal domains, followed by the association of three C-terminal domains (205). However, crystallography revealed that the Sin Nombre nucleocapsid N-terminal domain was monomeric and formed a coiled coil structure, and conserved hydrophobic residues participate in helix-helix
interaction that stabilize the coiled coil (248). Our NMR structure of the Andes virus N1–74
supports the crystallographic results; even at 1.4 mM, N1–74 remained monomeric in solution. Our
results, however, do not preclude the trimerization of full-length N protein in vivo by another
mechanism.
A feature of the N1–74 domain that had not been addressed in the literature is the role of many conserved polar residues whose side chains are pointed away from the coiled coil.
Furthermore, the majority of these surface-exposed residues are not involved in polar interactions
(Fig. 2D). Point mutations of these polar residues maintained the structural integrity and high
thermal stability of the coiled coil (Fig. 3 and Table 1). For example, Arg22, which forms a salt bridge with a conserved residue Glu55, can be mutated (R22F or R22M) without disrupting the
coiled coil structure of N1–74 (Table 1). R22F, which replaced arginine with a bulkier aromatic side
chain, decreased the overall melting temperature by 3 °C (Table 1). This change is likely
attributed to increased steric clash between phenylalanine and Glu55. However, the observation that R22M melts at a temperature comparable with that of wild type suggests that the salt bridge
between Arg22 and Glu55 does not play a significant role in helix-helix interaction and that hydrophobic interaction is the major force stabilizing the coiled coil. A mutation in a nonpolar
36 residue, Pro , which is at the turn connecting the two -helices of the coiled coil, had a Tm 45
approximately four degrees lower than wild type, which is consistent with a mutation that increases the number of conformations available at the Pro36 turn and destabilizes the overall
protein structure by uncoupling the helix-helix interaction. Nevertheless, all of the point mutations of the conserved surface residues maintained the coiled coil structure of N1–74 (Fig. 3 and Table
1).
Thus, there is no compelling structural reason for the high sequence conservation of
surface residues. Furthermore, these polar residues are clustered together on the surface of the N1–
74 domain and form distinct positively and negatively charged regions (Fig. 2D). We hypothesize that the reason for the clustering of conserved polar residues on the surface of N1–74 is that they are sites of molecular recognition involved in the proper function of the N protein. Our mutagenesis
and immunocytochemistry data suggest that point mutations in this group had a dramatic effect on the antibody recognition of the N protein with respect to its subcellular localization (Fig. 4).
During infection, nucleocapsids are trafficked to the cytoplasm (200) to assemble into mature virions (269). Mammalian cells transfected with the N protein alone show a granular pattern of immunofluorescence (174, 205). This localization pattern is thought to be necessary for
the nucleocapsid to perform its many functions in the establishment of an effective infection
(200). We questioned whether the conserved polar surface residues in the coiled coil domain are
important in the proper functioning of the N protein and reasoned that defects in the conformation or molecular recognition of the N protein will be manifested in the antibody recognition of the N
protein in the context of its subcellular localization. CD spectroscopy confirmed that the mutant forms of N1–74 maintained the structural integrity of the coiled coil structure (Fig. 3 and Table 1);
thus, the mutations altered only the surface property of the N protein. 46
Immunocytochemistry (Fig. 4) indicates that mutations in a conserved basic surface
formed by Arg22 and Lys26 show monoclonal antibody recognition depending on the subcellular
localization of the N protein. Polyclonal antibodies show that Arg22 and Lys26 mutants are present
in the cytoplasm and Golgi; however, only Golgi-associated mutant nucleocapsids are detected by
the monoclonal antibody (Fig. 4). Mutation of Arg22 or Lys26 changes the presentation of the N-
terminal coiled coil to the monoclonal antibody. This change is dependent on the subcellular localization of the N protein.
There are two possible scenarios that could account for this differential monoclonal antibody recognition of the Arg22 and Lys26 mutants. First, there may be a difference in the
conformation of the N-terminal coiled coil depending on whether the N protein is localized in the
cytoplasm or in the Golgi, and this conformational change upon binding to the Golgi exposes the
epitope, which is somewhere between residues 1–45 (comprising helix 1, the interhelical loop,
1–74 and part of helix 2 of the N coiled coil; Fig. 1B), thereby allowing the monoclonal antibody to
recognize the N protein associated with the Golgi. Second, the epitope may be masked differently
by molecular interactions when the N protein is localized in the cytoplasm or in the Golgi. In addition to self-association, several host proteins such as SUMO-1 (142, 183, 244), Ubc9 (244)
(142), Daxx (184), actin (153), microtubules (200), and MxA (245) were reported to bind the N
protein. Binding of the N protein with SUMO-1 and Ubc9 was required for localization of the N
protein in the perinuclear region (183, 244). Furthermore, because the N protein is not known to
be a membrane protein, its localization in the Golgi must involve interaction with a Golgi- associated protein. Any of these molecular interactions could potentially alter the epitope
presentation of the N1–74 coiled coil and needs to be experimentally verified.
In summary, our structural results revealed that the highly conserved polar residues in the
N-terminal coiled coil domain of the hantavirus nucleocapsid protein form distinct acidic and 47
basic surfaces, and point mutations of the conserved basic surface formed by Arg22 and Lys26 allowed a monoclonal antibody to distinguish between two populations of the N protein based on
its subcellular localization. Thus, in the Arg22 or Lys26 mutants, the conformation or molecular
interaction of the N protein is different when it is in the cytoplasm or in the Golgi, the site of viral
assembly and maturation.
FOOTNOTES
* This work was supported, in whole or in part, by National Institutes of Health Grants AI057160 and AI65359. This work was also supported by American Heart Association Grant 0755724Z (to R. N. D.); National Science Foundation Program Grant EF 0326999 (to S. C. S. J.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 These authors contributed equally to this work.
2 To whom correspondence should be addressed: Dept. of Molecular Biosciences, University of Kansas, 1200 Sunnyside Ave., Lawrence, KS 66045. Fax: 785-864-5294; E-mail: [email protected] .
3 The abbreviations used are: HCPS, hantavirus cardiopulmonary syndrome; HSQC, heteronuclear single quantum coherence; HMQC, heteronuclear multiple quantum coherence; N, nucleocapsid protein; NOE, nuclear Overhauser effect; NOESY, NOE spectroscopy.
ACKNOWLEDGEMENTS
We are grateful to Albert Rizvanov (University of Nevada, Reno) for constructing the N1– 74 expression plasmid, Evan Colletti (University of Nevada, Reno) and Mariana Bego (University of Nevada, Reno) for guidance in confocal microscopy, and Thenmalarchelvi Rathinavelan (University of Kansas), Gaya Amarasinghe (Iowa State University), and Edina Harsay (University of Kansas) for critical reading of the manuscript.
48
______
ADDENDUM TO CHAPTER 1:
The N-terminal Coiled Coil Domain of the Andes Hantavirus Nucleocapsid Protein is associated with Trimerization
Unpublished findings
______
INTRODUCTION
The majority of hantavirus nucleocapsid proteins (Npro) are found colocalized with the endoplasmic reticulum Golgi intermediate compartments (ERGIC) (217). A persistant question in hantavirus research is, that if the assembly site for hantaviruses is the Golgi, why are nucleocapsids rarely found there (31)? It was previously shown that mutation of R22 and K26 of the ANDV N protein masked an epitope responsible for binding the commercial antibodyAB34757 when the nucleocapsid was in the cytoplasm but not when it localizes to the Golgi (270). The use of mutagenesis and this antibody reveals that a subset of nucleocapsids is in fact localized in the Golgi. Presumably those nucleocapsids in the Golgi are in the process of assembly. The majority of Npro which localizes to the ERGIC is likely involved in genome replication, oligomerization, or cell protein binding. To gain further understanding of the processes that direct Npro to the
Golgi, Npro mutants were analyzed for their distribution and ability to oligomerize.
49
MATERIALS AND METHODS
Immunocytochemistry
The procedures for N protein expression and immunofluoresent staining are
similar to those described in Chapter 2. Following staining, slides were viewed at 40X.
20 fields of view were observed and each cell expressing Npro was scored as either
granular (small punctuate expression) or globular (highly expressed, dispersed throughout
cytoplasm). Totals for each Npro mutant and wild type were calculated as percentages of granular expression on each slide.
Analysis of Nucleocapsid Oligomerization
Presence of monomers, dimers and trimers was detected by transfecting 100mm dishes of
Cos-7 cells at 90% confluence with pcDNA-ANDV-N wild type and mutant plasmids using
Lipofectamine 2000 (Invitrogen). Cells were maintained at 37 °C and 5% CO2 for 2 days. At time of harvest cells were washed twice with PBS and 1% chilled formaldehyde was applied for
10 minutes. Cells were lysed in 1 ml lysis buffer (50 mM Tris-HCl [pH 7.4], 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% Tween 20, 10 µl protease inhibitor cocktail/ml). Cells were
removed from the flask by scraping and passed through a 22-gauge needle three times to dissociate cellular material. Cellular debris was removed by centrifugation at 4,000 x g for 10
min. The supernatant was transferred to a new 1.5ml tube. Normal mouse immunoglobulin G
(5ug/ml) was added to the supernatant, and the mixture was rotated for 30 min at 4°C. Lysates
were treated with polyclonal anti-Npro (Immunology Consultants Laboratory; no. RSNV-55) and rotated at 4 °C for 2 hours. Protein A/G agarose beads were added and rotated at 4 °C overnight.
The complexes were spun at 6,000rpm for 3 minutes. The precipitate was washed and spun with 50
ice-cold PBS 5 times. The protein complexes were removed from the beads by the addition of 2x
Laemmli sample buffer (Bio-Rad) containing 2-mercaptoethanol and then heated to 95°C for 5 min. Samples were analyzed by Western blotting.
Western blotting, was performed by separation of samples through a sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel. Gels were subsequently transferred
to a polyvinylidene difluoride (PVDF) membrane and treated with monoclonal anti Npro (Abcam
AB34757). After a 60 minute incubation,the membrane was washed with PBS and 0.5% tween three times. Goat anti mouse IgG – HRP conjugated antibodies were added and incubated for 60 minutes. Membranes were stained using alkaline peroxidase and visualized.
RESULTS
Alminiate et al. used both immunohistochemistry and a yeast two hybrid assay to show that truncation of the N-terminal PUUV/TULV Npro reduces trimerization and results in a globular distribution of Npro throughout the cytoplasm (205). In this study it was observed that when oligomerization of Npro is reduced there is a lower percentage of Npro localized in a punctuate pattern near the nucleus. Almost 25% of truncated Npro localizes in a highly expressed globular distribution that extends to the plasma membrane (205). To analyze the effect of single mutants on the full length ANDV Npro, these mutants were expressed in Cos-7 cells and their granular and globular distributions were measured (Fig. 1a). Mutation of residues in the acidic loop displayed a significant reduction in the amount of cells displaying a granular distribution
(Fig. 1b). The position 22 mutants were not considered in this assay.
It is suggested that reduced trimerization of truncated N is what accounts for the altered cellular distribution of Npro (205). To determine if mutation of the acidic loop in Npro would affect the ability of the protein to trimerize, plasmids expressing these mutants were expressed 51
FIGURE 1. Granular vs. Globular distribution of N in transfected Cos – 7 cells. A. 20 frames of cells were scored as either granular (pink arrows) or globular (yellow arrows. B. Distribution of Npro wild type and mutants in transfected cells is presented as percentages.
and cross-linked in Cos-7 cells. Co-immunprecipitation of these cells with anti- nucleocapsid antibodies revealed that equal amounts of dimers and trimers were present in cells expressing wild type and mutant Npro (Fig. 2). This demonstrates that Npro exists in these three forms in relatively equal amounts. Additionally, mutation of the N-terminal coil coil does not change oligomerization. Therefore, the trend towards a more globular distribution of Npro mutants in the acidic loop region does not correlate with oligomerization.
DISCUSSION
Npro oligomerization is predicted to be a significant factor in viral replication and
assembly as it is the trimeric form of Npro which binds the the panhandle strucure of the
viral 5’ and 3’ ends (207). It is also possible that trimeric Npro forms complexes around
the viral RNA much like the 5 subunits of histones do to give structure and protection to
the respective nucleic acid (271). Our cross-linking experiment revealed that Npro exists in monomer, dimer, and trimer forms in equal concentrations when transfected 52
individually. This correlates with what was previously seen in infected VeroE6 cells
(172). It remains unknown which form of Npro is in higher concentration in the virion.
It could be assumed that if the packaging of viral RNA into the budding virion requires
trimeric Npro, this form would dominate the assembly process.
FIGURE 2. Cross linking of ANDV N proteins individually expressed. Cos-7 cells were cross-linked then co-immunoprecipitated and a western blot was performed on the samples using anti-Npro antibodies. The marker (mk) demonstrates that Npro migrates as Trimers (~110kD), Dimers (~115kD) and monomers (~50kD). The IgG heavy chain is migrates at ~55kD. No antibody was used in the immunoprecipitation as the negative (-) control.
This preliminary data attempted to further define the requirement of conserved
polar residues in the N-terminal domain of Npro for oligomerization. A significant
change in the distribution of Npro mutants in the acidic loop region was observed.
However, this could not be correlated with trimerization using co-immunoprecipitation
assay with cross-linked proteins. At the time this work was being performed, Almanaite
et al published a model for the interaction between TULV-Npro monomers of the coiled
coil region to form trimers (272). The in silico model was built from the template of the
recently determined crystal structure of SNV-Npro N-terminal domain (273). In this 53
model acidic residues D35, D37, and D38 clustered to bring the loop of each monomer
together. This predictive model was supported by yeast two-hybrid analysis (272).
Residues R22, E29, E55, and R48 had stabilizing effects in the model and in the yeast
two hybrid assay (272). This study presents a significant role for the conseved acidic
loop in the coiled coil structure for oligomerization.
The co-immunoprecipitation assay used in this study was likely not sensitive
enough to detect differences in bindings between monomers. Changes in binding
affinitties were detected in the yeast two-hybrid assay. It is likely that single or double point mutations in the coiled coil region can not ablate binding altogether. The binding of RNA the binding domain (middle region: 175-217) would potentially enhance the formation of trimers(175, 207). The C-terminal domain also contributes to oligomerization (174). The effects of RNA and the C-terminal domain could compensate for any modification to the structure of the N-terminal domain. Further analysis should include multiple mutations of both domains to better understand the mechanism of hantavirus nucleocapsid oligomerization.
54
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CHAPTER 2:
The Middle Region of Hantavirus Nucleocapsid binds Vimentin
Daniel M Boudreaux, William H Welch, Stephen C St Jeor
Manuscript Submission in Progress
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INTRODUCTION
In the preliminary studies using co-immunoprecipitation of Andes nucleocapsid, vimentin
was identified as one of the potential binding partners. Vimentin is an intermediate filament (IF)
which is found in most cell types (274). Its function is to maintain cellular and tissue integrity
during cellular motility and to manage the position of intracellular organelles (274, 275, 276).
Consequently vimentin filaments attach to the nucleus, mitochondria, ER, the Golgi, and to the plasma membrane (276, 277). Vimentin is also responsible for functional transport of endosomes
and lysosomes (4). Additionally, vimentin is required to maintain the cell’s resilience to
mechanical stress (278).
The structure of vimentin enables it to be one of the most dynamic of all the cytoskeletal
components (274). Although the sequence similarity between the different IFs is only 20%, they
follow the same general structure (279). The IF monomer contains a globular N-terminal “head”
bound to a very long helical domain followed by a globular C-terminal tail (Fig 1B). The helical
region is divided into four fragments by short non-helical regions. The helices are characterized
by heptad repeats of non-hydrophobic residues that are stabilized when the filaments form dimers
and the residues are buried between each monomer (280). Dimerization forms a long coiled coil 55
structure which in turn forms tetramers, protofilaments, and eventually a large network of 10nm
filaments (Fig. 1B) (281, 282). The ability of this network to assemble and disassemble and the
individual flexibility of the linkers between the helices in each monomer contributes to the overall
elasticity of this cytoskeletal component (274). Assembly is driven by the arginine and lysine
rich N-terminal head region (283, 284). The assembled vimentin network is also tethered to
actin and microtubules to form an overall cytoplasmic matrix that coordinates the placement of
organelles within the cell (285).
FIGURE 1. Structure and Assembly of Intermediate filaments. A. Variation between filaments exists at the N-terminal “head” and C-terminal “tail” regions. The generally conserved long coiled-coil region in the middle has four domains of coiled coils (1A, 1B, 2A, 2B) seperated by short alpha-helical domains (290). B. Monomers interact at the coiled coil regions to make multiple contacts on the sides of the filament. The final filaments involve interaction between the head and tail regions. (Fig.s adapted from Minin and Moldaver 2008 (275))
The vimentin matrix provides an abundant medium to support multiple interactions with cellular proteinsM to promote formation of the cytoskeleton and coordinate the placement of organelles (274, 276). Plectin is the overall coordinator of these bindings (286). The N-terminal 56
region of plectin binds to both actin and vimentin (287, 288). The domain 5 of plectin is required for binding of proteins to vimentin (289) The two coiled coil regions possess charged residues which alternate between positive and negative charges every 2-3 residues (291).
Vimentin binding of phosphorylated extracellular signal regulated kinase (ERK) occurs through electrostatic interaction between these charges and prevents dephosphorylation of ERK (292).
The head region of vimentin binds to the C domain of phospholipase C (293). The head domain contains a high concentration of positively charged residues in beta sheets which suggests this type of motif may have an affinity for vimentin.
The parasitic nature of viruses suggests that intricate interactions between the viral components and the host cell’s organelles and cytoskeletal components occur to enable entry, replication, transport, and release of progeny virions (294). Vimentin filaments are utilized by viruses as a necessary aspect in the life cycle of the virus and have been reported to be used during replication by multiple viral families. Vimentin is speculated to be incorporated into virions of poxviruses because it was observed in high concentrations associated with the cytoplasmic membrane during the budding process (295). Cellular vimentin can be altered either by using acrylamide to alter the depolymerization of vimentin fibers or by siRNA which reduces expression of the protein fibers can be disrupted by acrylamide (296, 297). Alteration of vimentin reduces the replication of hepatitis C, human cytomegalovirus, and junin viruses (298,
299, 300). In turn viral infection can affect the arrangement and function of vimentin in the cell.
This effect has been observed for parvovirus, HIV, rabies virus, and hantavirus (152,301,302,
303, 304). As a part of viral infection inside the cell, viral proteins have been shown to bind directly to vimentin. This has been shown for virion-associated proteins of Theiler’s virus and of cowpea mosaic virus, structural proteins of reovirus, NSP1 of dengue virus and VP2 of bluetongue virus (305,306, 307, 308, 309). In the case of bluetongue virus, VP2 binds to 57
vimentin, causes vimentin rearrangement, and requires vimentin to replicate the virus (305, 310).
This research suggests the utilization of vimentin by viruses is generally conserved.
Vimentin was found to be rearranged into cages around the nucleocapsid protein (Npro) in cells infected by Hantavirus serotype HTNV but not Black Creek Canal Virus (BCCV), SEOV, or ANDV (201). In this report the majority of HTNV nucleocapsid proteins expressed either individually or by infected cells co-localized with the ERGIC. In some cells it was observed that
N localized to a distinct site that was surrounded by vimentin filaments. The distinction between
HTNV and other serotypes suggests that a specific motif within the N protein is responsible for this caging effect. We report here that vimentin binds to ANDV, PHV, and SNV nucleocapsids.
We confirmed that vimentin is not bound to HTNV N. Alignment of the serotypes which bound vimentin revealed conservation in several charged residues in the middle region of Npro which is the least conserved region in the protein. Mutation of these residues abolished vimentin binding.
We propose a model for the middle region of SNV Npro (SNV-N-Mid). The Head region of vimentin (VimH) was also modeled and joined to the previously NMR resolved structure for the helical fragment 1A. These two models were interactively docked to identify residues which are potentially important for the interaction. This study suggests that vimentin plays a significant role in hantavirus replication.
METHODS:
Cloning
Expression plasmids used in transfection assays were constructed by PCR amplification of reverse transcribed cDNA from cells infected with either PHV, HTNV, or SNV using Promega
MMLV RT-PCR kit. cDNA was then pcr-ed using Invitrogen High Fidelity Platinum Taq and primers which included the forward sequence for the start codon and the reverse complement 58
sequence which added a FLAG epitope to the codons just upstream of the stop codon for the
nucleocapsid gene. PCR products were gel purified and cloned into the pTARGET mammalian
expression vector using TA cloning ligation reaction. Clones produced from this ligation were
sequenced to confirm correct orientation and correct amino acid identity. The pcDNA-ANDV-N-
V5 plasmid was previously constructed similar to the pTARGET constructs by Albert Rizvanov.
Site-directed mutagenesis was performed using the Stratagene Quickchange XL mutagenesis kit.
Primers were designed using stratagene mutagenesis primer design calculator. Clones containing the desired mutation were selected through sequencing.
Sequence Alignment
Multiple sequence alignment was performed using ClustalW (311) Sequences for residues 141-330 of the hantavirus nucleocapsid was obtained from genbank accessions;
NP_941977 - HTNV, AAA47086 - PHV, AAA68038.1 - SNV, NP_604471 - ANDV,
AAA47826 - SEOV. The protein sequence for BCCV was translated from the nucleotide sequence accession L39949.
Immunocytochemistry
Immunocytochemistry was performed as reported (147). Briefly, Cos-7 cells (ATCC; no.
CRL-1651) were grown overnight in 24-well plates with coverslips at 37 °C and 5% CO2 in
Dulbecco's modified Eagle's medium containing 10% fetal bovine serum. The cells at 80% confluence were transfected using Lipofectamine 2000 (Invitrogen) with 0.8 µg of pcDNA3.1-
AND-Npro plasmid, which expresses full-length wild type or mutated N protein. At 48 h after transfection, the cells were washed with ice-cold phosphate-buffered saline and fixed at room temperature with methanol:acetone (3:1) for 10 min. The cells were incubated in 10 mM glycine for 30 min and permeabilized in phosphate-buffered saline with 0.1% Triton X-100 for 30 min. 59
Permeabilized cells were incubated with antibodies for 60 min at room temperature and washed for 5 min three times with 0.3% Tween in phosphate-buffered saline after each incubation. Goat serum (10%) was used as a blocking agent. Primary antibodies used were rabbit polyclonal anti- hantavirus nucleocapsid (1:1000) (Immunology Consultants Laboratory; no. RSNV-55), and
mouse-anti-vimentin (1:1000) (Zymed No. 18-0052). Secondary antibodies used were Alexa-
Fluor-488 (1:1000) (Invitrogen; no. A11008 and Alexa-Fluor-594 (1:1000) (Invitrogen; no.
A11005). Lastly, the cells were stained with 4',6-diamidino-2-phenylindole (Bio-Genex; no.
CS2010-06), mounted on slides, and visualized using a 60X objective on an Olympus FV1000 confocal microscope. The images were cropped and adjusted using Adobe Photoshop CS2.
Co-Immunoprecipitation and Western Blotting
Cos-7 cells (ATCC; no. CRL-1651) were grown overnight in 100mm dishes24-well at 37
°C and 5% CO2 in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum. The
cells at 80% confluence were transfected using Lipofectamine 2000 (Invitrogen) with 0.8 µg of
pcDNA3.1-AND-V5 plasmid, which expresses full-length wild type or mutated N protein. At 48
h post transfection, the cells were washed 2 times with ice cold phosphate buffered salin (PBS;
pH 74.) Cells were lysed in 1 ml lysis buffer (50 mM Tris-HCl [pH 7.4], 150 mM NaCl, 1 mM
EDTA, 1% Triton X-100, 0.1% Tween 20, 10 µl protease inhibitor cocktail/ml). Cells were removed from the flask by scraping and passed through a 22-gauge needle three times to dissociate cellular material. Cellular debris was removed by centrifugation at 4,000 x g for 10
min. The supernatant was transferred to a new 1.5ml tube. Normal mouse immunoglobulin G
(5ul/ml) was added to the supernatant, and the mixture was rotated for 30 min at 4°C. Lysates
were divided into 3 tubes, treated with antibody and rotated at 4°C for 2 hours; 450ul were treated
with either anti-V5 or anti-Flag, 450ul were treated with anti-vimentin, and 100ul was incubated
without antibody and considered as the “lysates”. 50 µl/ml of protein G plus agarose beads was 60
added and rotated at 4°C overnight. The complexes were spun at 6,000rpm for 3 minutes. The precipitate was washed and spun with ice-cold PBS 5 times. The protein complexes were removed from the beads by the addition of 2x Laemmli sample buffer (Bio-Rad) containing 20%
2-mercaptoethanol and then heated to 95°C for 5 min. Samples were analyzed by Western
blotting.
Western blotting, was performed by separation of samples through a sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel. Gels were subsequently transferred
to a polyvinylidene difluoride (PVDF) membrane and reacted with antibodies as described in
Results and Materials and Methods. For electrophoreses, 15 µl of lysate (out of a total of 800 µl) was used for the lysate lanes and the remaining lysate was used for immunoprecipitations. In the
final step of the immunoprecipitation, beads were resuspended in 100 µl of gel loading buffer and
30 µl was loaded in each well.
Modelling and Docking
Secondary structure and hydrophobicity probabilities were predicted for the SNV-Npro
mid section (141-330) and Vimentin Head Region amino acids 1-100 (VimH) using JPRED,
SSPro, SOPM, GOR4, and ProfSec (312, 313, 314, 315, 316). Modeling was performed using
the Sybyl8.0 software package. Three templates were generated using Fugue and threaded using
the Orchestrar program (317). Each initial model was analyzed for their overall energy, van der
Waals bump violations, pseudoenergy as predicted by the Sybyl 7.2 protable protein analysis and the formation of a single chain with a minimum of gapped, unmodeled regions. The template from chain A of homoserine dehydrogenase (PDB id: 1ebf) was chosen for SNV-N-Mid. A gap between 222 and 262 filled using joined using a the Sybyl 8.0 loop search tool. A loop from the
PDB was selected based on the following hierarchy; homology, vander wal bump violations, and 61
secondary structure match to JPRED prediction. The model produced by orchestrar produced a
D-chiral Ile157. A mutational switch from a glycine back to isoleucine corrected the chirality.
The PDB structure for the endonuclease I-DMOI (PDB id: 1b24) was selected as a template for the VimH.
Staged minimization and dynamics simulations were performed in the Amber99
Forcefield to determine the most energetically favorable positions for atoms in each molecule.
Staging occurred in order by; first minimizing the sidechains, running dynamics on all atoms except the backbone of predicted alpha helix or beta sheets, minimized under the same conditions, and finally minimizing the entire molecule. Following minimization, torsional constraints were enforced into the phi and psi angles to maintain alpha helix and beta sheets and an additional molecular dynamics simulation followed by minimization. Constraints added were
residues of SNV-N-Mid for helixes were; 161-168, 324-335, 318-322, 279-292, 228-244, 204-
210, 185-192, 262-269 and beta sheets for SNV-N-Mid 275-277. For VimH, constraints were added to residues for helixes; 6-14, 41-54, 91-98 and beta sheets; 35-38, 78-80. Torsional constraints were maintained throughout docking procedures in order to maintain the most likely secondary structure and reduce computational strain during dynamics.
The minimized VimH structure was joined to the NMR resolved structure for fragment
1A of vimentin by guiding amino acid 100 of two VimH into proximity with amino acids 101of each chain (A and B) of the 3G1e structure (318). The VimH were run in molecular dynamics to find better conformations for this structure. The molecule was minimized by maintaining torsional constraints on VimH and maintaining Fragment 1A as an aggregate prior to vimentin docking. This formed a protein consisting of the N-terminal residues 1-141 (VimH-1A) of the totatl 466 amino acids. 62
Docking between SNV-N-Mid and VimH-1A was performed visually using Molcad
representations of the electrostatic surface of both molecules. The negatively charged surface of
residues D279 and D282 of SNV-N-Mid and the overall positively charged surface of VimH was
given preference as potential docking mechanism. Following multiple docking attempts
preliminary energy calculations were performed. The dock of the lowest energy was selected for
further dynamics maintaining the position of fragment 1A and the torsional constraints of both
molecules. The interaction energy of this dock is derived from the total energy equation:
Total Energy = energy of protein 1 + energy of protein 2 + interaction energy
To form the equation:
Interaction energy = (total energy of complex) – (energy of VimH-1A + energy of SN-N-Mid)
The effect of the mutation at D279 and D282 was analyzed by mutating these sidechains on the
minimized docked complex. This mutant was run in dynamics and interaction energies were
calculated.
RESULTS
Rearrangement of Vimentin Filaments by Hantavirus Nucleocapsid is Genotype Specific
The finding that SNV nucleocapsid binds vimentin and that HTNV-N is capable of rearranging vimentin into cages that surround the protein prompted a further analysis of the binding and localization of vimentin (refer to the prologue section) (201). The four serotypes were chosen as representatives of their geographic regions and pathogenicity. The epitope-tagged nucleocapsids for ANDV (V5 Tag), PHV (Flag Tag), SNV (Flag Tag), and HTNV (Flag Tag) were transiently transfected into cells and microscopy was performed at 1,3, and 5 day intervals 63
(Fig. 2). As expected the Npro of SNV, ANDV, and PHV was granularly dispersed throughout
the cytoplasm. A partial colocalization with vimentin was observed. Notably, HTNV
FIGURE 2. Immunocytochemistry of Hantavirus genotypes. A. Cos-7 cells at confluencies of 60-100% were transfected with a plasmid expressing either SNV Npro, ANDV Npro, PHV Npro, or HTNV Npro. Two days post transfection cells were fixed and double labeled with anti hantavirus Npro (red) and anti vimentin (green). The cell nuclei were stained blue using 4’,6- diamidino-2-phenylindole. The majority of expressed HTNV Npro were observed to localize to distinct area which colocalizes with the Golgi (not shown). The presence of HTNV causes a curving of vimentin so that the protein is caged by vimentin as displayed by arrows. B. Z- stacking of HTNV to display that vimentin surrounds the nucleocapsid protein in both the ZX and ZY planes displayed by arrows.
Npro was the only genotype that caused vimentin to remodel into cages around the protein. This
finding in combination with previous studies indicates that the property of vimentin 64
rearrangement by N protein is exclusive to HTN-N because it is not observed in other Old World hantaviruses (SEOV), New World Hantaviruses (ANDV, BCCV, and SNV), and non-pathogenic viruses (PHV) (201).
Genotype specificity of the Nucleocapsid-Vimentin Binding
Previously vimentin was identified as a potential binding partner for SNV-Npro in co- immunoprecipitation assays followed by MALDI-TOF identification (reference the prologue section). To confirm the binding and determine if other serotypes also interact with vimentin individually expressed Npro from four genotypes were co-immunoprecipitated 2 days post- transfection. It was observed that SNV, ANDV, PHV all bound vimentin but HTNV did not.
Additionally the highly variable region suspected to contain the sequence differences responsible for effect on vimentin of the SNV-N-mid (141-330) was tested for its ability to bind vimentin.
Co-immunoprecipitation of the four genotypes and SNV-N-Mid reveals that HTNV lacks the ability to bind vimentin and the SNV-N-Mid is the region involved in the binding (Fig. 3). This binding correlates viruses which do not cause vimentin rearrangement with vimentin binding.
HTNV possesses a unique property in that it does not bind vimentin but it causes vimentin to rearrange.
A Simple Model for SNV-N Middle Region
The finding that the SNV-N-middle region is responsible for vimentin binding prompted a further mapping of potential residues as a vimentin binding domain. The N- and C- terminal regions are known to contain domains responsible for oligomerization (173, 174, 272). Amino acids in this region are likely cooperatively bound to each other to present a surface representative of a trimer complex. While the C- and N- terminal domains are predicted to be in 65
close contact with each other leaving little surface exposure for interaction with other proteins,
the middle
FIGURE 3. Co-immunoprecipitation of Hantavirus Genotypes and SNV-N-Mid. Cos-7 cells were transfected with pTARGET -Npro-Flag for 2 days each expressing either SNV, ANDV, HTNV, PHV and truncated SNV-N-Mid proteins. Lysates were either run directly on western blot, or immunoprecipitated with either monoclonal anti-Flag or with monoclonal anti- Vimentin antibodies. Immunoprecipitates were run on western blots and probed with either anti- Flag or anti-vimentin antibodies. The IgG heavy chain runs at ~50kD.
region is thought to be free from self-association and available for interaction with RNA and other proteins. This region possesses an RNA binding domain (175-217) and a SUMO-1 interacting domain (188-191) (142, 175, 319). NMR structure determination was attempted on the SNV-N-Mid residues 141-298 without resolution which suggests this structure may exist in 66
multiple conformations in water/solvent and may adapt to a single conformation in a cellular
environment where cytoskeletal components or other cellular proteins could scaffold it.
FIGURE 4. Model for the SNV-Npro middle region. A. Shaded ribbon diagram displaying the secondary structure built from the template PDB: 1ebf residues 141-330. Secondary structure includes alpha helices (purple), beta sheets (yellow), and unstructured regions (cyan). B. Space Fill diagram showing residues which are aliphatic (green), polar (white), and surface clusters of acidic (red) and basic (blue) residues.
A model is proposed here to predict the surface residues of SNV-N-mid which could potentially interact with vimentin (Fig. 4). The template 3G1E homoserine dehydrogenase was identified through a Fugue search. Orchestrar was used to build the model from this template. A loop search was performed to complete a gap between resides 222 and 262. Following correction of a Ile157 in D-chirality the molecule was run through a series of staged minimizations and molecular dynamics. The final structure generally contains two large domains which are stabilized by the internal helix 317-329. Both domains are primarily alpha helical with multiple clusters of charged residues on the surface (Fig. 4). The alpha helices in the model correlate with the consensus alpha helices from 4 secondary structure prediction programs (Fig. 5A). 67
Additionally the RNA binding domain is presented as a grouping of positively charged amino acids in proximity with a deep cleft which presents a reasonable domain for attraction of negatively charged RNA molecules. The Sumo-1 binding domain is exposed on the surface in a protruding bulge with a few aspartic acid residues (188-191).
68
Determination of Vimentin Binding Domain
The majority of the known binding between cellular proteins and vimentin involve
electrostatic interactions (288, 292, 293). To further map the vimentin binding domain it was
reasoned that both charged residues on the surface of the SNV-N-Mid model and residues which
are conserved in SNV, ANDV, PHV, BCCV, and SEOV, but differed in HTNV would be
FIGURE 6. Co-immunoprecipitation of Vimentin with Mutant Nucleocapsids. Cos-7 cells were transfected with pTARGET-SNV-Npro-Flag for 2 days. Lysates were either run directly on western blot, or immunoprecipitated with either monoclonal anti-Flag or with monoclonal anti- Vimentin antibodies. Immunoprecipitates were run on western blots and probed with either anti- Flag or anti-vimentin antibodies. The IgG heavy chain runs at ~50kD. 69
responsible for the interaction. Clusters of charged residues that were identified on the model
include; D161-D162, K173-R175-H176, R197-R199, D237-E238, D279-D282, and R288-H302.
Multiple sequence alignment was performed to identify whether these residues are conserved
(Fig. 5B). From this analysis several residues were selected to alter the charge of the sidechains
but maintain the general size of the sidechain so as not to alter any vander Wals forces. Mutants designed include; ∆D161K/D162K, ∆K173D/R175E/H176D, ∆R197E/R199E, ∆D237K/E238R,
∆D279K/D282K, ∆R288E/H302D (Fig. 5B).
FIGURE 7. Helix-Loop-Helix motif A. Shaded ribbon diagram displaying the secondary structure from the model for SNV-N-Mid. Residues 275-277 have a tendency to form a beta sheet through molecular dynamics simulations B. Space Fill diagram showing the acidic cluster 279-282 along the first helix and part of the loop. Residues are colored as aliphatic (green), polar (white), and surface clusters of acidic (red) and basic (blue) residues.
The effect of mutations on the full length SNV-Npro wild-type and mutants was analyzed by co-immunoprecipitation of cells which expressed Npro mutants for 2 days. The 70
∆D279K/D282K mutation was found to disrupt the vimentin binding (Fig. 6). This suggests that
electrostatic interactions are at least partially responsible for vimentin binding. These aspartic
acids are located on the model at a loop formed between two helices (Fig. 7). This helix loop
helix motif may contain a beta sheet in the loop region because both the model run in dynamics
and the secondary structure server SSPro3 predicts beta for amino acids between 270-275 (Fig.
5A).
Modeling of the Vimentin Head region and the Joining to Fragment 1A
The interaction between vimentin and SNV-Npro was disrupted by mutation of two
aspartic acids to lysines. To further investigate the interaction the vimentin primary sequence
was searched for clusters of positively charged residues which may form charge-charge
interactions with the aspartic acids of SNV-Npro. The helices in the middle coiled-coil domain of vimentin contain charged residues which alternate from positive to negative as the helix turns. It is thought that the alternation promotes binding of monomers to stack together and form a large filamentous matrix. Because these charges are engaged in monomer to monomer interactions they would not be exposed for interaction with SNV-Npro. The tail region does not contain any prominent clusters of charges either. Remarkably the head region is highly basic with a pI of
11.5. Therefore it was hypothesized that the aspartic acids of SNV-N-mid would pair with basic clusters on the vimentin head (VimH).
To test the mechanism of binding, a model for the VimH and the first coiled-coil
(fragment 1A) was designed to be a substrate for SNV-N-mid docking. There is no available structure for the VimH region. The template I-DMOI, Intron-encoded endonuclease, PDB
ID:1B24 was used to develop a model for VimH (amino acids 1-100) in a similar manner as described for SNV-N-Mid with the exception that the constructed model formed a single chain and did not require loop searching and no amino acids had unfavorable chirality. The structure 71
was run in a staged minimization and molecular dynamics simulations and constraints were
enforced on predicted helices; 6-14, 41-54, 91-98 and beta sheets; 35-38, 78-80. The completed model formed a compact structure which contained a combination of alpha helix and beta sheets
(Fig. 8).
FIGURE 8. Primary Sequence of the Vimentin Head. Sequence of VimH aligned with Jpred, SSPro(3), SOPM, GOR4, Profsec, secondary structure predictions and the secondary structure that was modeled (Model_SS). Secondary structure is predicted as either alpha helices (H) or beta sheets (E).
Two VimH moleculess were joined to fragment 1A and molecular dynamics were performed to determine the most rational conformation formed from this peptide bond. The two charged VimH appear to repel each other through the dynamics simulation and settle in a conformation which places them apart. The overall structure, termed VimH-1A is generally similar to that of many intermediate filaments with a head region and long filamentous helix middle region (274). It is predicted that from position 141 the helix region would repeat and extend 4-5 times the length of the helix modeled here and would form an extensive coiled coil structure until the tail region begins. 72
A Simple Model for the mechanism of SNV-N-Mid binding to Vimentin
The models for both SNV-N-Mid and VimH-1A were visually docked by displaying them as MolCad charged surface residues and guiding Asp279 and Asp282 into multiple positively charged regions. Initial interaction energies were calculated and the best dock was selected (Fig. 10). This dock was run in molecular dynamics for 1 picosecond and minimized for
10,000fs. The final dock of the two structures revealed a tight binding between several
FIGURE 9. Model of VimH-1A. A. Shaded ribbon diagram displaying the secondary structure built from the template PDB: 1B24 for the head region, residues 1-100 and joined to fragment 1A PDB: 3G1E residues 101-141. The displayed secondary structure includes; alpha helices (purple) beta sheets (yellow), and undefined loops (cyan). The full vimentin structure is predicted to extend to the right from residue 141 to 410 and the with a primarily coiled coil structure followed by the tail at residues 411-466. B. Space Fill diagram showing acidic (red) and basic (blue) surface clusters.
surfaces on SNV-N-mid (Fig. 10B). Contacts were made between groupings of SNV-N-Mid residues; (I215, I216, R213 S217) (G224, I223, F225, S226, F227 F228, Q271, M270, D273),
(D278, I280, D282, E281), V304, W305, V306, F307) and vimentin. These interactions are both 73
electrostatic and hydrophobic (Fig. 10C, 10D). The overall interaction energy of this complex -
206 kcal/mol. This suggests a favorable interaction between these two molecules when docked.
FIGURE 10. Docking of the SNV-Npro Middle Region to VimH-1A. A. Initial dock guiding SNV-Nmid residues D279, E281, and D282 to VimH residues R4 and R12. Displayed as a space fill diagram showing aliphatic (green), polar (white), acidic (red) and basic (blue) surface clusters. B. The complex of SNV-Nmid (dark orange) with the VimH-1A (green) after 1 picosecond of molecular dynamics. C. Based on the 3 angstrom proximity of side chains after the dynamics run, charge-charge interactions are predicted to form between SNV-N-Mid residues D273, D271, D279, E281, D282 and the Vimentin residues R4, R12, R113, R122, K129, E136. Sidechains are displayed as in a ball and stick representation showing aliphatic (green), polar (white), acidic (red) and basic (blue) surface cluster. The backbones of SNV-Nmid (dark orange) and vimentin (green) are displayed as a ribbon tube diagram D. Based on the 3 angstrom proximity of side chains after the dynamics run, hydrophillic interactions are predicted to form between SNV-N-Mid residues I215, I216, S217, I223, g224, F225, S226, F227, F228, M270, V277, V304, W305, V306, F307 and the Vimentin residues V5, S8, S9, M13, Y37, Y39, G40, L43, F114. Hydrophillic (blue) and hydrophobic (gold) surface clusters sidechains are displayed in a ball and stick representation.
74
The significance of mutant D279K-D282K was considered by mutating the side chains of the
SNV-Npro-Mid after the structure of the dock in Fig. 10b. The mutant and wild type were run in
dynamics for 0.01 picoseconds. Interaction energies were calculated in a Boltzmann distribution.
A difference of -73.4 kcal/molwas measured between the mutant and wild type (table 1). As the
mutant is unable to bind in co-immunoprecipitation assays it is suggested that this loss of energy
is correlates with loss of binding in cell culture assays.
Docking of SNV-Mid Interaction Energy to VimH-1A (kcal/mol)
WT -206 ±10
∆D279K/282K -132 ±13
Difference -73
TABLE 1 Interaction energies of wild type and mutant SNV-Mid docked to VimH-1A. These energies are for qualitative ranking only. They do not represent the complete thermodynamic cycle of binding. Data obtained from 0.01 picoseconds of molecular dynamic simulations of the complex.
DISCUSSION
Hantavirus nucleocapsid is the most abundant viral protein in the virion and in infected cells. This protein has been reported to interact with itself and with multiple cellular proteins
(150). Additional cellular partners were potentially identified using MALDI-TOF analysis of co- immunoprecipitated SNV infected cells (see prologue). This study confirmed the finding that
SNV-Npro binds to vimentin. The binding domain was localized to the highly variable middle region (141-330). Molecular modeling and co-immunoprecipitation indicate that both electrostatic and hydrophobic interactions are involved in vimentin binding. The negatively charged loop region at residues 258-295 is involved in the interaction. The mechanism is defined 75
by the interactive docking of two molecular models together. Both of these models were the best
fit given the capability of the Fugue and Orchestrar programs. NMR attempts on the SNV-N-Mid resulted in an unresolvable spectrum of 1H and 15N chemical shifts. This suggests that the
unligated protein may exist in multiple conformations. To conserve computational energy the
models were run through relatively short molecular dynamic simulations. Most of the secondary
structure built by orchestrar was also predicted through multiple secondary prediction servers
which each use different algorithms. It is likely that the secondary structure in the model is
consisitent but the overall conformation may shift depending on the influence of cellular binding
partners. Here SNV-N-Mid was allowed to interact with vimentin through molecular dynamics.
The adjustments to the conformation were minimal. A more realistic structure of SNV-N-Mid and the mechanism of vimentin binding will be gained by solvating the molecular area and running an extended molecular dynamics simulation for several nanoseconds. Alternatively, a second attempt at NMR of just the helix-loop-helix region may give a more resolvable spectrum.
The multiple sequence alignment of the middle region of hantavirus genotypes reveals a
4-6 amino acid insert in mild or non-pathogenic genotypes at position 246 (PUUV, TULV,
Topografav virus, and Thailand virus not shown and Fig. 5). The insert contains mostly hydrophilic residues. This region was modeled to be in a long loop which is surface exposed.
The addition of residues from non-pathogenic viruses could create a binding site for a cellular protein which may contribute to these viruses non-pathogenic phenotype. Further modeling of each of these genotypes will reveal potential structural differences that may contribute to the understanding of hantavirus pathogenesis.
The SNV-N-Mid model includes the RNA binding domain (residues 175-217) and the
SUMO-1 binding domain (residues 188-191) (175, 183). Because this region contains a series of positively charged residues it was thought that it would contain a canonical RNA binding motif 76
which could be found in other structures. BLAST searching of the RCSB for a good template did
not reveal any potential targets with reliable E values. The RNA-binding domain of hantaviruses
may be a novel motif. This region was modeled using the template for homoserine
dehydrogenase. It forms a cleft which places several lysines and arginines on the rim of the cleft.
Phe205 was shown to play a significant role in RNA binding (175). The sidechain of this residue
is in the deepest part of the cleft. It has also been predicted that the nucleocapsid has an affinity
for the panhandle region which forms an imperfect double stranded RNA helix from base pairing
between the 3’ and 5’ ends (207). Measurement of this cleft determined that it was too small for
a dsRNA helix to fit. A brief docking simulation revealed that single-stranded RNA has a
favorable interaction energy for this region (data not shown). The location of this region in the
model is on the opposite side of the vimentin binding surface. It is feasible that this protein could
bind both simultaneously. If that is true, it would suggest that vimentin provides a matrix to
stabilize Npro during transcription/packaging processes.
The relationship of nucleocapsids with vimentin is strikingly different between SNV and
HTNV. Here we showed that mutation of D279K and D282K disrupts the binding of Npro to
vimentin enough to prevent co-immunoprecipitation of each other. HTNV in fact possesses
positively charged residues in this region which may prevent the binding in infected cells. HTNV
also causes vimentin to rearrange into cages around the Npro (200). There are two possible
explanations for this. HTNV Npro may bind accessory cytoskeletal proteins differently than
other genotypes which would ultimately affect vimentin causing the caging effect. A comparison
of protein binding partners of SNV-Npro and HTNV-Npro would support this possibility.
Secondly, the model SNV-Nmid binds to the head region very close to the sequence on the
vimentin head (8-SSYRRXFGG-17). This region is required for the initiation of depolymerization of the vimentin filament matrix (283). If SNV-Npro binds this region it could inhibit the depolymerization process, thereby preventing vimentin rearrangement. The lack of 77
binding in HTNV would permit rearrangement and promote the caging. The overall effect of
preventing depolymerization would cause the cells to be more rigid and divide and slower rates.
Could SNV infected cells be more rigid than HTNV infected cells? Would that affect viral
replication rates? Further consideration is needed to investigate this possibility.
The function of vimentin binding by Npro is unknown. Vimentin may provide a matrix
for Npro to position itself while it performs its multiple functions. Vimentin has traditionally
been thought of as a static filamentous matrix that provides flexibility to cells (285). Many
organelles are postioned by interaction with vimentin (276, 277). Recently it was proposed that
Bunyamwera family members rearrange many cellular components to form their own unique
organelle inside the cell called a viroplasm or viral factory (320). Vimentin may play a role in
stabilizing this organelle or may be a component of the inner lamina of the viroplasm. It has also
been demonstrated that cytoskeletal motors like dynein can direct transport along vimentin
filaments (321). The possibility that Npro uses vimentin to travel through the cell is currently under investigation. Lastly, Npro may bind vimentin in an effort to disrupt the cell’s immune response to vimentin. During infection vimentin may be used as a matrix to stabilize interactions in immune signaling processes. Npro may in fact have a higher affinity for vimentin then these allowing it to outcompete the cellular interactions supported by vimentin.
A vital question from this study is, how would disruption of vimentin affect viral
replication? A very preliminary study was performed by adding varying concentrations of
acrylamide in VeroE6 cells pre- and post- SNV infection. The replication process was monitored
by immunocytochemistry to observe the expression of Npro through a time course of 1, 2, and 4
days. This method did not reveal that acrylamide, which alters the polymerization of vimentin
filaments, had any effect on the temporal expression of Npro. Refinement of this experiment will
include the use of plaque assays to monitor overall virus production, shorter time points and more 78
specific doses of acrylamide. The use of siRNA to reduce the presence of vimentin in cells is
also under consideration.
Taken together this work has offered a potential model for vimentin binding as well as
modeling regions for RNA binding, SUMO-1 binding, and a potential pathogenic marker. The modeling is currently undergoing further refinement. With further work it is hoped that a greater understanding of the structure of this highly reactive viral protein will be gained.
79
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CHAPTER 3:
The Hantavirus Glycoprotein G1 Tail Contains Dual CCHC-type Classical Zinc Fingers
D. Fernando Estrada, Daniel M. Boudreaux, Dalian Zhong, Stephen C. St. Jeor, and Roberto N. De Guzman
J. Biol. Chem., Vol. 284, Issue 13, 8654-8660, March 27, 2009
______
ABSTRACT
Hantaviruses are distributed worldwide and can cause a hemorrhagic fever or a cardiopulmonary syndrome in humans. Mature virions consist of RNA genome, nucleocapsid protein, RNA polymerase, and two transmembrane glycoproteins, G1 and G2. The ectodomain of G1 is surface-
exposed; however, it has a 142-residue C-terminal cytoplasmic tail that plays important roles in
viral assembly and host-pathogen interaction. Here we show by NMR, circular dichroism spectroscopy, and mutagenesis that a highly conserved cysteine/histidine-rich region in the G1 tail of hantaviruses forms two CCHC-type classical zinc fingers. Unlike classical zinc fingers, however, the two G1 zinc fingers are intimately joined together, forming a compact domain with a unique fold. We discuss the implication of the hantaviral G1 zinc fingers in viral assembly and host-pathogen interaction.
80
INTRODUCTION
Many viruses in the family Bunyaviridae, which consists of five genera (Hantavirus,
Orthobunyavirus, Nairovirus, Phlebovirus, and Tospovirus), cause emerging zoonotic infections
in humans (322). Examples are the La Crosse encephalitis orthobunyavirus, Rift Valley fever phlebovirus, and the Crimean-Congo hemorrhagic fever nairovirus (tospoviruses are plant
pathogens). Hantaviruses use rodents as their primary reservoir, and although some (e.g. Prospect
Hill virus) are nonpathogenic to humans, others (e.g. Andes virus) can cause either the hantavirus cardiopulmonary syndrome or the hemorrhagic fever with renal syndrome in humans (322).
Annually, over 150,000 cases of hantaviral infections are reported worldwide (323) with mortality rates reaching as high as 40% (324).
Bunyaviridae viruses are enveloped and have three genomic RNA molecules: the small
(S), medium (M), and large (L) segments, and four viral proteins: the RNA polymerase, the nucleocapsid (Npro) protein, and the membrane glycoproteins, G1 and G2 (322). The ectodomains of G1 and G2 are glycosylated, form a heterodimer on the viral surface, and function as the viral spike proteins (322). In G1 and G2, the N termini form the ectodomains, followed by
single pass transmembrane helices, then the C termini or cytoplasmic tails project within the
virions. Bunyaviridae viruses lack a matrix protein (325),which link the membrane to the
ribonucleoprotein among enveloped viruses (326). Based on this observation, it was suggested
that the cytoplasmic tails of the viral glycoproteins might bind the viral ribonucleoprotein (327).
Indeed, recent results have shown that the G1 tail binds the viral ribonucleoprotein in phlebovirus
(328) and is required for packaging the genome in orthobunyavirus (329). These data suggest that the G1 tail plays a critical role in viral assembly. 81
Other reports suggest that among hantaviruses, the G1 tail is important in host-pathogen
interaction. The G1 tail of human pathogenic hantaviruses inhibits the cellular interferon response
(170, 171) against viral infection by disrupting protein-protein interactions (171). In nonpathogenic hantaviruses, by contrast, the interferon response is activated (170, 330). The G1 tail contains conserved immunoreceptor tyrosine-based activation motifs, which are involved in protein-protein interactions in the cellular immune response to viral infection (167). Further, the
G1 tail of pathogenic hantaviruses is ubiquitinated and proteasomally degraded (331), which is
thought to regulate the activity of the G1 tail (331), whereas the nonpathogenic hantavirus G1 tail
is stable.
The G1 tail varies in length from 78 residues in orthobunyaviruses to 142 residues among hantaviruses. Sequence alignment shows a region of conserved cysteine and histidine residues in the G1 tail of Bunyaviridae. Further, this region was predicted to form a RING finger motif in the
G1 tail of hantavirus (169). Here, we show by NMR that the conserved cysteine/histidine region
in the G1 tail of hantaviruses forms two classical ββ -fold zinc fingers(332, 333, 334, 335) and not a RING finger structure as suggested earlier (169). We also discuss the implication of our
structural findings of the hantavirus G1 tail in the context of viral assembly and host pathogen
interaction.
MATERIALS AND METHODS
Protein Expression and Purification
The cysteine/histidine-rich region (residues 543–599) of the G1 tail of the Andes virus
(strain 23) and Prospect Hill virus was subcloned into pET-21a (Novagen) as a C-terminal fusion
to a His6-tagged GB1 domain separated by a TEV protease cleavage site. GB1 is the B1 immunoglobulin-binding domain of Streptococcus protein G (336), and a GB1 expression plasmid (obtained from Peter E. Wright, Scripps Research Institute, La Jolla, California) was used 82
in the subcloning. Isotopically (15N or 15N, 13C) labeled protein was overexpressed in bacteria as follows: Freshly transformed Escherichia coli BL21(DE3) was grown in 1 liter M9 minimal
media supplemented with 0.1 mM ZnSO4 before and after induction. The cells were grown at 37
°C, induced with 1 mM isopropyl-β-D-thiogalacto-pyranoside at A600 = 0.8, and protein
expression was continued at 15 °C overnight (to a final A600 of 2.0). The cells were centrifuged,
2 resuspended in buffer A (20 mM Tris-HCl, pH 8.0, 20 mM NaCl, 1 mM DTT, 0.1 mM ZnSO4),
and lysed by sonication. Cellular debris was removed by centrifugation, and to the supernatant
was added one-tenth volume of 1% polyethyleneimine (pH 8) to precipitate the nucleic acids.
Following centrifugation, the supernatant was applied to a 5-ml HiTrap Q column (GE
Healthcare), and bound protein was eluted with a 100 ml linear gradient of buffer B (20 mM Tris-
HCl, pH 8.0, 0.5 M NaCl, 1 mM DTT, 1 mM ZnSO4). Fractions containing the fusion protein were pooled and dialyzed against TEV digestion buffer (50 mM Tris-HCl, pH 8.0, 20 mM NaCl,
1 mM DTT, 1 mM ZnSO4). TEV digestion was carried out at 25 °C for 16 h with 0.16 mg of
recombinant TEV protease (337) per 10 ml of fusion protein. The His6-tagged GB1 domain was removed by passing the digest through a 1-ml nickel affinity column (I1408; Sigma); purified
G1543–599 was recovered in the flow-through. Recombinant G1543–599 zinc finger retained two extra
N-terminal amino acids (Gly-His) resulting from the subcloning.
Site-directed mutagenesis was performed using the QuikChange kit (Stratagene). In total,
7 cysteine and 5 histidine residues (4 native His residues and the cloning artifact, His542) were
mutated individually to serine or phenylalanine, respectively, and confirmed by DNA sequencing.
Mutants H542F, H553F, H552F, H590F, and C594S were expressed as soluble proteins and were
purified by nickel affinity chromatography as previously described (338). Mutants C548S,
C551S, C555S, H564F, C568S, C573S, and C576S were expressed as inclusion bodies (despite
the presence of the GB1 solubility tag) and were purified as follows. Inclusion bodies were 83
resuspended at room temperature in buffer (50 mM Tris-HCl, pH 8.0, 100 mM NaCl, 8 M urea, 1
mM DTT, 0.1 mM ZnSO4, and 0.1 mM phenylmethanesulfonyl fluoride). Solubilized protein was
dialyzed into buffer (20 mM Tris-HCl, pH 8.0, 0.5 M NaCl, 6 M urea, 1 mM DTT, and 0.1 mM
ZnSO4), loaded into a 5-ml nickel affinity column, and eluted with buffer (20 mM Tris-HCl, pH
8.0, 20 mM NaCl, 6 M urea, 1 mM DTT, 0.1 mM ZnSO4, 1 M imidazole). Eluted protein was refolded using stepwise dialysis to remove urea. None of the mutant fusion proteins was cleaved.
The two-dimensional 1H-15N HSQC of GB1 in the fusion protein was used as a marker to determine the refolding of the fusion protein.
RNA Binding Assay
Andes virus (strain 23) was used to inoculate a T175 flask of confluent monolayer of
Vero E6 cells at a multiplicity of infection of 0.1 plaque-forming unit/cell and incubated for 14 days in a Biosafety Level 3 environment. The cells were harvested, and total RNA was extracted
using TRIzol (15596-018; Invitrogen), ethanol-precipitated, and resuspended in water to a final concentration of 300 ng/µl. The presence of viral and cellular RNA was confirmed by reverse
transcription-PCR. Total cellular and viral RNA was incubated at room temperature for 15 min with increasing amounts of Andes G1 zinc finger protein and a known RNA-binding protein,
PACT (339) (a gift from Dr. Gaya Amarasinghe, Iowa State University) in binding buffer (10 mM
NaPO4, 10 mM NaCl, pH 7.6). Samples were mixed with an equal volume 50% glycerol and
loaded in a 0.7% agarose Tris-borate gel for electrophoretic mobility shift assay. The gel was run
at 70 V for 50 min in Tris-borate buffer, pH 8.3. The gel was visualized by staining with SYBR
Green II RNA specific dye (Invitrogen).
84
CD Spectroscopy
The GB1 tag was removed in all samples for CD spectroscopy. Each sample contained 5–
10 µM protein in buffer (10 mM NaPO4, pH 7.0, 10 mM NaCl, 1 mM DTT, 0.1 mM ZnSO4). CD spectra were acquired in triplicate using a JASCO J-815 Spectropolarimeter at 25 °C and 50
nm/min scan rate. Titration with EDTA and ZnSO4 were applied to the same sample.
NMR Spectroscopy
NMR data were acquired at 25 °C using a Bruker Avance 800 MHz spectrometer
equipped with a cryoprobe, processed with NMRPipe (340) and analyzed with NMR-View (341).
For NMR structure determination, the G1 zinc-binding domain of the Andes virus was used.
15 15 13 Typical NMR samples contained 1 mM N- or N, C-labeled protein in buffer (10 mM NaPO4,
pH 7.0, 10 mM NaCl, 1 mM DTT, 0.1 mM ZnSO4) dissolved in 10% D2O or 100% D2O.
Backbone assignments were obtained from two-dimensional 1H-15N HSQC (342) and three-
dimensional HNCA (343), CBCA(CO)NH (343), HNCACB (344), and HNCO (345). Secondary structures were identified from the C , Cβ, C', and H chemical shifts (346). Side chain assignments were obtained from two-dimensional 1H-13C HMQC (256), three-dimensional
HBHA(CO)NH (347), and three-dimensional 13C-edited HMQC-NOESY (348) (mixing time
2+ (tmix) = 120 ms). The tautomeric ring assignments of Zn -coordinated histidines were identified
by 15N HMQC (349). NOE cross-peaks were identified from three-dimensional 15N-edited
13 NOESY-HSQC (350) (tmix = 120 ms) and three-dimensional C-edited HMQC-NOESY (348)
(tmix = 120 ms).
Structure calculation
NOE distance restraints were classified into upper bounds of 2.7, 3.5, 4.5 and 5.5Å and lower bound of 1.8 Å based on peak volumes. Backbone dihedral angles in the α-helical regions 85
were restrained to φ (-60±20°) and ψ (-40±20°). Initial structures were generated by torsion angle dynamics in CYANA (351), followed by molecular dynamics and simulated annealing in
AMBER7 (Case et al., 2002); first in vacuo, then with the generalized Born (GB) potential to account for the effect of solvent during structure calculation. Tight distance restraints that imposed tetrahedral Zn2+-coordination to Cys and His residues were used in the CYANA
FIGURE 1. The G1 tail of Hantaviruses, Nairoviruses, and Orthobunyaviruses (genera of Bunyaviridae) contains a cysteine/histidine-rich region with two CCHC arrays. Structure determination of the Andes virus dual CCHC-region revealed a novel zinc finger domain. Shown are the secondary structures ( -helices and β-strands), zinc-coordinating residues (blocked), the two CCHC motifs (boxed), conserved residues (gray), and residue numbers for the Andes virus G1 sequence. Sequence alignment was generated using CLUSTALW and formatted with ESPript 2.2.
calculations (352). Structural calculations were also done without Zn2+ restraints to confirm that the domain could fold from NOE-derived restraints only. CYANA and AMBER structure calculation protocols have been described elsewhere (353). Iterative cycles of AMBER calculations followed by refinement of NMRderived restraints were performed until the structures 86
converged with low restraint violations and good statistics in the Ramachandran plot. A family of
twenty lowest energy structures was analyzed using PROCHECK (354) and graphics were
generated using Pymol (355).
RESULTS
Protein Expression
Sequence analysis of the G1 cytoplasmic tail of hantaviruses revealed two highly
conserved CX2CX12–13-HX3C (where X is any amino acid) motifs, which suggested the presence of
two CCHC-type zinc fingers (Fig. 1). Expression of the Andes virus G1 zinc fingers (residues
543–599) in E. coli resulted in cell death, with cell density reaching only A600 of 0.9 after
induction at A600 of 0.8, suggesting that the zinc finger was toxic to E. coli. Thus, the zinc finger
domain was expressed as a GB1 fusion protein. The GB1 tag contained His6 for nickel affinity
purification and a TEV protease cleavage site to recover the native G1 zinc finger domain. The
fusion protein was overexpressed in soluble form in E. coli, purified under native conditions, and digested with TEV protease to obtain the G1 zinc finger domain.
Zn2+ Is Required for Proper Folding
CD spectrum of the Andes virus G1 zinc finger showed a folded -helical domain with
local minima at 209 and 222 nm (Fig. 2). Titration of EDTA to a final concentration of 1.25 mM
caused a spectral shift to 205 nm, indicating a partial loss of secondary structure. However, the
minimum at 222 nm remained despite EDTA treatment, suggesting that although the global fold
is disrupted by removal of zinc ion, some residual helical structure remained. Subsequently,
titrating ZnSO4 back into the solution resulted in increased -helical content, suggesting
restoration of the global fold. 87
FIGURE 2. CD spectroscopy and titration with EDTA and ZnSO4 of recombinant Andes virus G1 tail CCHC-region (residues 543–599), which was expressed and purified under native conditions, showed that Zn2+-binding is required for the proper folding of this domain. Native G1 tail zinc finger domain showed a folded CD spectrum (open squares). Titration with an excess of EDTA resulted to an unfolded peak (at 204 nm) and reduced the helical peak (at 222 nm) (closed squares). The addition of 2.5 and 5 mM ZnSO4 yielded folded CD spectra (triangles and circles).
NMR data were also used to confirm the requirement for Zn2+ coordination on the proper folding of the zinc finger domain. The Andes virus and the Prospect Hill virus zinc-binding domains purified under native conditions showed well dispersed and sharp peaks in their two- dimensional 1H-15N HSQC spectra (supplemental Fig. S1). After treatment with excess EDTA,
peaks in the HSQC of the Andes virus zinc-binding domain deteriorated, showed residual peaks
that differ markedly from each other with respect to peak intensities and sharpness, and displayed a collapse of the amide side chains (supplemental Fig. S1). In the presence of excess EDTA, the
HSQC spectrum of the Prospect Hill virus showed a collapse of the backbone and side chain
amide peaks (supplemental Fig. S1), which indicated that the protein was unfolded. 88
NMR Structure Determination
We determined the NMR structure of the Andes virus zinc finger domain. The Andes virus G1 zinc finger domain showed an excellent well dispersed two-dimensional 1H-15N HSQC
(Fig. 3). Nearly complete backbone assignments were obtained from three-dimensional HNCA,
CBCA(CO)NH, HNCACB, and 15N-edited NOESY-HSQC. Analysis of the C , H , Cβ, and C'
secondary chemical shifts (supplemental Fig. S2) supported the presence of two short -helices and two random coil regions flanking the central domain (346). Side chain assignments were completed using two-dimensional 1H-13C HMQC, three-dimensional HBHA-(CO)NH, and three-
dimensional 13C-edited HMQC-NOESY. There were four conserved histidines (at positions 552,
553, 564, and 590) that could potentially coordinate Zn2+ ion; however, long distance NOEs
(between Cys548-His564 and Cys573-His590) indicated that His564 and His590 were involved in Zn2+
coordination. A two-dimensional 15N HMQC (349) spectrum showed Zn2+ coordination through the His564 N 1 and His590 N 2 atoms (supplemental Fig. S3). Manual analysis of three-dimensional
15N- and 13C-edited NOESY spectra identified 859 unambiguous interproton distance restraints.
The NOE restraints together with 24 and 15 dihedral angle restraints and zinc coordination restrains (Table 1) were used in structure calculation and refinement with CYANA and AMBER.
The 20 lowest energy NMR structures converged into a family of structures (Fig. 4A) with low restraint violations and good Ramachandran plot statistics (Table 1).
89
Structure of Individual Zinc Finger
Each G1 zinc finger folded similarly to the ββ fold of classical zinc fingers. In the first
CCHC array (ZF1), residues Met546–Cys555 formed a β-hairpin that encompassed the first two
coordinating cysteines (Cys548 and Cys551; Fig. 4B). Asp549 and Val550 formed the loop apex with
the coordinating cysteines on either side of the β-hairpin. The structured region terminated at
559 Lys where helix 1 began and folded back toward the β-hairpin and allowed the completion of
564 568 ZF1 with His and Cys on the interior face of helix 1. 90
FIGURE 3. The Andes virus G1 tail zinc finger domain (residues 543–599) shows a well dispersed two-dimensional 1H-15N HSQC spectrum, which facilitated the acquisition of additional NMR data sets and allowed resonance assignments and NMR structure determination of this domain. Shown are the complete assignments for backbone and side chain amides.
In ZF2, the β-hairpin (Gly571–Thr580) contained the first two coordinating cysteines
(Cys573 and Cys576; Fig. 4, B and C). The coordination site on the loop was partly formed by the
positioning of Pro574 between the two. Strong Cys573 H to Pro574 H NOEs indicated that Pro574 was in the trans configuration. A structured loop followed the β-hairpin and terminated at Glu584,
where helix 2 began, and folded back toward the β-hairpin to complete ZF2 by coordinating
His590 and Cys594 to the Zn2+ ion.
91
A Novel Dual CCHC Zinc Finger
Unlike classical ββ zinc fingers, which fold independently of each other forming a
"beads-on-a-string" configuration, the two G1 zinc fingers interacted with each other, forming a
compact structure in which the two zinc atoms were located a mere 10 Å apart (Fig. 4). Two
short, parallel helices of 8 and 9 residues in length were linked by a 15-residue β-hairpin
568 583 extending between Cys of helix 1 and Thr of helix 2. Another loop preceded helix 1 and an
unstructured tail of 7 residues followed helix 2. Both zinc coordination sites were formed at the
junction of a loop and the face of a proximal -helix. Structural searches using DALI (356) and
TM-align (357) returned no homologous structures; thus, the G1 zinc finger domain has a novel fold.
Mutations of Zn2+-coordinating Residues
To confirm the Zn2+ coordination topology indicated by the NMR structure, we created point mutants in each of the cysteine and histidine residues within the Andes virus zinc finger
domain. Of the 8 residues expected to coordinate zinc, only C594S and H590F expressed as
soluble proteins, the rest (C548S, C551S, H564F, C568S, C573S, and C576S) could be expressed only as inclusion bodies despite the presence of the GB1 solubility tag. This result suggested that zinc coordination was necessary for stabilizing the overall fold of the zinc finger domain. For
further analysis, all of the inclusion bodies were solubilized overnight in 8 M urea, purified by nickel affinity chromatography, and refolded by stepwise dialysis to remove urea. Refolding of
the zinc finger domain was determined by the proper refolding of the attached GB1 tag using two- dimensional 15N HSQC, which served as a control to show that the refolding conditions would have properly refolded a native protein. The spectra of the mutated G1 zinc finger domain in the
GB1 fusion proteins consisting of C548S, C551S, H564F, C573S, C576S, H590F, and C594S all 92
showed narrowly dispersed spectra consistent with an unfolded domain (supplemental Fig. S4).
These results suggested that the two zinc fingers did not fold independently of each other
(supplemental Fig. S4). Of these eight positions, only C568S showed any peak dispersion at all
(supplemental Fig. S4). In each instance, the peaks corresponding to the attached GB1 tag were well dispersed, thus indicating that the fusion protein was refolded properly (supplemental Fig.
S4). These results suggested that, in the dual zinc finger domain, mutation of a Zn2+-coordinating residue in either ZF1 or ZF2 lead to the unfolding of the entire domain.
Mutations of Non-Zn2+-coordinating Residues
The domain contains three histidines (His552, His553, and His542, the cloning artifact) and a
cysteine (Cys555) (Fig. 1) that are not involved in Zn2+ coordination. To eliminate the possibility that Zn2+ could coordinate these other cysteine and histidine residues, we generated four additional point mutants corresponding to H542F, H552F, H553F, and C555S. Three of the four mutants (H552F, H553F, and H542F) gave a dispersed spectrum consistent with a folded domain
(supplemental Fig. S5). Only the C555S mutant gave an unfolded spectrum (supplemental Fig.
S5). Analysis of the structure reveals that the side chain of Cys555 was oriented toward the interior
of the structure and therefore played a role in stabilizing the hydrophobic core of the overall
domain. These data hence confirmed that Zn2+ was coordinated to the predicted zinc finger residues (Cys548, Cys541, Cys568, Cys574, Cys576, and Cys594 and His564 and His590).
Hantaviral G1 Zinc Fingers Does Not Bind RNA
Classical ββ -fold zinc fingers are well known nucleic acid-binding motifs (332, 333,
334, 335). However, our attempts to verify the ability of the Andes virus G1 zinc finger domain 93
FIGURE 4. The NMR structure of the Andes virus G1 tail zinc-binding domain reveals two classical ββ fold zinc fingers that are joined together. A, stereoview of the superposition of 20 lowest energy NMR structures. B and C, ribbon structures of the lowest energy NMR structure showing the residues involved in the first (ZF1) (B) and second (ZF2) (C) zinc fingers. Shown are the cysteine and histidine residues (yellow) that coordinate Zn2+ ions (gray) as well as the secondary structures ( 1- 2, β1-β2). The dual hantaviral G1 zinc fingers interact with each other and form a single domain with a novel fold as revealed by DALI (356) and TM-align (357) structural homology searches.
to bind RNA by electrophoretic mobility shift assay revealed that under the conditions used, the
G1 zinc finger domain did not bind RNA obtained from Andes virus-infected Vero E6 cells
(supplemental Fig. S7). Although a known RNA-binding protein PACT (339) showed smearing 94
of the RNA bands, which suggested nonspecific PACT-RNA interaction, increasing amounts of
the Andes virus G1 zinc finger failed to demonstrate even nonspecific binding of RNA
(supplemental Fig. S7).
DISCUSSION
The G1 tail of Bunyaviridae viruses is important in viral assembly (328) (329) and host- pathogen interaction (170) (171) (330) (167) (331). Our results showed that a conserved cysteine/histidine-rich region in the hantavirus G1 tail (Fig. 1) required Zn2+ binding to fold properly (Fig. 2). This region formed an independently folded domain that gave excellent NMR
data (Fig. 3) and NMR structure determination revealed dual CCHC-type zinc fingers where Zn2+ ligands were sequential and nonoverlapping (Fig. 4). The folding of each G1 zinc finger is related to the classical ββ zinc finger fold (358), which are among the most abundant protein motifs in eukaryotic genomes (reviewed in Ref. (359).
Implication of the Zinc Finger Structure in the Biology of Hantavirus
It has been suggested that the conserved cysteine-histidine region in the G1 tail of
hantaviruses forms a RING finger motif (169). This assumption is based on the following observations: (i) the G1 tail is ubiquitinated and proteasomally degraded as part of the host-
pathogen interaction of hantaviruses (331), (ii) RING fingers are structural domains of ubiquitin ligases, which are part of the ubiquitin degradation pathway, and (iii) some viruses contain RING
finger motifs that are involved in the ubiquitin degradation pathway as part of their host evasion
mechanism (169).
Instead of forming a RING finger motif (169), however, our results showed that the conserved cysteine/histidine region in the G1 tail of hantaviruses formed a dual classical type ββ
-fold zinc fingers. Classical zinc fingers are well known DNA- and RNA-binding domains (334) 95
(335) (360). Recent reports indicate that proteins containing classical zinc fingers are also
involved in protein-protein interaction (359) (361) (362) (363). Thus, instead of functioning as a domain of a ubiquitin ligase (as a RING finger), the classical ββ -fold of the hantaviral zinc
fingers suggests nucleic acid binding and/or protein-protein interaction. This is consistent with the
observations that the G1 tail is important in binding the ribonucleoprotein during viral assembly
of Bunyaviridae (328) (329).
Thus, the ββ -fold implies that the hantaviral zinc fingers may interact with the RNA
genome or the protein component of the ribonucleoprotein during viral assembly. Our electrophoretic mobility shift assay showed that the hantaviral zinc fingers did not interact with
RNA (supplemental Fig. S7). Additionally, the hantaviral zinc domain has a theoretical pI of 5.8,
which is too acidic to be a nucleic acid-binding motif. Further, many of the dual zinc fingers that have been characterized to date (RING, MYND, and LIM) are involved in protein-protein interaction (reviewed in Ref. (363). Therefore, a similar protein binding function for the hantaviral zinc finger is likely rather than RNA binding. It has also been suggested by others that during the assembly of hantaviruses, the G1 tail binds the nucleocapsid protein (150), which is a key component of the viral ribonucleoprotein. Efforts are now underway to identify the protein
binding partners of the hantaviral zinc fingers.
Unique Properties of Hantaviral Zinc Fingers
Although hantaviral zinc fingers have classical ββ zinc finger fold (Fig. 4), they differ from classical zinc fingers in two aspects. First, the two hantaviral zinc fingers fold together as a single domain, which is likely due to a short 4-residue linker between the two zinc fingers.
Commonly, classical zinc fingers fold independently of each other, forming a beads-on-a-string
configuration. However, multiple classical zinc fingers can interact with each other when bound 96
to DNA (360) or RNA (334) (335). Another example of a dual classical ββ zinc finger that folds
together as one unit is the yeast Zap1 transcription factor (364). Second, the folding of one
hantaviral zinc finger affects the folding of the other zinc finger. For example, mutations in
cysteine and histidine residues that disrupted the first or second zinc finger disrupted the folding of the entire dual zinc finger domain (supplemental Fig. S4). Because classical zinc fingers fold independently of each other, disrupting the folding of one zinc finger domain does not affect the folding of the other zinc fingers.
Other Viral Zinc Fingers
Among viruses, the CCHC zinc fingers of the nucleocapsid proteins of retroviruses (365)
have been studied extensively because of their critical role in binding and packaging the RNA genomes. Examples of zinc fingers in viral glycoproteins, however, are scarce. Our structure
presented here is the first atomic resolution structure of a zinc finger domain from a viral
glycoprotein. The sequence homology of the hantaviral zinc finger region with other
Bunyaviridae (Fig. 1) also suggests that that the G1 tail in nairoviruses and orthobunyaviruses will
also form zinc finger motifs. Therefore, our results form the structural framework for future
studies aimed at elucidating the precise role of the G1 tail in the viral assembly and immune
evasion of Bunyaviridae.
FOOTNOTES
The atomic coordinates and structure factors (code 2K9H) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).
* This work was supported, in whole or in part, by National Institutes of Health Grant AI065359 (to S.C.S.J.) and AI057160 (to R. N. D.). This work was also supported by American Heart Association Grant 0755724Z (to R. N. D.), National Science Foundation Grant 0326999 (to S. C. S.), the Madison and Lila Self Graduate Fellowship (to D. F. E.), and the Reno Cancer Foundation (to D.M.B.). The costs of publication of this article were defrayed in part by the 97
payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 To whom correspondence should be addressed: Dept. of Molecular Biosciences, University of Kansas, 1200 Sunnyside Ave., Lawrence, KS 66045. Fax: 785-864-5294; E-mail: [email protected].
2 The abbreviations used are: DTT, dithiothreitol; HSQC, heteronuclear single quantum coherence; HMQC, heteronuclear multiple quantum coherence; NOE, nuclear Overhauser effect; NOESY, NOE spectroscopy.
ACKNOWLEDGEMENTS
We are grateful to Yu Wang (University of Kansas), Peter Gegenheimer (University of Kansas), Brian Lee (Southern Illinois University), and Gaya Amarasinghe (Iowa State University) for helpful discussion.
98
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ADDENDUM TO CHAPTER 3:
The Zinc Finger Motif of Andes Hantavirus Gn has the potential to bind to the viral RNA panhandle
Unpublished findings
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INTRODUCTION
The Andes hantavirus is a member of the bunyaviridae family (128). All members of the
bunyaviridae family contain a unusually long cytoplasmic tail in glycoprotein Gn (often termed
G1) (31, 131). The hantavirus cytoplasmic tail (~135 amino acids) has been suggested to play a
role in assembly by binding to the nucleocapsid (366) Though this interaction has never been
proven it reasons that a binding between Npro and Gn would connect Npro and the viral RNA to
the membrane bound glycoproteins Gn and Gc to form a mature virion. In fact three other
members of the Bunyaviridae family; Tomato Spotted Wilt Virus, Uukuniemi Virus,
Bunyamwera Virus possess an interaction between the Gn cytoplasmic tail and Npro which is significant in the assembly process (328, 329, 367). These members all share a common dual
CCHC motif in their cytoplasmic tail which may be responsible for the Gn to Npro interaction
(unpublished findings).
It was recently determined that the conserved dual CCHC motif in the Andes hantavirus
Gn cytoplasmic tail forms a structure of two zinc fingers (368). This zinc finger follows the classical helix-loop-beta-loop-beta pattern (368). Zinc finger motifs are reported to bind either
RNA or DNA and in some rare cases they bind proteins (334, 335, 359, 360, 361, 362, 363). 99
Early growth response protein 1 (EGR) is a good representative of a zinc finger containing
protein which binds DNA (369). This protein makes several contacts between the arginine and
lysine sidechains of the helix and the first loop in the zinc finger and the major groove of double
stranded DNA. EGR contains three CCHH motifs in which each zinc finger is connected by 8-10
amino acid loop regions. Each zinc finger binds around the major groove in a sequence-specific
manner which is critical for EGR’s role as a transcription factor (370, 371).
The dual zinc fingers of Andes (ANDV-Gn-ZF) contain only 4 amino acids between the two cysteines. In this structure the zinc fingers are uniquely bound together into one globular structure. Each zinc finger is incapable of flexing around a DNA helix to interact with the major groove. Furthermore ANDV-Gn-ZF contains only one lysine and no arginines. Its theoretical pI is 5.8 and is much too acidic to interact with nucleic acids. Furthermore, it was shown that his motif cannot bind to either viral or cellular RNA (368). The absence of Gn as a binding partner for Npro in SNV infected cells (see Prologue Section) and the inability of the Gn zinc finger to bind viral RNA does not explain how Npro and RNA could interact at the assembly site to associate the RNA with the budding viral membrane.
The influenza matrix protein M1 selectively binds and exports viral RNA out of the nucleus for packaging into the virion at the cell membrane through interaction with basic residues up and downstream of the zinc finger motif (372, 373). The influenza M1 zinc finger itself is not necessary for RNA binding or for viral replication (374). This prompted an analysis of the amino acid sequences of Gn cytoplasmic tail that flank the zinc finger motif. Here it is presented that the protein sequence which includes the regions up and downstream of the two zinc fingers (Gn-
ZF-Arms) is rich in lysines. Using a modified RNA-ChIP assay and molecular modeling it was demonstrated that Gn has potential to bind viral RNA with a specific affinity for the dsRNA panhandle of the S segment (ANDV-S-PanH). 100
MATERIALS AND METHODS
RNA-ChIP Assay
Cos-7 cells in a 100mm dish were transfected with the pAD-ANDV-M plasmid using lipotectamine 2000 (Invitrogen). This plasmid expresses Gn, Gc, and negative sense M segment viral RNA (article in press). Cells were incubated for 3 days and expression of G1 was confirmed. Cells were fixed for 10 min with 1% formaldehyde, washed twice with 1x PBS, and lysed for 15 min in lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 1% Triton X-100, 0.1% Tween
20, 1 mM EDTA). Samples were sonicated to shear RNA ( 1000 bp), and fragments were diluted
sixfold with ChIP buffer (12.5 mM Tris, pH 8, 200 mM NaCl, 1% Triton X-100) and precleared with mouse immunoglobulin G-AC (Santa Cruz Biotechnology) at 4°C for 30 min. For each immunoprecipitation, 10ul of polyclonal anti-Gn antibody was incubated with the lysate at 4°C
overnight. No-antibody control immunoprecipitations were also performed. Protein A/G-Plus- agarose beads (Santa Cruz Biotechnology) were blocked with various amounts of yeast ribosomal
tRNA and bovine serum albumin at 4°C overnight and then washed with ChIP buffer. The blocked and washed protein G-Plus beads were incubated with the lysate at 4°C for 1 h. The beads were washed once with low-salt buffer (0.1% SDS, 0.1% Triton X-100, 2 mM EDTA, 20 mM
Tris, pH 8, 150 mM NaCl), once with high-salt buffer (0.1% SDS, 0.1% Triton X-100, 2 mM
EDTA, 20 mM Tris, pH 8, 500 mM NaCl), and twice with Tris-EDTA (TE). Beads were
resuspended in TE, incubated with DNAse I at 37°C for 30 min, and then incubated with
proteinase K and 10% SDS at 37°C for 4 h, followed by incubation at 65°C overnight. For input
control samples, NaCl was added to the sonicated lysate to a final concentration of 0.3 M and incubated at 65°C overnight. After the antibody, no-antibody, and immunoprecipitated was incubated, RNA was extracted using Trizol per manufacturer’s instructions (Invitrogen). cDNA 101
was producting using MMLV RT step with random primers (Promega). DNA was then used as a template for PCR with primers specific for the ANDV-M segment.
Alignment
Multiple sequence alignment was performed using ClustalW (311) Sequences for residues 141-330 of the hantavirus nucleocapsid was obtained from genbank accessions;
NP_604472 – ANDV, AAB87908 – LECV, AAC54561 –NY1V, AAG28741 – SEOV,
CAB42098 – TOPV, NP_942586 – TULV, CAB43026 –PUUV, X55129 – PHV, AAB87913 –
MACV, AAC42202 SNV, GQ120966 – HTNV.
Molecular Modeling
Secondary structure and hydrophobicity probabilities were predicted for the sequence of
Gn-ZF-Arms using JPRED, SSPro, SOPM, GOR4, and ProfSec (312, 313, 314, 315, 316).
Modeling was performed using the Sybyl8.0 software package. Loop searches were performed to model the N-terminal arm (residues 525-542) and the C-terminal arm (residues 600-627).
Templates were choosen based on their sequence homology, van der Waals bump violations, and secondary structure matching those predicted byJPRED, SSPro, SOPM, GOR4, and ProfSec.
The rotavirus capsid protein VP6 (PDB Id: 1qhd) was selected for the N-terminal arm. The
Pyrococcus furiosus protein 403030 (PDB Id: 1xx7) was selected for the C-terminal arm. These loops were connected to the NMR determined structure of the ANDV-Gn-ZF (residues 543-599).
The sidechains of Phe100 andTyr618 required mild rotation to correct van der Waals bump violations.
Staged Minimization and Dynamics simulations were performed in the Amber99
Forcefield to determine the most energetically favorable positions for atoms in each molecule.
Staging occurred in order by; first minimizing the sidechains, running dynamics on all atoms 102
except the backbone of predicted alpha helix or beta sheets, minimized under the same conditions, and finally minimizing the entire molecule. Following minimization, torsional constraints were enforced into the phi and psi angles to maintain alpha helix and beta sheet structure to run an additional molecular dynamics simulation followed by minimization.
Constraints added to residues: 526 -541, 558-568, 583-591, 599-604, and 614-622 for alpha helix and to residues 546-548, 553-556, 571-573, and 577-579 for beta sheets. Torsional constraints were maintained throughout docking procedures in order to maintain the most likely secondary structure and maintain structured regions during dynamics.
Double and single stranded RNA was built using Sybyl8.0 build function. The double stranded RNA panhandle from ANDV–S segment (ANDV-S-PanH) was constructed by building
RNA bases 1-22 of the 5’end as a double stranded helix. Because the building of a dsRNA helix produced perfectly matched pairs, those bases on the 3’ end (bases 1849-1879) which are mismatched were mutated to match the correct 3’end sequence. Additional single stranded RNA was built and joined to the RNA helix. The ends of the single stranded regions were maintained as an aggregate through molecular dynamics simulations.
Docking
Docking between Gn-ZF-Arms and RNA was performed interactively by guiding the
Lys559 which is at the center of the zinc finger region near the major groove of dsRNA or along the ssRNA. A series of dynamic simulations was performed by keeping the RNA as an aggregate and either maintaining the arms or the two zinc fingers as an aggregate. A final dynamics simulation was run keeping the protein backbone as an aggregate and allowing the RNA and sidechains to move in response to each other. The interaction energy of docking attempts is derived from the total energy equation: 103
Total Energy = energy of protein 1 + energy of protein 2 + interaction energy
To form the equation:
Interaction energy = (total energy) – (energy of RNA + energy of Gn-ZF-Arms)
RESULTS
Full Length G1 transiently binds the M segment in vivo
Initial efforts to demonstrate the potential for viral RNA binding by the ANDV-Gn-ZF involved the use of the pAD-ANDV-M plasmid. This vector uses a bidirectional promoter system to produce; [1] (-) sense viral RNA from the polymerase I promoter which is cleaved by the polymerase I terminator and [2] (+) sense mRNA for viral protein expression from the polymerase II promoter (cytomegalovirus immediate early promotor sequence) and polyadenylated at the 5’ end. This system has been used successfully using the cDNA of the entire influenza genome inserted into eight plasmids which produce all viral components into cells where the virus can assemble (375). Here it is used to express Gn and the (-) sense M segment viral RNA into cells. The interaction between these protein and RNA is tested using a modified assay from the original ChIP assay procedure (376). Co-immunoprecipitation of Gn revealed that M segment RNA could be bound to the protein in transfected cells (Fig. 1). This result suggests that Gn might bind RNA instead of binding to Npro which prompts a further analysis of the zinc finger domain within Gn. 104
FIGURE 1. RT-ChIP assay of pAD-ANDV-M transfected cells. Cos-7 cells were harvested 3 days post transfection with pAD-ANDV-M. A modified RT-ChIP assay was performed using primers for a 330 bp region of ANDV M segment. cDNA samples include the transfected cells before co-immunoprecipitation (Input), the co-IP using polyclonal Gn antibody (Antibody), negative control for pulldown (No Ab), water controls for the RT and the PCR reaction (H20pcr, H20rt), and a pcr of the plasmid pAD-ANDV-M (pAD-AM) as a positive control.
Model of the dual zinc finger and lysine-rich flanking arms (Gn-ZF-Arms)
The linker between the two zinc fingers is too short to allow both CCHC motifs to separate and interact with two sections of the major groove of a double stranded RNA like classical zinc fingers do (Fig. 3A, 3B). Based on the finding that RNA binding properties of influenza M1 are determined by basic residues near the zinc finger and the zinc finger itself is not responsible for RNA binding, the possibility that ANDV-Gn might bind RNA in a similar manner was explored. Analysis of the cytoplasmic tail of ANDV-Gn revealed a high concentration of conserved lysines which flank the dual zinc finger motif (Fig. 2). The lysine-rich region also contains several aspartic acid and glutamic acids which alternate between the lysines. Overall the predicted pI for the Gn-ZF-Arms protein is 8.8. The addition of the flanking regions made this protein more basic from the original pI of the zinc finger region alone (pI = 5.8). 105
To gain insight into the structure of these regions a predictive model was developed.
Secondary structure prediction revealed that the N-terminal region from 525 to 543 and the C- terminal region 600 to 608 were likely to form an alpha helix structure (Fig. 2C). Using Sybyl8.0 loop search program templates were identified which would match the secondary structure 106
predictions. The flanking regions were added and the whole structure was run through staged minimization and molecular dynamics simulations. The final structure formed a globular region for the two zinc finger motifs with the two flanking regions extended outward like arms
FIGURE 3. Addition of N- and C- terminal arms to ANDV-Gn-ZF is predicted to form an dsRNA binding motif. A. NMR structure of the zinc finger region. Arrow points to the short linker (569-573) which maintains each zinc finger in close proximity. B. The NMR structure of zif 268 (PDB Id: 1g2d) a three CCHH motif structure with 8-10 amino acids (arrows) between each motif structure. C. Ribbon diagram showing the secondary structure of the model constructed using loop searches. Secondary structure includes alpha helices (purple), beta sheets (yellow), and unstructured regions (cyan). D. Space Fill diagram of the Gn-ZF-Arm model shows the residues which are aliphatic (green), polar (white), and surface clusters of acidic (red) and basic (blue) residues. Note the high concentration of basic residues along the periphery of the arms.
(Fig. 3C) These arms did not prefer to interact with each other or with the zinc finger throughout molecular dynamics simulations. There are alternating positive and negative sidechains along both helices which prefer to be surface exposed (Fig. 3D). 107
The Gn-ZF-Arms model interacts with double stranded RNA
The overall positive charge on both arms suggested that these structures may have affinity for RNA. Visual measurement of the space between the two arms suggested that a dsRNA helix much like the panhandle structure of the 3’ and 5’ ends hantavirus viral RNA segments. To test if the Gn-ZF-Arms protein would have an affinity for dsRNA a helix for a dsRNA helix was built and visually docked to the major groove of the RNA (Fig. 4A, 4B). A series of molecular dynamics simulations were performed to allow the dual zinc finger motif and the K-rich arms to interact with the RNA. The final complex was run in dynamics together and the interaction energy was calculated to be -635 kCal/mol (Fig. 4C, 4D). In general the overall negative energy of this complex suggests a favorable interaction between a dsRNA helix and Gn-
ZF-Arms.
The Gn-ZF-Arm protein has affinity for the ANDV-S panhandle
Members of the bunyaviridae family contain viral RNA segments in a stem-loop structure which is formed by semi-perfect base-pairing of the 5’ and 3’ ends (Fig. 5A, 5B) (179, 377, 378).
Base-pairing forms a panhandle structure that is characteristic of a RNA double helix. It is thought that the unpaired bases would create a bulge in the helix and make it imperfect (179).
Variation in nucleotide sequences at the 3’ and 5’ viral RNA ends between hantavirus segments and genotypes can create a variety of panhandle helix and bulge structures (179). Because the 3’ end of the segment is upstream of the gene it has been suggested that the variation in the structure would result in altered promoter strengths (378). Another possibility is that the structures are recognized with different affinities by the nucleocapsid’s RNA binding domain and possibly the
RNA binding domain of Gn-ZF-Arms. To predict if the Gn-ZF-Arms protein would interact with different affinities to a dsRNA helix with unpaired RNAs causing a bulge structure the ANDV-S segment 5’ and 3’ end structure was constructed and the protein was docked to it.. The first and 108
FIGURE 4 Molecular docking of Gn-ZF-Arms to dsRNA. Ribbon diagram of the secondary structure of Gn-ZF-Arms to include alpha helices (purple), beta sheets (yellow), and unstructured regions (cyan) and dsRNA (red). Direct view (A) and Side View (B) shows the initial dock guiding the two helices into the major groove of the dsRNA. A series of molecular dynamic simulations was performed and the protein was attracted to the RNA as seen in direct view (C) and side view (D).
last 40 nucleotides of the ANDV-S region were built as a RNA double helix with some single stranded portions. Performing molecular dynamics on this structure maintained the overall helix structure of the first 25 nucleotides but allowed bulging to occur where unmatched nucleotides caused an expanded, bulge of the helix (Fig. 5C)
To test if the Gn-ZF-Arms protein has an affinity for the bulging structure of the AND-S panhandle several docks were aligned along the major groove of the panhandle structure to create
9 separate docking attempts. Another dock was attempted on the single stranded region. Because the zinc finger region alone was previously shown not to bind RNA, this region was docked to the 109
FIGURE 5 The ANDV-S viral panhandle. A. Schematic diagram of the entire ANDV-S segment displaying the 3’ end (position 1), the start codon (position 40) the stop codon (position 1329) and the 5’ end (1871). B. The RNA sequence of the panhandle region showing unmatched bases at 1-11, and 18. C Conformation adopted in the dsRNA as a result of unmatched base pairs. The phosphodiester backbone (red) is distorted and some bases (displayed in CPK color scheme) protrude out of the helix
RNA as a negative control. Interaction energies were calculated throughout the molecular dynamics runs to gain a boltzman distribution of several conformations that complex exists (Fig.
6). Dock #4 which placed the K559 of the zinc finger near the RNA base G19 and was rotated such that K534 was near A10 and K606 was near U1854, had a significantly higher interaction 110
FIGURE 6 Interaction energies and percentage of protein residues within 3A of the RNA for 11 docking attempts of Gn-ZF-Arms with the ANDV-S-panhandle. For attempts 1-8, the protein was guided into the major groove of the RNA double helix. Attempt 9 guided the globular zinc finger motif onto the position where dsRNA ended and the single strands split away. The arms of attempt 9 were in contact with the single strands. Attempt 10 docked the protein onto single stranded RNA. Attempt 11 involved the zinc finger protein alone without the arms docked to double stranded RNA. These energies are for qualitative ranking only. They do not represent the complete thermodynamic cycle of binding.
energy than the rest of the docking attempts (Fig. 7A, 7B). In this complex there were 28 amino acids, of which 6 are positively charged, were within 3A of the RNA suggesting that van der
Waals forces contribute in some part to the interaction energy (Fig. 7B, 7C). The positioning allowed the loop between the two helices in the C-terminal arm to rotate which the helix of residues 614-622 near the beginning of the single stranded RNA structure. Arginine 620 was located near the single stranded RNA (Fig. 7B, 7C). The interaction energy calculated here is for 111
qualitative interpretation only. In general the energy of dock #4 is almost twice that of the dock using only the ZF without the arms (Fig. 6). Taken together this suggests that Gn-ZF-Arms protein may in fact be an RNA binding protein with affinity for the hantavirus 3’ and 5’ panhandle structures.
FIGURE 7. The Gn-ZF-Arms protein initially docked with the ZF region on top of the second bulge (g18). A. Docking attempt #4 shown as a shaded ribbon with alpha helices (purple), beta sheets (yellow), and unstructured regions (cyan) and dsRNA (red). B. Ball and stick representation of all RNA (red) within 3A of positively charged residues (blue) on Gn-ZF- Arm protein. A total of 28 out of 100 amino acids on the protein were within 3A of the RNA.
DISCUSSION
This preliminary work indicates that the ANDV Gn cytoplasmic tail has the potential to bind viral RNA with specificity for the second bulge in the panhandle. This finding is based on 112
the interaction between predictive models for the flanking regions to the zinc finger and the panhandle region of ANDV-S. In vivo experiments such as the ChIP assay have been problematic because it is difficult to distinguish binding of Npro to Gn or viral RNA to Gn in infected cells. For this reason the use of the pAD-ANDV-M vector was used to express both Gn and viral RNA in cells. The lack of a reliable monoclonal antibody to Gn has prevented confident results from the ChIP assay. Further attempts to develop a suitable assay to analyze protein-
RNA interactions in vivo will be useful to analyze the ability of Gn to bind viral RNA.
In vitro assays have indicated that that a purified protein expressing the ANDV-Gn 524 to
610 with a maltose binding protein tag is capable of binding to purified RNA from infected cells
(data not shown). This assay is a modified version of electrophoretic mobility shift assay for the detection of RNA. It demonstrates that this protein binds RNA in a dose-dependent manner.
Because the source of RNA is infected cells the possibility exitsts that ANDV-Gn could bind cellular RNA. Experiments are ongoing which involve using purified viral RNA, mutagenesis of lysines in the flanking regions, and NMR.
The molecular modeling performed in this study was attempted to predict if the cytoplasmic tail of Gn could have potential as an RNA binding domain. Based on the overall negative interaction energy between the protein and RNA, the interaction is predicted to be favorable. The addition of a solvent to the complex and extending the length of the molecular dynamics simulations is in progress. This will give a better resolution to the structural properties of the Gn-ZN-Arms RNA binding potential.
If Gn is indeed proven to contain an RNA binding domain, a significant contribution to the current thought on hantavirus assembly will be made. Influenza matrix protein M1 is an example of a protein which is bound to the membrane, the nucleoprotein and the viral RNA for the purpose of coordinating viral RNA packaging into the budding virion (224, 379). The 113
genomes of both hantaviruses and influenza viruses are composed of segmented negative stranded RNA with eight and three segments respectively. A burning question in influenza research is what is the process for packaging one copy of each of the eight genomic segments into the assembly site to form a complete and infectious virion (224)? Current evidence indicates that genetic elements in the 3’ and 5’ ends of each segment and specific regions of the viral protein provide specific interactions that “selectively” package the viral RNA genome into the assembly site (380, 381). This is in contrast to the previous thought that no interactions between RNA and proteins existed and RNA was “randomly” packaged into the virion (382). This study asks; Can the proposed interaction between Gn-ZF-Arms and the viral RNA panhandle act to selectively package one of each of the three hantavirus viral segments? What is the specificity of the RNA – protein interaction? Furthermore, would this specificity exclude the possibility that functional viruses could be packaged as reassortants between segments of one genotype having a unique panhandle structure and a different genotype with its own unique panhandle structure? Further research is ongoing to determine the specificity of the Gn – viral RNA interaction.
114
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CONCLUSION
______
The functions of three motifs in hantavirus proteins have been investigated. The N-
terminal domain of the Andes nucleocapsid forms a coiled coil of two long (~30a.a.) helices
which are connected by an acidic loop. Based on NMR and modeling the SNV, PUUV, and
TULV nucleocapsids also form a coiled coil structure indicates that the structure is conserved and
may have an essential function for the virus (248, 272). Residues in the acidic loop contribute to
the normal trafficking of the protein in the cytoplasm and the formation of oligomers (272). This
motif could have a significant impact on the assembly and cell regulation properties of the
nucleocapsid and viral replication overall.
The SNV middle domain of the nucleocapsid protein is highly variable amongst
hantavirus genotypes. Because the N-terminal and C-terminal domains are involved in
oligomerization (ref) the middle region is likely to behave more as a single monomer with more
surface exposed residues and the potential for interaction with cellular proteins. The RNA
binding domain has been mapped to the middle region of the nucleocapsid (175). It reasons that
this region would be presented to the inside surface of the trimer complex to coordinate the RNA
between the trimers and protect it from cellular degradation (247) . With the exception of the
RNA binding domain and a potential SUMO-1 binding domain, this large region (141-330) has been mostly unmapped (183). A helix-loop-helix was identified in positions 258-295 of SNV nucleocapsid which makes contacts with the cytoskeletal intermediate filament, vimentin through 115
the use of molecular dynamics simulations. The interaction between nucleocapsids of most hantavirus genotypes and vimentin appears to be conserved. HTNV nucleocapsid uniquely does not bind vimentin but it does cause vimentin to rearrange in the cytoplasm to surround the nucleocapsid into cages (201). HTNV N significantly contains positively charged residues in the loop of the helix-loop-helix region. This further supports the requirement of the SNV motif for binding to vimentin. Because multiple viruses require functional vimentin for the production of infective virus in cells (305, 306, 307), the association of nucleocapsids with vimentin is likely a critical event in the process of hantavirus infection.
Many viruses demonstrate that the packaging of ribonucleoprotein complexes into the membrane near the site of assembly occurs through interaction of the viral capsid protein and the viral membrane bound proteins. The identification of a conserved dual CCHC motif in cytoplasmic tail of the membrane bound ANDV glycoprotein Gn suggested that it could be a nucleic acid binding motif. It was hypothesized that Gn would interact with viral RNA to coordinate the packaging of the RNP into the budding viral membrane. NMR determined that the dual CCHC motifs bound zinc to form two classical zinc finger folds. A significant difference between the ANDV zinc finger motif and most zinc fingers is the number of residues that link the two CCHC domains together. Most zinc fingers have a long enough linker to allow two (or in some cases three) zinc fingers to flex around double stranded DNA to make multiple contacts with the major groove. The ANDV zinc finger has a short linker. The individual zinc fingers are not positioned to interact with nucleic acids. Further analysis of the regions near the
ANDV zinc finger reveals two lysine-rich regions. By the use of molecular modeling a mechanism for zinc finger to viral RNA interaction is proposed. Multiple contacts between the viral RNA 3’ and 5’ ends of and the helixes up and downstream of the glycoprotein Gn were formed during molecular dynamic simulations. The potential binding of glycoprotein Gn to viral
RNA is inferred to coordinate each of the three viral RNA segments into the packaging process. 116
Another potential role of the zinc finger to viral RNA interaction may be in the replication/transcription processes. Though minigenome synthesis of viral RNA does not require
Gn, the possibility of its role in replication should not be disregarded (155).
The threat of hantaviruses as an emerging pathogen and a potential to cause outbreaks requires further research and development of antiviral drug treatments. The ultimate purpose of this dissertation was to describe regions in hantavirus proteins which are essential for viral replication in order to identify potential targets for antiviral development. While the efforts reported in this dissertation offer the possibility of discovering a good target there remains much work to do. Future research should include a more thorough analysis of the function of these motifs, a testing of potential inhibitors of viral infection, and an analysis of the requirement of these motifs for the production of infective virus.
Further analysis on the structure and function of the three motifs would be useful. Both the nucleocapsid middle region and the zinc finger flanking regions need to have NMR performed to gain a better view of their conformations. NMR efforts so far have given a poor spectrum which is too difficult to interpret. This suggests that the proteins exist in multiple conformations in a water environment. The addition of vimentin with nucleocapsid peptides or RNA with the
Zinc Finger peptides may stabilize the conformation enough to gain a resolved NMR spectrum.
Both models offer enough reliability that they can be used to design point mutants to be tested in in vivo assays (ChIP, co-immunoprecipitation). The initial mutants designed to map the vimentin binding site on the nucleocapsid considered that only electrostatic forces were involved. The model suggests that hydrophobic interactions may also play a role. This possibility should be tested further. Additionally the lysine-rich regions that flank the zinc finger should also be mutated to determine their requirement for viral RNA binding. 117
A better method for understanding the effect of a motif during infection is the development of a reverse genetics system that would permit the manipulation of protein sequences in the live virus. This involves a group of plasmids with promoters that will express the viral proteins and RNA in cells where assembly and formation of the virion can occur.
Because the formation of the virus is initiated by plasmids, standard molecular biology techniques can be used to alter the genes of the virus to produce mutant viruses by design. To date no reverse genetics system has been successful for hantaviruses. Other members of the bunyavirus family have developed this system (383, 384, 385, 386). Reverse genetics systems have been used to develop attenuated viruses as vaccine candidates, develop reassortants to identify pathogenic markers in pathogenic and non-pathogenic genotypes, and to identify essential motifs in the virus required for replication (387). The development of a reverse genetic system for hantaviruses would greatly enhance the understanding of its biology. Obstacles to this endeavor include; matching a cell culture types with the species of the promoter sequences on the plasmid, selecting a single culture or co-culture of cell types which would express the RNAs and proteins for a long enough time to permit assembly, and expressing all proteins and viral RNA in the cytoplasm so that they will interact and assemble. Solutions to these challenges are currently being evaluated.
An alternative method for testing the function of motifs in the proteins of the hantavirus would be to affect the binding of the motif using inhibitors. The process of oligomerization requires both the N- and C-terminal ends of the nucleocapsid. A useful experiment would be to overexpress only the N-terminal coiled coils in order to interact with full length nucleocapsid monomers. If expression was high enough the truncated protein might prevent complete oligomerization and thus reduce the ability of the virus to assemble. To disrupt vimentin, acrylamide and siRNA have shown to be very effective at reducing the ability of viruses to replicate (296, 298, 299, 300). The use of these inhibitors would further support the involvement 118
of vimentin to hantavirus replication. Zinc finger inhibitors are currently a strong candidate for a antiviral treatment for HIV (388). These are sequence specific nucleotide analogues which are conjugated with heavy metals to add stability and increase the affinity for the zinc finger (389).
The use of these inhibitors against hantavirus infections is worth consideration in order to both define the mechanisms of these motifs and to be considered as potential antiviral treatments.
119
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APPENDIX I
Supplementary Material for Chapter 1
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Fig. S1. Sequence alignment of hantavirus nucleocapsid N1-74 coiled coil domain with the conserved hydrophobic (gray) and polar (yellow) heptads highlighted. The sequences are arranged according to hantaviral species that cause: (top) HCPS (Hantavirus CardioPulmonary Syndrome), (middle) nonpathogenic or mild form of HFRS (Hemorrhagic Fever with Renal Syndrome) and (bottom) severe form of HFRS. The hantavirus species are: AND, Andes; SNV, Sin Nombre; LAN, Laguna Negra; MUL, Muleshoe; BAY, Bayou; RIO, Rio Mamore; ELM, El Moro Canyon; NYV, New York; TUL, Tula; ISL, Isla Vista; PHV, Prospect Hill; PUU, Puumala; TOP, Topografov; KHA, Khabarovsk; SEO, Seoul; THA, Thailand; HTN, Hantaan; DOB, Dobrava.
147
Fig. S2. Secondary C, H, C’, and Cchemical shifts show that the first 33 residues (shaded), which are the His-tag from pET151, lack secondary structure whereas the Andes virus N1-74 region consists of two-helices.
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Fig. S3. Immunocytochemistry of full length N protein with mutations in the N1-74 coiled coil domain. Cos-7 cells were transfected with will type and mutant N protein, and doubly stained with (A) monoclonal and polyclonal anti-N antibodies, and (B) monoclonal anti-N and Golgi-specific antibodies. The anti-N monoclonal antibody was AB34757 from Abcam (Cambridge, Mass.).
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APPENDIX II
Supplementary Material for Chapter 3
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Fig. S1. The proper folding of the hantavirus G1 tail zinc finger domain depends on Zn2+- binding. 1H-15N HSQC spectra of (A) Andes virus and (B) Prospect Hill virus G1 zinc finger domains before (black peaks) and after (red peaks) addition of 4 mM EDTA. In the Andes zinc finger, addition of EDTA resulted in the deterioration of the HSQC peaks, with peaks that are sharp and other peaks with reduced intensities, as well as collapse of asparagine and glutamine side chain resonances. In the Prospect Hill virus, addition of EDTA collapsed the backbone and side chain peaks (at 7.8-8.6 and 6.8-7.6 1H ppm, respectively) into a characteristic HSQC of an unfolded protein. We determined the NMR structure of theG1 zinc finger domain from the Andes virus, which is pathogenic to humans (whereas Prospect Hill virus is not). 151
Fig. S2. Secondary C, H, C’, and Cchemical shifts of the Andes virus G1 zinc finger domain. Shown are the CCHC-zinc coordination ligands (red bars) and the secondary structures (-strands and -helices).
Fig. S3. The tautomeric states of the Zn2+-coordinating histidines (His590 and His564) were determined by 2D 1H-15N HMQC following the method of Pelton et al. (Protein Sci., 1993, 2, 543-558). This spectrum was acquired using an 15N-labeled protein in buffer (10 mM NaPO4 pH 7.0, 10 mM NaCl, 1 mM DTT, 0.1 mM ZnSO4) in 100% D2O with the following acquisition parameters: 15N carrier frequency (195 ppm), 15N sweep width (70 ppm), number of scans (32), number of 15N complex points (128).
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Fig. S4. Effect of point mutations in the Zn2+-coordinating cysteine and histidine residues. 1H-15N HSQC spectra of GB1-zinc finger domain fusion proteins with point mutations in the eight Zn2+-coordinating residues. Cysteine was mutated into serine and histidine into phenylalanine. Spectra of GB1 fusion proteins (black peaks) are overlayed with the spectrum of free GB1 tag (red peaks)
153
Fig. S5. Effect of point mutation in histidine and cysteine residues that do not coordinate Zn2+ ion. The 2D 1H-15N HSQC spectra of histidine point mutants (H542F, H552F, and H553F) showed the characteristic well dispersed and distinct peaks of a folded protein. The spectrum for the C555S mutant also showed well dispersed peaks, however, there is a region in the middle of the spectrum that is characteristic of an unfolded protein. C555 is a non-Zn2+-coordinating residue, however, it forms part of the hydrophobic core, and the C555S mutation caused an unfolding in some regions of the zinc finger domain. Shown are the 1H-15N HSQC spectra of GB1-zinc finger fusion proteins (black peaks) overlayed with the spectrum of free GB1 tag (red peaks). 154
Fig. S6. The structures (top panel) and zinc binding topology (bottom pane) of zinc fingers (A) Andes virus G1 zinc finger domain is different from that of (B) RING domain (PDB 1CHC) and (C) LIM domain (PDB 1M3V). The cartoon depiction of zinc binding topology was adapted from Gamsjaeger et al. (Trends Biochem. Sci., 2007, 32, 63-70) 155
Fig. S7. The hantaviral zinc finger domain does not bind RNA. (A) Electrophoretic mobility shift assay of total RNA (from VeroE6 cells 14 days post-infection with Andes virus), a known RNA-binding protein (PACT) expressed as a fusion protein with the maltose binding protein (MBP), and the Andes virus zinc finger domain (ZF). The smearing of the RNA bands by MBP- PACT showed nonspecific RNA binding, while the RNA bands remained unchanged with the zinc finger domain. (B) RT-PCR using primers specific to beta actin and the S-segment of the Andes virus genome (ANDV-S) confirmed the presence of cellular and viral RNA. Controls were uninfected VeroE6 cells, water, and plasmid DNA with the S segment of the Andes virus genome (pGEM-S).