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Publicly Accessible Penn Dissertations
2018
Enhancement Of Ebola Virus Infection By Seminal Amyloid Fibrils
Stephen Michael Bart University of Pennsylvania, [email protected]
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Recommended Citation Bart, Stephen Michael, "Enhancement Of Ebola Virus Infection By Seminal Amyloid Fibrils" (2018). Publicly Accessible Penn Dissertations. 2681. https://repository.upenn.edu/edissertations/2681
This paper is posted at ScholarlyCommons. https://repository.upenn.edu/edissertations/2681 For more information, please contact [email protected]. Enhancement Of Ebola Virus Infection By Seminal Amyloid Fibrils
Abstract The 2014-2016 West Africa epidemic was the worst Ebola virus outbreak on record, with the infected numbering over 28,000 and the dead more than 11,000. Among those who survived the disease, Ebola virus was found to persist in numerous anatomical locations and be shed in semen. During the West Africa outbreak, numerous occurrences of Ebola sexual transmission were documented. While sexual transmission has the potential to compromise public health eradication efforts and reignite outbreaks, no molecular details of host factors influencing this phenomenon are known. Amyloid fibrils present in healthy individuals’ semen have been suggested to be a host factor that aids in sexual transmission of viruses including HIV-1. To understand if seminal amyloid fibrils increase infection by Ebola virus, a recombinant virus expressing the Ebola glycoprotein was preincubated with the type species of the seminal amyloids, SEVI. When present, SEVI resulted in a nearly 20-fold increase in infection of physiologically relevant target cells. SEVI and other seminal amyloids were also found to enhance authentic Ebola virus infection by 20- to 40-fold in cell culture. Mechanistically, SEVI was found to increase viral attachment then subsequent internalization of virus through macropinocytosis, a previously unknown ability to enhance uptake by this mechanism critical for Ebola virus entry. Finally, a new ability of SEVI to enhance viral stability during incubation at physiologic temperatures and under desiccating conditions is described, a finding important for transmission biology. Together, these functions of seminal amyloids represent a first step owart d understanding Ebola virus sexual transmission at a molecular level and provide potential therapeutic targets to prevent transmission by this route.
Degree Type Dissertation
Degree Name Doctor of Philosophy (PhD)
Graduate Group Cell & Molecular Biology
First Advisor Paul Bates
Subject Categories Virology
This dissertation is available at ScholarlyCommons: https://repository.upenn.edu/edissertations/2681 ENHANCEMENT OF EBOLA VIRUS INFECTION BY SEMINAL AMYLOID FIBRILS
Stephen Michael Bart
A DISSERTATION
in
Cell and Molecular Biology
Presented to the Faculties of the University of Pennsylvania
in
Partial Fulfillment of the Requirements for the
Degree of Doctor of Philosophy
2018
Supervisor of Dissertation
______
Paul Bates, PhD Professor of Microbiology
Graduate Group Chairperson
______
Daniel S. Kessler, PhD Associate Professor of Cell and Developmental Biology
Dissertation Committee
Ronald N. Harty, PhD, Professor of Pathology and Microbiology Sara Cherry, PhD, Professor of Microbiology Susan R. Weiss, PhD, Professor of Microbiology Carolina B. Lopez, PhD, Associate Professor of Microbiology and Immunology
ENHANCEMENT OF EBOLA VIRUS INFECTION BY SEMINAL AMYLOID FIBRILS
COPYRIGHT
2018
Stephen Michael Bart
This work is licensed under the Creative Commons Attribution- NonCommercial-ShareAlike 3.0 License
To view a copy of this license, visit https://creativecommons.org/licenses/by-nc-sa/3.0/us/
This thesis is dedicated in memory of my brother, my uncle, and my Grandpap, who were here to
start my graduate school journey but not to see its end.
iii
ACKNOWLEDGMENTS
During my graduate career, I’ve been able to rely on the support of so many people both at Penn and in my life. There’s no way I could thank all of them in this limited space, but here’s an attempt.
Thank you to Paul for taking me as a student and providing a fantastic opportunity for me to learn and grow by independently pursuing my interests, while all the time being there for support.
And thank you to all of the members of the Bates lab during my tenure: Ken, Nate, Luis, MJ, Ben,
Courtney, Shannon, Lavinia, Patricia, Tomaz, and Trevor, as well as our Doms lab friends
Zahra and Amber. Even as everyone left to pursue bigger and better things, your help, friendship, and kindness were always appreciated, as were our Taco Bell lunches and zoo/Sabrina’s trips whenever Paul wasn’t around. A special thanks to MJ Drake for being a mentor and friend from my rotation (when she helped hide my mistakes from Paul) until now. The members of the Hensley
(especially Seth, Shannon, Amy, and Elinor) and Weiss labs (especially Jill and Courtney) also get special shoutouts for letting me borrow reagents and/or procrastinate by visiting and talking to them.
Special thanks to my CAMB friends, including my roommates David, Steve, and Devin,
Terra, Colleen, Somdutta, Bobby, Christin, and Chris; there’s no one else I’d rather have spent the last five years with. Courtney (again), Mary Margaret, and Nadia have also been fantastic, especially when it comes to talking about feelings and being motivators to be healthier (but pointing out that it’s hard, usually via Facebook memes). I also want to acknowledge my friends from back home, some of whom I’ve known since I was four: Megan, Dylan, Kristen, Dollar, and Gwen have always been there and will always be a major part of my life.
Some other important people: my committee members, Ron, Sara, Susan, and Carolina for all taking the time to take a genuine interest in my life, research, and career to help at each step of the way. Arnaldo for being a great friend and mentor, and taking a chance with me during my summer program at Penn as an undergrad, and Sparky for giving me a great experience in his lab.
Jim has been a great mentor as I embarked on a journey through amyloid biology. The CAMB
iv coordinators, Meagan, Kathy, Anna, and Christina have all been beyond helpful. As I started my journey into public health, numerous people have been invaluable – Hillary, Ami, Eileen, and
Kristen being extraordinary.
Lastly, and most importantly, I want to thank my family. We have been through a rough time the last five years, but my Mom and Dad have remained supportive all throughout, even though I’m not sure they completely understand what I do, exactly. There’s not nearly enough space here to list my extended family, but both of my sets of grandparents and my aunts and uncles have always been incredibly supportive of my education. My brother Matt may no longer be with us, but he will always be a lasting part of my life.
And thanks to you, if you plan on reading this whole story about Ebola and semen. I spent a lot of time trying to make sure that the figures were exactly right, pixel by pixel, so of course I can’t wait to see whatever glaring mistake I made as soon as it’s printed.
v ABSTRACT
ENHANCEMENT OF EBOLA VIRUS INFECTION BY SEMINAL AMYLOID FIBRILS
Stephen Michael Bart
Paul Bates
The 2014-2016 West Africa epidemic was the worst Ebola virus outbreak on record, with the infected numbering over 28,000 and the dead more than 11,000. Among those who survived the disease, Ebola virus was found to persist in numerous anatomical locations and be shed in semen.
During the West Africa outbreak, numerous occurrences of Ebola sexual transmission were documented. While sexual transmission has the potential to compromise public health eradication efforts and reignite outbreaks, no molecular details of host factors influencing this phenomenon are known. Amyloid fibrils present in healthy individuals’ semen have been suggested to be a host factor that aids in sexual transmission of viruses including HIV-1. To understand if seminal amyloid fibrils increase infection by Ebola virus, a recombinant virus expressing the Ebola glycoprotein was preincubated with the type species of the seminal amyloids, SEVI. When present, SEVI resulted in a nearly 20-fold increase in infection of physiologically relevant target cells. SEVI and other seminal amyloids were also found to enhance authentic Ebola virus infection by 20- to 40-fold in cell culture.
Mechanistically, SEVI was found to increase viral attachment then subsequent internalization of virus through macropinocytosis, a previously unknown ability to enhance uptake by this mechanism critical for Ebola virus entry. Finally, a new ability of SEVI to enhance viral stability during incubation at physiologic temperatures and under desiccating conditions is described, a finding important for transmission biology. Together, these functions of seminal amyloids represent a first step toward understanding Ebola virus sexual transmission at a molecular level and provide potential therapeutic targets to prevent transmission by this route.
vi TABLE OF CONTENTS
ACKNOWLEDGMENTS ...... IV
ABSTRACT ...... VI
LIST OF TABLES ...... IX
LIST OF ILLUSTRATIONS ...... X
CHAPTER 1 – EBOLA VIRUS ...... 1 Section 1.1 – Ebola virus epidemiology ...... 1 Section 1.2 – Ebola virus biology ...... 5 Section 1.3 – Ebola virus disease ...... 8 Section 1.4 – Post-Ebola syndrome and persistence ...... 9 Section 1.5 – Ebola sexual transmission...... 11 Section 1.6 – Ebola virus disease treatment and prevention ...... 12 Section 1.7 – Bibliography ...... 15
CHAPTER 2: SEMINAL AMYLOID FIBRILS...... 26 Section 2.1 – Introduction to seminal amyloids ...... 26 Section 2.2 – Mechanism of enhancement by seminal amyloids ...... 27 Section 2.3 – Seminal amyloids and function ...... 29 Section 2.4 – Targeting amyloid fibrils ...... 30 Section 2.5 – Bibliography ...... 32
CHAPTER 3: ENHANCEMENT OF EBOLA VIRUS INFECTION BY SEMINAL AMYLOID FIBRILS ...... 35 Section 3.1 – Abstract...... 36 Section 3.2 – Significance Statement ...... 37 Section 3.3 – Introduction ...... 38 Section 3.4 – Materials and Methods ...... 40 Section 3.5 – Results ...... 45 Section 3.5.1 – Seminal amyloids enhance rVSV-EboGP-mCherry infection ...... 45 Section 3.5.2 – SEVI enhances EBOV VLP binding and internalization ...... 48 Section 3.5.3 – SEVI-mediated enhancement maintains EBOV entry requirements ...... 51 Section 3.5.4 – SEVI alters rVSV-EboGP-mCherry physical characteristics ...... 51 Section 3.5.5 – Seminal amyloids enhance infection by authentic EBOV ...... 55 Section 3.6 – Discussion ...... 57 Section 3.8 – Supplemental Information...... 60 Section 3.9 – Acknowledgments ...... 64 Section 3.10 – Bibliography ...... 65
CHAPTER 4: DISCUSSION AND FUTURE DIRECTIONS ...... 71 Section 4.1 – Summary ...... 71 Section 4.2 – Future directions ...... 73 Section 4.2.1 – Ex vivo infections ...... 73
vii Section 4.2.2 – Enhancement in the context of other viruses...... 74 Section 4.2.3 – Stabilization against other environmental factors ...... 75 Section 4.2.4 – Mechanisms of enhancement at the cellular and molecular level ...... 77 Section 4.2.5 – Implications for antiviral development ...... 79 Section 4.3 – Concluding remarks ...... 80 Section 4.4 – Materials and methods for preliminary work ...... 81 Section 4.4 – Bibliography ...... 82
APPENDIX – IDENTIFICATION OF HOST FACTORS THAT RESTRICT ZIKA VIRUS INFECTION ...... 84 Section 1.1 – Introduction ...... 85 Section 1.2 – Materials and Methods ...... 86 Section 1.3 – Results ...... 87 Section 1.4 – Bibliography ...... 89
viii LIST OF TABLES
Supplementary Table 3.1. Peptide sequences ...... 61
ix LIST OF ILLUSTRATIONS
Figure 1.1. Ebola virus outbreaks, 1976-present ...... 2 Figure 1.2. Affected countries, 2014-2016 Ebola outbreak...... 3 Figure 1.3. Schematic of EBOV genome organization and virion structure ...... 6 Figure 1.4. Schematic of EBOV entry into cells ...... 7 Figure 1.5. Summary of evidence of filovirus persistence in semen ...... 9 Figure 2.1. Model of enhancement of HIV-1 infection by SEVI ...... 28 Figure 3.1. Seminal amyloids enhance rVSV-EboGP-mCherry infection ...... 47 Figure 3.2. SEVI enhances rVSV-EboGP-mCherry binding, internalization, and cellular macropinocytosis...... 50 Figure 3.3. SEVI alters rVSV-EboGP-mCherry physical characteristics...... 53 Figure 3.4. Seminal amyloids enhance EBOV infection ...... 56 Figure 3.5. Model of SEVI-mediated enhancement of EBOV infection ...... 59 Figure 3.S2. SEVI enhances VLP binding and internalization ...... 61 Figure 3.S1. SEM1 and PAP85-120 exhibit autofluorescence in the mCherry channel .61 Figure 3.S3. Macropinocytosis and cathepsin inhibitors do not exhibit cytotoxicity ...... 62 Figure 3.S4. SEVI does not promote increased stability following treatment with dilute bleach...... 62 Figure 3.S6. α-synuclein does not enhance rVSV-EboGP-mCherry desiccation tolerance ...... 63 Figure 3.S5. No differential thermal degradation occurs over six hours at room temperature in the presence or absence of SEVI ...... 63 Figure 4.1. SEVI does not dramatically increase ZIKV infection ...... 74 Figure 4.2. SEVI does not reduce neutralization sensitivity to anti-EboGP antibodies ...76 Figure A.1. Generation of IFNAR1 knockout cells...... 91 Figure A.2. Generation of SAM cells for screening...... 92 Figure A.3. Schematic for screening A549 SAM cells...... 93
x CHAPTER 1 – EBOLA VIRUS
Ebola virus was first identified in 1976 in the Central African country of Zaire (now
Democratic Republic of the Congo). Named for the nearby Ebola River, the previously unknown virus caused a devastating hemorrhagic disease in the area of the village of Yambuku, killing almost
90% of those infected. Over the next four decades, the five Ebolavirus species caused sporadic and localized outbreaks before the 2014-2016 West African outbreak that infected tens of thousands.
Section 1.1 – Ebola virus epidemiology
Ebolaviruses, which are Biosafety Level 4 pathogens due to their high pathogenicity, have collectively caused 29 documented outbreaks in humans worldwide (Figure 1.11–3). Four of the five viral species in the Ebolavirus genus cause Ebola virus disease (EVD) in humans4. Ebola Zaire
(EBOV) has caused most identified outbreaks, including both the initial 1976 outbreak and the
2014-2016 West African epidemic, and is the most lethal. Ebola Sudan (SUDV) has also caused considerable morbidity and mortality, while Ebola Bundibugyo (BDBV) and Ebola Tai Forest (TAFV) have caused smaller, limited outbreaks. The fifth Ebolavirus, Ebola Reston (RESTV), was identified in monkeys exported into the United States from the Philippines and causes asymptomatic human infections that have been identified only by serology5. The Ebolavirus genus, along with its sister genera Marburgvirus (which contains the eponymous Marburg virus, MARV) and Cuevavirus
(which contains a single species identified only by sequencing of European bats and never isolated6), comprise the Filoviridae family. Discounting RESTV, Ebolavirus outbreaks have originated exclusively in sub-Saharan Africa, though spread to Europe and North America has occurred. Case-fatality ratios for the various outbreaks involving human-to-human transmission have ranged from 36-90%2. Most outbreaks have been relatively small and contained to remote areas, with the West Africa EBOV outbreak being the exception.
1 Figure 1.1. Ebola virus outbreaks, 1976-present. Ebola virus disease outbreaks (excluding accidental laboratory exposures) are indicated by colored circles at the site of the index case, with larger circles indicating more cases. Countries reporting at least one human Ebola virus case are colored in dark gray.
The 2014-2016 West African outbreak is by far the deadliest Ebola outbreak on record.
The official totals for the outbreak, which are almost certainly underestimates of the true disease burden, tally 28,652 cases and 11,325 deaths, a case-fatality ratio (CFR) of 39.5%2. The index case of the outbreak is believed to be a 2-year-old child who died in December 2013 in Meliandou,
Guinea7. This region of Guinea has been heavily deforested and mined, and it is hypothesized that habitat destruction may have exposed the EBOV reservoir to the human population8. The virus spread to the boy’s family and to others from surrounding villages before reaching the neighboring countries of Liberia and Sierra Leone by March 20149. Over the next few months, the virus began spreading through major population centers, including the capital cities of Monrovia, Liberia;
Conakry, Guinea; and Freetown, Sierra Leone. Poor sanitation within urban slums contributed greatly to the spread of the virus10. West Africa has not historically been a site of widespread Ebola outbreaks in the past, and the most heavily affected countries were ill equipped to respond to the outbreak.
2 The virus spread to several nearby countries, including Senegal, Nigeria, and Mali, as well as outside Africa, with cases being diagnosed in Spain, Italy, the United Kingdom, and the United
States11. These countries exhibited very limited viral spread relative to the most heavily affected countries of Guinea, Liberia, and Sierra Leone (Figure 1.2).
The particular strain of EBOV causing the outbreak was named the Makona strain and was found to be 97% similar to EBOV isolates from the Democratic Republic of the Congo (DRC) and
Gabon7,12. The strain clusters phylogenetically with the other Zaire ebolavirus species, with about
400 nucleotide differences across the 19 kb genome separating it and the 1976 EBOV-Mayinga strain7. Genomic sequencing confirmed that the outbreak was monophyletic and appeared to initiate from a single introduction of the virus into the human population followed by sustained human-to-human transmission9.
EBOV is transmissible from the beginning of the symptomatic phase, and the infected can transmit virus even after death. EBOV is highly infectious; as few as 1-10 aerosolized particles are
Figure 1.2. Affected countries, 2014-2016 Ebola outbreak. Map showing the countries reporting cases of Ebola virus disease during the outbreak initiating in West Africa. Darker shades of blue indicate higher numbers of confirmed EVD cases.
3 lethal in non-human primate models of EBOV infection13. The main route of transmission is through contact with infected bodily fluids. Those caring for infected relatives and those involved in preparation of bodies for burial are at particular risk for EBOV transmission; the latter was particularly important during the West African outbreak14. This method of exposure is thought to have played a major role in the spread of the virus before public health measures alleviated this transmission route.
The reservoir for Ebolaviruses between outbreaks has yet to be definitively identified, though evidence suggests that fruit bats are the most likely culprit. EBOV RNA and antibodies have been detected among African bat populations, and seroprevalence of EBOV antibodies increases in bat populations during human outbreaks15-17. Further, MARV, a related filovirus, has been isolated from African bat species18. Notably, the index case of the West Africa outbreak was reported to have been playing in a hollow tree that harbored a large bat population before his symptoms began19. Bats do not show symptoms of disease despite high levels of EBOV in the blood20. Virus has been found in various wild animal carcasses, including gorillas, chimpanzees, and duikers, indicating that infection is not limited to humans21. EBOV outbreaks among chimpanzees and gorillas have led to large declines in their populations22,23. Swine have been documented as hosts for RESTV in addition to monkeys as previously described24. The role of domestic animals as vectors of EBOV transmission is unclear. During a 2002 outbreak of EBOV in
Gabon, 30% of dogs were seropositive for EBOV, though the specificity of the assay used is questionable25. No disease was documented in these animals, and in vitro experiments suggest that canine cells are less susceptible to EBOV infection than primate cells (Box 1)26, leaving the role of domestic animals in EBOV transmission an open question.
While EBOV has clearly evolved on a genetic level between the 1976 outbreak and the present, there have been no notable changes in viral characteristics over time27. However, due to the high degree of person-to-person transmission in the 2014-2016 outbreak, it was hypothesized that mutations may have arisen in the virus as it adapted to humans rather than its natural host.
4 One such mutation, at position 82 in the EBOV glycoprotein, arose during the outbreak and was subsequently found to increase infectivity of the virus in culture28–30. The increase in infectivity, however, comes at a cost to the virus, resulting in decreased glycoprotein stability under physiological temperatures30. Bats have a higher physiological temperature than humans (due to the higher metabolic rate necessary for flight), potentially explaining why the mutation has not been previously seen in outbreaks with less-extensive human-to-human EBOV transmission.
Section 1.2 – Ebola virus biology
The EBOV virion is a long, filamentous, enveloped particle of variable length (600 to 1400 nm) and a uniform diameter (80 nm)31. The EBOV genome comprises a single strand of negative- sense RNA divided into 7 genes encoding 9 proteins31 (Figure 1.3). The genome is packaged within a ribonucleoprotein (RNP) structure composed of nucleoprotein (NP), minor nucleoprotein (VP30), and the polymerase complex (L/VP35). The RNP is enclosed within a matrix composed of the matrix protein VP40 and the minor matrix protein VP24. The glycoprotein (GP1/GP2, or EboGP) is the only virion surface protein and effects attachment to target cells and fusion of the virion and cellular membranes. EboGP is thus the target of the neutralizing antibody response in infected individuals. Additionally, cells infected with EBOV produce two other nonstructural products from
5 Figure 1.3. Schematic of EBOV genome organization and virion structure. the glycoprotein gene, the secreted glycoprotein (sGP) and small secreted glycoprotein (ssGP).
These products share a portion of sequence with EboGP; mRNA production for each is a result of mRNA editing by polymerase slippage or stuttering, with sGP being the main gene product32,33.
The function of these soluble glycoproteins is unclear, but they may assist in evading the host antibody response34,35. EboGP and VP40 together are sufficient to produce filamentous virus-like particles (VLPs) which can mimic the entry process for authentic EBOV36–38.
EBOV undergoes a unique and complex entry process to infect cells39 (Figure 1.4). The first step of viral entry is attachment to the surface of cells by generally nonspecific mechanisms with cellular attachment factors40. Characterized interactions include binding of carbohydrates on
EboGP to C-type lectins such as DC-SIGN and DC-SIGNR41 and direct or indirect binding of phosphatidylserine (PS) in the virion membrane with cellular receptors. These cellular PS receptors include members of the Tyro3 family of receptor tyrosine kinases (e.g. Axl) and the T cell immunoglobin and mucin domain family (e.g. TIM-1)42–46. These attachment factors are abundant on dendritic cells (DCs) and macrophages, which represent important early cell types for EBOV infection. Interestingly, it is hypothesized that the PS-mediated attachment process reflects
6 “apoptotic mimicry,” as apoptotic blebs also present PS on their outer lipid leaflet and are cleared by antigen-presenting cells such as DCs and macrophages47. Intact cells employ flippases to maintain PS on only the inner leaflet of the plasma membrane and avoid recognition through this mechanism.
Attachment to the surface of the cell triggers the virion internalization by macropinocytosis, a relatively nonspecific uptake mechanism involving actin dynamics and membrane ruffling38,48,49.
Within the cellular endosomal network, the virion requires the activity of the Rab GTPases Rab5 and Rab7 to traffic to a low pH compartment49. There, cellular cathepsin B/L proteases process
EboGP, removing part of the glycoprotein and exposing a receptor-binding domain to permit the glycoprotein to interact with the cellular cholesterol transporter Niemann-Pick C1 (NPC1) within the late endosome50–54. This binding event is necessary to trigger a conformational change within the glycoprotein, which results in the fusion of virion and cellular membranes and the release of the
RNP into the cytoplasm. Within the cytoplasm, the polymerase transcribes and replicates the genome, leading to production of new EBOV particles budding from the plasma membrane.
Figure 1.4. Schematic of EBOV entry into cells.
7 Section 1.3 – Ebola virus disease
When EBOV was first described in 1976, the disease it caused was termed Ebola hemorrhagic fever (EHF) due to the severe bleeding seen in some patients. Because only a fraction of individuals exhibit hemorrhagic symptoms, EHF has been rechristened Ebola virus disease
(EVD). The incubation period lasts 2-21 days4. Individuals can transmit the virus as soon as they are symptomatic, and the first phase of disease is marked by non-specific symptoms typical of viral infections (chills, fever, myalgia, etc)4,55. The second phase encompasses additional symptoms including lethargy, nausea, vomiting, abdominal pain, anorexia, diarrhea, coughing, headache, and hypotension4,55. Hemorrhage is observed in a minority of cases4,55. In severe cases, multi-organ failure affecting the cardiovascular, respiratory, and renal systems; hepatic injury; and hypotensive shock can contribute to death 6-16 days after onset of symptoms4,55. Viral RNA and antigen can be detected in the blood during the symptomatic phase of disease, and antibodies against the virus can be detected by serology during and after symptomatic disease56,57.
In non-human primate models, the first cells in which EBOV antigen can be detected are
DCs and other cells of the monocyte/macrophage lineage58. In a guinea pig model of infection, macrophages positive for the EBOV genome can be detected by in situ hybridization (ISH) throughout the disease course in multiple anatomical sites59. Trafficking of these cells to lymph nodes and through blood is hypothesized to be a mechanism for spread of EBOV throughout the body to other target cells and organs60. Understanding of human pathology is limited by the paucity of autopsies due to biosafety concerns61. Hepatocytes are a major target cell for EBOV infection, and hepatic necrosis is observed in fatal EBOV cases61. Other cell types found infected by EBOV in humans include epidermal DCs, endothelial cells, and connective tissue fibroblasts in the skin61.
The only cells characterized as resistant to EBOV infection are lymphocytes, though they are severely depleted during infection in a mechanism potentially involving TLR462–65. Severe cases of disease are marked by massive releases of pro-inflammatory cytokines and vasodilators, which likely contributes to pathology66. Recent retrospective analysis of existing guinea pig model tissues indicates that numerous cell types are infected that have not been previously appreciated, including
8 (among others) the endocardium, Schwann cells of the peripheral nervous system, smooth muscle of the vaginal wall, and the epithelium of the vagina and penis67. Infection at these previously overlooked sites may play critical roles in persistent infection (Section 1.4) and sexual transmission
(Section 1.5).
Some proportion of individuals exposed to EBOV develop an antibody response while experiencing no or only mild symptoms. While determination of this fraction is complicated by disparate serological profiles among African populations, recent estimates suggest that a small percentage (2-11%) of those serologically positive for EBOV did not show symptoms of disease68,69.
Individuals are apparently able to transmit virus to others even in the absence of any symptoms70.
Section 1.4 – Post-Ebola syndrome and persistence
A constellation of symptoms, referred to as post-Ebola syndrome, has been described among EVD survivors. These symptoms are variable and include headache, ocular pain and vision loss, impotence, hearing loss, arthralgia, myalgia, extreme fatigue, anorexia, and neurological complications. Among a small cohort of survivors, all reported at least one post-Ebola symptom, with headache being most common and reported by a majority of subjects71. As early as 1977, when a researcher accidentally infected himself with SUDV and fell ill with EVD, it was known that
Ebolaviruses could persist even after recovery from disease72. Even in the absence of symptoms
Figure 1.5. Summary of evidence of filovirus persistence in semen.
9 and after EBOV has been cleared from blood, EBOV can persist in immune privileged sites including the central nervous system, eye, mammary glands, and the reproductive system. EBOV
RNA has been detected in semen 2.5 years after disease (Figure 1.5)73, and persistence in vaginal fluids has been documented 33 days post-symptom onset74.
The issue of persistence came to the forefront during the 2014-2016 West Africa epidemic, during which it was appreciated that persistent virus could cause significant issues including uveitis, meningoencephalitis, and sexual transmission. Ocular problems, including uveitis, blindness, cataracts, and retinal scarring, can be debilitating and correlate with inflammation from persistent infection75. One man exhibited a change in eye color associated with persistent EBOV in his eye months after his recovery from acute disease76. A woman in the UK suffered from meningoencephalitis due to a resurgence of EBOV that was genetically identical to the virus she had survived months before, even though she had developed EBOV antibodies77. Perhaps most worrying, however, is the potential for transmission of persistent virus to reignite an outbreak in the absence of surveillance mechanisms. A case of suspected transmission from mother to child by breastfeeding was reported involving a mother who had not shown symptoms of EVD but harbored
EBOV RNA in her breastmilk70. Further, studies prior to, during, and after the 2014 outbreak have demonstrated that filoviruses could persist and be shed in semen for weeks to months following infection. For the first time, several cases of EBOV sexual transmission were reported during the
2014 outbreak, and cohort studies assessed the length of time that EBOV could be detected in semen (Figure 1.572–74,78–93). EBOV RNA has been detected in semen up to 965 days following infection, though the presence of EBOV RNA does not necessarily imply the presence of infectious virus74. Cultivation of EBOV from semen is complicated by semen’s cytotoxic character and contamination with bacteria and fungi90,94, but infectious virus has been identified from semen collected up to 233 days following EVD by injection into SCID mice90.
The cell types that sustain persistent EBOV infection have not been well defined in humans. Persistent infection likely occurs in tissues that were infected during acute disease but failed to clear the virus. Relevant to persistence and shedding of EBOV in semen, EBOV antigen
10 has been detected in the seminiferous tubules, testicular endothelium, and blood vessels of the male reproductive system in an EBOV case61. Infection of monkeys shows replication within the interstitial cells of the testis95. Monkeys that survived EBOV infection either naturally, after vaccination, or after treatment with investigational antivirals showed evidence of viral persistence96.
Mirroring reports of disease in humans, EBOV RNA and antigen was observed in these archived rhesus macaque brain, eye, and epididymis samples weeks after infection96. Immunofluorescence microscopy indicated that tissues infected with persistent EBOV had high levels of infiltration by
CD68+ cells, which are cells of the monocyte/macrophage lineage and known targets for EBOV infection96. Both EBOV genome and antigenome were detected by fluorescence in situ hybridization in a macaque epididymis sample and EboGP was detected both inside and outside of cells by immunofluorescence96. These data suggest ongoing replication and perhaps virion production at the time of death.
Section 1.5 – Ebola sexual transmission
In 1968, a doctor returned to Marburg, Germany after contracting an unknown hemorrhagic disease in Africa. Two months after he recovered, he transmitted the virus, then christened MARV, to his wife by sexual transmission and initiated a small outbreak93. His semen was found to be infectious in a guinea pig model93. Sexual transmission of EBOV had never been rigorously demonstrated before the 2014 Ebola outbreak, when several substantiated incidents of male-to- female EBOV sexual transmission of this persistent virus were reported. The first documented occurrence was in Liberia in 2015 when a man who survived EVD sexually transmitted EBOV to his wife 180 days following his illness79. While no infectious virus was isolated from his semen,
EBOV RNA was detected79. The wife’s virus was found to be highly similar to the husband’s virus, and phylogenetic analysis showed that the wife’s virus clustered not with the currently circulating
EBOV isolates but instead with sequences from the village when the husband was ill79. EBOV sexual transmission seems to be an uncommon event, as another woman with whom the husband had sexual relations apparently did not contract the virus79. The finding that EBOV could be sexually transmitted for months after recovery from the virus led the WHO to extend their guidance to EVD
11 survivors to use safer sex practices for one year after their disease. Genomic sequencing data have been used to identify transmission chains with evolution rates that are much lower than would be expected, a marker for transmission of persistent EBOV. This strategy has been used to identify previously overlooked clusters of EBOV sexual transmission97.
While most sexual transmission events have not been extensively documented and remain relatively uncommon, published cases demonstrate potential major public health concerns. A modeling study predicted that a three-month infectious period could extend the outbreak by 83 days, though many of the model criteria were estimated in the absence of reliable data98.
Importantly, once the virus has been transmitted from a persistently infected source, it can be spread through normal mechanisms of contact with bodily fluids. This raises the specter of sexual transmission reigniting an outbreak in an area declared free of EBOV transmission, a scenario that occurred in Guinea in early 2016. In December 2015, Guinea was declared Ebola-free, but several months later in March 2016, a cluster of seven cases was identified that further spread into
Liberia81. Independent genomic sequencing and epidemiological investigations converged on the conclusion that the outbreak was due to sexual transmission of persistent EBOV in a man who had
EVD 470 days prior81. Notably, this man had observed the WHO guidelines and abstained from unprotected sexual contact for one year after his disease, suggesting that the guidelines were insufficient to prevent reignition of EBOV transmission chains. The guidelines also suggest that male survivors should have their semen tested for the presence of EBOV until negative, and screening programs have been set up to provide counseling and monitor seminal persistence among survivors99.
Section 1.6 – Ebola virus disease treatment and prevention
Treatment for EVD is primarily supportive in nature and no approved therapies exist. Trials for some specific therapeutics were conducted during the West Africa outbreak, though issues of sample size arose as the outbreak waned in addition to ethical concerns100,101. The standard of care for EVD, hydration and electrolyte replacement therapies, counteracts the effects of vascular leak and diarrhea102. Because survivors of EVD develop neutralizing antibodies against EboGP,
12 plasma from surviving individuals (convalescent plasma) has been considered as an experimental treatment since the 1970s72. In an uncontrolled case series of 8 patients treated with convalescent plasma in a 1995 EBOV outbreak in the DRC, 7 survived; the CFR of 12.5% among treated patients was much lower than the overall outbreak CFR of 80%103. However, a recent trial of 99 patients showed no statistically significant difference in risk of death among EVD patients treated with convalescent plasma compared to controls, though there may have been a benefit among pediatric patients104. Notably, the plasma used for this trial was not tested for neutralizing capacity before infusion. Cocktails of monoclonal antibodies against EboGP have yielded promising results in preclinical trials in non-human primates and were used both under compassionate use settings and in trials during the outbreak. ZMapp, a cocktail of three human-mouse chimeric monoclonal antibodies, was administered to several patients outside of Africa; a majority survived101. A clinical trial demonstrated that treatment with ZMapp resulted in a 40% decrease in relative mortality among EVD patients, though this difference did not reach predetermined benchmarks to consider the treatment superior to standard of care105. A cocktail of three mouse monoclonal antibodies,
ZMAb, has been used in Europe to treat two patients, both of whom survived101. No further data is available on its efficacy in humans.
Numerous small molecule treatments have been developed to inhibit EBOV replication, but few have been tested in humans. Favipiravir, which is licensed in Japan to treat influenza, has been described to also have activity against other negative-strand viruses by targeting the viral RNA- dependent RNA polymerase106. Most EBOV patients treated in Europe received favipiravir. A non- randomized, historically controlled trial suggested that favipiravir may be efficacious in patients with high viral titers107. A clinical trial is ongoing to observe the effects of favipiravir in men whose semen remains positive for EBOV after recovery108. Another therapy, TKM-Ebola, is composed of multiple short interfering RNA molecules (siRNAs) targeting the EBOV genome. While phase I trials were halted due to cytokine abnormalities among participants, two patients in the US received the drug along with convalescent plasma and both recovered109. The efficacy of the treatment, however, is unknown. The last treatment used in humans during the 2014 outbreak is brincidofovir, a drug that
13 targets DNA virus DNA polymerases and shows activity against EBOV in vitro through an unclear mechanism110. Brincidofovir was used to treat one patient in the US and entered phase II trials during the outbreak, but the trials were halted due to declining case numbers101,111.
Finally, several vaccine candidates have arisen over the past few decades. The most advanced use viral vectors engineered to express EboGP. A phase III trial of a vaccine composed of vesicular stomatitis virus with its own glycoprotein replaced with EboGP showed 100% efficacy
(95% CI 74.7-100%) when contacts of infected individuals were vaccinated immediately after identification in a ring vaccination strategy112. However, numerous adverse events have been identified among those vaccinated113. Another vector vaccine, which uses a chimpanzee adenovirus to express EboGP followed by a boost with modified vaccinia Ankara expressing
EboGP, has not completed phase III trials but effectively produces both humoral and cellular responses in study participants114.
Given the high morbidity and mortality associated with EVD, the continued development of antiviral agents – both therapeutic and prophylactic – is of high priority.
14 Section 1.7 – Bibliography
1. Mylne, A. et al. A comprehensive database of the geographic spread of past human
Ebola outbreaks. Sci. Data 1, 140042 (2014).
2. Outbreaks Chronology: Ebola Virus Disease | Ebola Hemorrhagic Fever | CDC. (2017).
Available at: https://www.cdc.gov/vhf/ebola/outbreaks/history/chronology.html. (Accessed:
14th February 2018)
3. WHO | Ebola virus disease. WHO Available at:
http://www.who.int/mediacentre/factsheets/fs103/en/. (Accessed: 14th February 2018)
4. Feldmann, H. & Geisbert, T. W. Ebola haemorrhagic fever. Lancet Lond. Engl. 377, 849–
862 (2011).
5. Cantoni, D., Hamlet, A., Michaelis, M., Wass, M. N. & Rossman, J. S. Risks Posed by
Reston, the Forgotten Ebolavirus. mSphere 1, (2016).
6. Negredo, A. et al. Discovery of an ebolavirus-like filovirus in europe. PLoS Pathog. 7,
e1002304 (2011).
7. Baize, S. et al. Emergence of Zaire Ebola virus disease in Guinea. N. Engl. J. Med. 371,
1418–1425 (2014).
8. WHO | Ground zero in Guinea: the Ebola outbreak smoulders – undetected – for more
than 3 months. Available at: http://www.who.int/csr/disease/ebola/ebola-6-months/guinea/en/.
(Accessed: 21st February 2018)
9. Gire, S. K. et al. Genomic surveillance elucidates Ebola virus origin and transmission
during the 2014 outbreak. Science 345, 1369–1372 (2014).
10. Snyder, R. E., Marlow, M. A. & Riley, L. W. Ebola in urban slums: the elephant in the
room. Lancet Glob. Health 2, e685 (2014).
11. 2014 Ebola Outbreak in West Africa - Case Counts | Ebola Hemorrhagic Fever | CDC.
Available at: https://www.cdc.gov/vhf/ebola/outbreaks/2014-west-africa/case-counts.html.
(Accessed: 21st February 2018)
15 12. Kuhn, J. H. et al. Nomenclature- and database-compatible names for the two Ebola virus
variants that emerged in Guinea and the Democratic Republic of the Congo in 2014. Viruses
6, 4760–4799 (2014).
13. Franz, D. R. et al. Clinical recognition and management of patients exposed to biological
warfare agents. JAMA 278, 399–411 (1997).
14. Dietz, P. M., Jambai, A., Paweska, J. T., Yoti, Z. & Ksaizek, T. G. Epidemiology and Risk
Factors for Ebola Virus Disease in Sierra Leone—23 May 2014 to 31 January 2015. Clin.
Infect. Dis. 61, 1648–1654 (2015).
15. Pourrut, X. et al. Large serological survey showing cocirculation of Ebola and Marburg
viruses in Gabonese bat populations, and a high seroprevalence of both viruses in Rousettus
aegyptiacus. BMC Infect. Dis. 9, 159 (2009).
16. Olival, K. J. & Hayman, D. T. S. Filoviruses in bats: current knowledge and future
directions. Viruses 6, 1759–1788 (2014).
17. Pourrut, X. et al. Spatial and temporal patterns of Zaire ebolavirus antibody prevalence in
the possible reservoir bat species. J. Infect. Dis. 196 Suppl 2, S176-183 (2007).
18. Towner, J. S. et al. Isolation of Genetically Diverse Marburg Viruses from Egyptian Fruit
Bats. PLoS Pathog. 5, (2009).
19. Marí Saéz, A. et al. Investigating the zoonotic origin of the West African Ebola epidemic.
EMBO Mol. Med. 7, 17–23 (2015).
20. Swanepoel, R. et al. Experimental inoculation of plants and animals with Ebola virus.
Emerg. Infect. Dis. 2, 321–325 (1996).
21. Rouquet, P. et al. Wild Animal Mortality Monitoring and Human Ebola Outbreaks, Gabon
and Republic of Congo, 2001–2003. Emerg. Infect. Dis. 11, 283–290 (2005).
22. Walsh, P. D. et al. Catastrophic ape decline in western equatorial Africa. Nature 422,
611–614 (2003).
23. Bermejo, M. et al. Ebola outbreak killed 5000 gorillas. Science 314, 1564 (2006).
16 24. Barrette, R. W. et al. Discovery of swine as a host for the Reston ebolavirus. Science
325, 204–206 (2009).
25. Allela, L. et al. Ebola virus antibody prevalence in dogs and human risk. Emerg. Infect.
Dis. 11, 385–390 (2005).
26. Han, Z. et al. Ebola virus mediated infectivity is restricted in canine and feline cells. Vet.
Microbiol. 182, 102–107 (2016).
27. Olabode, A. S., Jiang, X., Robertson, D. L. & Lovell, S. C. Ebolavirus is evolving but not
changing: No evidence for functional change in EBOV from 1976 to the 2014 outbreak.
Virology 482, 202–207 (2015).
28. Diehl, W. E. et al. Ebola Virus Glycoprotein with Increased Infectivity Dominated the
2013–2016 Epidemic. Cell 167, 1088–1098.e6 (2016).
29. Hoffmann, M. et al. A Polymorphism within the Internal Fusion Loop of the Ebola Virus
Glycoprotein Modulates Host Cell Entry. J Virol 91, e00177-17 (2017).
30. Wang, M., Lim, S.-Y., Lee, S. & Cunningham, J. Biochemical Basis for Increased Activity
of Ebola Glycoprotein in the 2013–16 Epidemic. Cell Host Microbe 21, 367–375 (2017).
31. Feldmann, H., Sanchez, A. & Geisbert, T. W. Filoviridae: Marburg and Ebola viruses. in
Fields Virology (eds. Knipe, D. M. & Howley, P. M.) 1, 923–956 (Wolters Kluwer | Lippincott
WIlliams & Wilkins, 2013).
32. Feldmann, H., Volchkov, V. E., Volchkova, V. A. & Klenk, H. D. The glycoproteins of
Marburg and Ebola virus and their potential roles in pathogenesis. Arch. Virol. Suppl. 15, 159–
169 (1999).
33. Mehedi, M. et al. A new Ebola virus nonstructural glycoprotein expressed through RNA
editing. J. Virol. 85, 5406–5414 (2011).
34. Audet, J. & Kobinger, G. P. Immune evasion in ebolavirus infections. Viral Immunol. 28,
10–18 (2015).
35. Mohan, G. S., Li, W., Ye, L., Compans, R. W. & Yang, C. Antigenic subversion: a novel
mechanism of host immune evasion by Ebola virus. PLoS Pathog. 8, e1003065 (2012).
17 36. Noda, T. et al. Ebola virus VP40 drives the formation of virus-like filamentous particles
along with GP. J. Virol. 76, 4855–4865 (2002).
37. Kallstrom, G. et al. Analysis of Ebola virus and VLP release using an immunocapture
assay. J. Virol. Methods 127, 1–9 (2005).
38. Nanbo, A. et al. Ebolavirus is internalized into host cells via macropinocytosis in a viral
glycoprotein-dependent manner. PLoS Pathog. 6, e1001121 (2010).
39. Davey, R. A. et al. Mechanisms of Filovirus Entry. Curr. Top. Microbiol. Immunol. 411,
323–352 (2017).
40. Hunt, C. L., Lennemann, N. J. & Maury, W. Filovirus entry: a novelty in the viral fusion
world. Viruses 4, 258–275 (2012).
41. Simmons, G. et al. DC-SIGN and DC-SIGNR bind ebola glycoproteins and enhance
infection of macrophages and endothelial cells. Virology 305, 115–23 (2003).
42. Brindley, M. A. et al. Tyrosine kinase receptor Axl enhances entry of Zaire ebolavirus
without direct interactions with the viral glycoprotein. Virology 415, 83–94 (2011).
43. Hunt, C. L., Kolokoltsov, A. A., Davey, R. A. & Maury, W. The Tyro3 receptor kinase Axl
enhances macropinocytosis of Zaire ebolavirus. J. Virol. 85, 334–347 (2011).
44. Moller-Tank, S., Kondratowicz, A. S., Davey, R. A., Rennert, P. D. & Maury, W. Role of
the phosphatidylserine receptor TIM-1 in enveloped-virus entry. J. Virol. 87, 8327–8341
(2013).
45. Jemielity, S. et al. TIM-family proteins promote infection of multiple enveloped viruses
through virion-associated phosphatidylserine. PLoS Pathog. 9, e1003232 (2013).
46. Shimojima, M. et al. Tyro3 family-mediated cell entry of Ebola and Marburg viruses. J.
Virol. 80, 10109–10116 (2006).
47. Amara, A. & Mercer, J. Viral apoptotic mimicry. Nat. Rev. Microbiol. 13, 461–469 (2015).
48. Mulherkar, N., Raaben, M., Torre, J., Whelan, S. & Chandran, K. The Ebola virus
glycoprotein mediates entry via a non-classical dynamin-dependent macropinocytic pathway.
Virology 419, 72–83 (2011).
18 49. Saeed, M., Kolokoltsov, A., Albrecht, T. & Davey, R. Cellular entry of ebola virus involves
uptake by a macropinocytosis-like mechanism and subsequent trafficking through early and
late endosomes. PLoS Pathog 6, e1001110 (2010).
50. Chandran, K., Sullivan, N. J., Felbor, U., Whelan, S. P. & Cunningham, J. M. Endosomal
proteolysis of the Ebola virus glycoprotein is necessary for infection. Science 308, 1643–1645
(2005).
51. Carette, J. E. et al. Ebola virus entry requires the cholesterol transporter Niemann-Pick
C1. Nature 477, 340–343 (2011).
52. Côté, M. et al. Small molecule inhibitors reveal Niemann-Pick C1 is essential for Ebola
virus infection. Nature 477, 344–8 (2011).
53. Haines, K., Burgt, N., Francica, J., Kaletsky, R. & Bates, P. Chinese hamster ovary cell
lines selected for resistance to ebolavirus glycoprotein mediated infection are defective for
NPC1 expression. Virology 432, 20–8 (2012).
54. Spence, J. S., Krause, T. B., Mittler, E., Jangra, R. K. & Chandran, K. Direct Visualization
of Ebola Virus Fusion Triggering in the Endocytic Pathway. mBio 7, e01857-01815 (2016).
55. Haaskjold, Y. L. et al. Clinical Features of and Risk Factors for Fatal Ebola Virus Disease,
Moyamba District, Sierra Leone, December 2014-February 2015. Emerg. Infect. Dis. 22,
1537–1544 (2016).
56. Towner, J. S. et al. Rapid diagnosis of Ebola hemorrhagic fever by reverse transcription-
PCR in an outbreak setting and assessment of patient viral load as a predictor of outcome. J.
Virol. 78, 4330–4341 (2004).
57. Gupta, M., MacNeil, A., Reed, Z. D., Rollin, P. E. & Spiropoulou, C. F. Serology and
cytokine profiles in patients infected with the newly discovered Bundibugyo ebolavirus.
Virology 423, 119–124 (2012).
58. Geisbert, T. W. et al. Pathogenesis of Ebola hemorrhagic fever in cynomolgus
macaques: evidence that dendritic cells are early and sustained targets of infection. Am J
Pathol 163, 2347–70 (2003).
19 59. Connolly, B. M. et al. Pathogenesis of experimental Ebola virus infection in guinea pigs.
J. Infect. Dis. 179 Suppl 1, S203-217 (1999).
60. Schnittler, H.-J. & Feldmann, H. Marburg and Ebola hemorrhagic fevers: does the
primary course of infection depend on the accessibility of organ-specific macrophages? Clin.
Infect. Dis. 27, 404–406 (1998).
61. Martines, R., Ng, D., Greer, P., Rollin, P. & Zaki, S. Tissue and cellular tropism,
pathology and pathogenesis of Ebola and Marburg viruses. J Pathol. 235, 153–174 (2015).
62. Iampietro, M. et al. Ebola virus glycoprotein directly triggers T lymphocyte death despite
of the lack of infection. PLoS Pathog 13, e1006397 (2017).
63. Wool-Lewis, R. J. & Bates, P. Characterization of Ebola virus entry by using pseudotyped
viruses: identification of receptor-deficient cell lines. J. Virol. 72, 3155–3160 (1998).
64. Reed, D. S., Hensley, L. E., Geisbert, J. B., Jahrling, P. B. & Geisbert, T. W. Depletion of
peripheral blood T lymphocytes and NK cells during the course of ebola hemorrhagic Fever in
cynomolgus macaques. Viral Immunol. 17, 390–400 (2004).
65. Geisbert, T. et al. Apoptosis induced in vitro and in vivo during infection by Ebola and
Marburg viruses. Lab Invest 80, 171–86 (2000).
66. Wauquier, N., Becquart, P., Padilla, C., Baize, S. & Leroy, E. M. Human fatal zaire ebola
virus infection is associated with an aberrant innate immunity and with massive lymphocyte
apoptosis. PLoS Negl. Trop. Dis. 4, (2010).
67. Cooper, T. K. et al. Histology, immunohistochemistry, and in situ hybridization reveal
overlooked Ebola virus target tissues in the Ebola virus disease guinea pig model. Sci. Rep. 8,
1250 (2018).
68. Mulangu, S. et al. Serologic Evidence of Ebolavirus Infection in a Population With No
History of Outbreaks in the Democratic Republic of the Congo. J. Infect. Dis. 217, 529–537
(2018).
20 69. Glynn, J. R. et al. Asymptomatic infection and unrecognised Ebola virus disease in
Ebola-affected households in Sierra Leone: a cross-sectional study using a new non-invasive
assay for antibodies to Ebola virus. Lancet Infect. Dis. 17, 645–653 (2017).
70. Sissoko, D. et al. Ebola Virus Persistence in Breast Milk After No Reported Illness: A
Likely Source of Virus Transmission From Mother to Child. Clin. Infect. Dis. Off. Publ. Infect.
Dis. Soc. Am. 64, 513–516 (2017).
71. Scott, J. T. et al. Post-Ebola Syndrome, Sierra Leone. Emerg. Infect. Dis. 22, 641–646
(2016).
72. Emond, R. T., Evans, B., Bowen, E. T. & Lloyd, G. A case of Ebola virus infection. Br
Med J 2, 541–544 (1977).
73. Fischer, W. et al. Ebola Virus RNA Detection in Semen More than Two Years After
Resolution of Acute Ebola Virus Infection. Open Forum Infect Dis ofx155- (0).
doi:10.1093/ofid/ofx155
74. Rodriguez, L. L. et al. Persistence and genetic stability of Ebola virus during the outbreak
in Kikwit, Democratic Republic of the Congo, 1995. J. Infect. Dis. 179 Suppl 1, S170-176
(1999).
75. Shantha, J. G., Crozier, I. & Yeh, S. An update on ocular complications of Ebola virus
disease. Curr. Opin. Ophthalmol. 28, 600–606 (2017).
76. Shantha, J. G. et al. Long-term Management of Panuveitis and Iris Heterochromia in an
Ebola Survivor. Ophthalmology 123, 2626–2628.e2 (2016).
77. Jacobs, M. et al. Late Ebola virus relapse causing meningoencephalitis: a case report.
Lancet Lond. Engl. 388, 498–503 (2016).
78. Rowe, A. K. et al. Clinical, Virologic, and Immunologic Follow‐Up of Convalescent Ebola
Hemorrhagic Fever Patients and Their Household Contacts, Kikwit, Democratic Republic of
the Congo. J Infect Dis 179, S28–S35 (1999).
79. Mate, S. E. et al. Molecular Evidence of Sexual Transmission of Ebola Virus. N. Engl. J.
Med. 373, 2448–2454 (2015).
21 80. Arias, A. et al. Rapid outbreak sequencing of Ebola virus in Sierra Leone identifies
transmission chains linked to sporadic cases. Virus Evol 2, vew016 (2016).
81. Diallo, B. et al. Resurgence of Ebola Virus Disease in Guinea Linked to a Survivor With
Virus Persistence in Seminal Fluid for More Than 500 Days. Clin Infect Dis 63, 1353–1356
(2016).
82. Green, E. et al. Viraemia and Ebola virus secretion in survivors of Ebola virus disease in
Sierra Leone: a cross-sectional cohort study. Lancet Infect. Dis. 16, 1052–1056 (2016).
83. Soka, M. J. et al. Prevention of sexual transmission of Ebola in Liberia through a national
semen testing and counselling programme for survivors: an analysis of Ebola virus RNA
results and behavioural data. Lancet Glob. Health 4, e736-743 (2016).
84. Sow, M. S. et al. New Evidence of Long-lasting Persistence of Ebola Virus Genetic
Material in Semen of Survivors. J. Infect. Dis. 214, 1475–1476 (2016).
85. Uyeki, T. et al. Ebola Virus Persistence in Semen of Male Survivors. Clin Infect Dis 62,
1552–1555 (2016).
86. Barnes, K. G. et al. Evidence of Ebola Virus Replication and High Concentration in
Semen of a Patient During Recovery. Clin Infect Dis 65, 1400–1403 (2017).
87. Deen, G. et al. Ebola RNA Persistence in Semen of Ebola Virus Disease Survivors —
Preliminary Report. New Engl J Med. 151014140118009 (2015).
doi:10.1056/NEJMoa1511410
88. Keita, M. et al. Unusual Ebola Virus Chain of Transmission, Conakry, Guinea, 2014–
2015. Emerg Infect Dis 22, (2016).
89. Purpura, L. J. et al. Ebola virus RNA in semen from an HIV-positive survivor of Ebola.
Emerg. Infect. Dis. 23, 714 (2017).
90. Sissoko, D. et al. Persistence and clearance of Ebola virus RNA from seminal fluid of
Ebola virus disease survivors: a longitudinal analysis and modelling study. Lancet Glob. Heal
5, e80–e88 (2017).
22 91. Subtil, F. et al. Dynamics of Ebola RNA Persistence in Semen: A Report From the
Postebogui Cohort in Guinea. Clin. Infect. Dis. 64, 1788–1790 (2017).
92. Bausch, D. G. et al. Assessment of the risk of Ebola virus transmission from bodily fluids
and fomites. J. Infect. Dis. 196, S142–S147 (2007).
93. Martini, G. & Schmidt, H. [Spermatogenic transmission of the ‘Marburg virus’. (Causes of
‘Marburg simian disease’)]. Klin Wochenschr 46, 398–400 (1968).
94. Kiessling, A. A. Isolation of human immunodeficiency virus type 1 from semen and
vaginal fluids. Methods Mol. Biol. Clifton NJ 304, 71–86 (2005).
95. Baskerville, A., Fisher-Hoch, S. P., Neild, G. H. & Dowsett, A. B. Ultrastructural pathology
of experimental Ebola haemorrhagic fever virus infection. J. Pathol. 147, 199–209 (1985).
96. Zeng, X. et al. Identification and pathological characterization of persistent asymptomatic
Ebola virus infection in rhesus monkeys. Nat Microbiol 2, nmicrobiol2017113 (2017).
97. Blackley, D. et al. Reduced evolutionary rate in reemerged Ebola virus transmission
chains. Sci Adv 2, e1600378–e1600378 (2016).
98. Abbate, J., Murall, C., Richner, H. & Althaus, C. Potential Impact of Sexual Transmission
on Ebola Virus Epidemiology: Sierra Leone as a Case Study. PLoS Negl. Trop D 10,
e0004676 (2016).
99. Bausch, D. & Crozier, I. The Liberia Men’s Health Screening Program for Ebola virus:
win-win-win for survivor, scientist, and public health. Lancet Glob. Heal 4, e672–e673 (2016).
100. Adebamowo, C. et al. Randomised controlled trials for Ebola: practical and ethical issues.
Lancet Lond. Engl. 384, 1423–1424 (2014).
101. Trad, M. A. et al. Ebola virus disease: An update on current prevention and management
strategies. J. Clin. Virol. Off. Publ. Pan Am. Soc. Clin. Virol. 86, 5–13 (2017).
102. Lyon, G. M. et al. Clinical care of two patients with Ebola virus disease in the United
States. N. Engl. J. Med. 371, 2402–2409 (2014).
23 103. Mupapa, K. et al. Treatment of Ebola hemorrhagic fever with blood transfusions from
convalescent patients. International Scientific and Technical Committee. J. Infect. Dis. 179
Suppl 1, S18-23 (1999).
104. van Griensven, J. et al. Evaluation of Convalescent Plasma for Ebola Virus Disease in
Guinea. N. Engl. J. Med. 374, 33–42 (2016).
105. PREVAIL II Writing Group et al. A Randomized, Controlled Trial of ZMapp for Ebola Virus
Infection. N. Engl. J. Med. 375, 1448–1456 (2016).
106. Oestereich, L. et al. Successful treatment of advanced Ebola virus infection with T-705
(favipiravir) in a small animal model. Antiviral Res. 105, 17–21 (2014).
107. Sissoko, D. et al. Experimental Treatment with Favipiravir for Ebola Virus Disease (the
JIKI Trial): A Historically Controlled, Single-Arm Proof-of-Concept Trial in Guinea. PLoS Med.
13, e1001967 (2016).
108. Tolerance and Activity Evaluation of High Doses of Favipiravir Against Ebola Virus in the
Semen - Full Text View - ClinicalTrials.gov. Available at:
https://clinicaltrials.gov/ct2/show/NCT02739477. (Accessed: 22nd February 2018)
109. Kraft, C. S. et al. The Use of TKM-100802 and Convalescent Plasma in 2 Patients With
Ebola Virus Disease in the United States. Clin. Infect. Dis. Off. Publ. Infect. Dis. Soc. Am. 61,
496–502 (2015).
110. Florescu, D. F. & Keck, M. A. Development of CMX001 (Brincidofovir) for the treatment of
serious diseases or conditions caused by dsDNA viruses. Expert Rev. Anti Infect. Ther. 12,
1171–1178 (2014).
111. Florescu, D. F. et al. Administration of Brincidofovir and Convalescent Plasma in a
Patient With Ebola Virus Disease. Clin. Infect. Dis. Off. Publ. Infect. Dis. Soc. Am. 61, 969–973
(2015).
112. Henao-Restrepo, A. M. et al. Efficacy and effectiveness of an rVSV-vectored vaccine
expressing Ebola surface glycoprotein: interim results from the Guinea ring vaccination
cluster-randomised trial. Lancet Lond. Engl. 386, 857–866 (2015).
24 113. Agnandji, S. T. et al. Phase 1 Trials of rVSV Ebola Vaccine in Africa and Europe. N. Engl.
J. Med. 374, 1647–1660 (2016).
114. Ewer, K. et al. A Monovalent Chimpanzee Adenovirus Ebola Vaccine Boosted with MVA.
N. Engl. J. Med. 374, 1635–1646 (2016).
25 CHAPTER 2: SEMINAL AMYLOID FIBRILS
Amyloid fibrils are traditionally considered pathologic in nature, being associated with degenerative diseases including Alzheimer’s disease, prion diseases, and amyloidosis. In these cases, proteins adopt a fold that renders them remarkably stable and insoluble, leading to toxic protein aggregation. It has recently been discovered that amyloid fibrils are present in healthy individuals and may play an important physiological role. However, like many existing host factors, viruses may hijack them to facilitate their own transmission and replication.
Section 2.1 – Introduction to seminal amyloids
The role of semen in sexual transmission of viruses has long been unclear. Semen had traditionally been thought to act only as a passive vehicle for the virus, but studies have suggested that it may play direct roles in enhancing human immunodeficiency virus type 1 (HIV-1) infection by pH neutralization1,2, opsonization3, and recruitment of target cells to the female genital tract4,5.
To identify additional pro-viral mechanisms, a library of peptides and small proteins derived from human semen was screened for factors that modulate HIV-1 infection6. This screen led to the identification of fractions that increased HIV-1 infection, and mass spectrometry identified those fractions as containing a peptide derived from prostatic acid phosphatase (PAP)6. PAP is a major proteinaceous component of semen and is present at 1-2 mg/mL; a 39-amino acid peptide,
PAP248-286, was recovered at a concentration of 35 μg/mL in semen6. Unexpectedly, when this peptide was chemically synthesized, it had no effect on viral infection. It was not until the solutions became turbid after short-term agitation or incubation that a pronounced enhancement in viral infectivity was noted6. Interestingly, when the turbid solution was analyzed, it was found that the
PAP248-286 peptides had oligomerized into amyloid fibrils that were termed semen-derived enhancer of viral infection (SEVI)6. Amyloid formation was confirmed by electron microscopy and increases in fluorescence upon incubation with the amyloid-binding dyes thioflavin T and Congo red6.
SEVI was found to dramatically enhance the ability of HIV-1 to infect reporter cell lines and primary cells6. The enhancement effect was found to be greater at low virus concentrations, a
26 conclusion supported by viral titration experiments indicating a 105-fold enhancement in viral titer6.
Because HIV-1 infectivity is limited by poor attachment to host cells, likely contributing to the finding that less than 0.1% of viral particles are infectious in vitro7,8, this level of enhancement may be very significant in terms of disease transmission. The presence of SEVI fibrils was found to reduce the number of virions required to establish a productive infection in cell culture by several orders of magnitude, from 103-105 down to 1-3 virions6.
Subsequent studies have found that SEVI is not the only amyloid present naturally within semen. Another fragment of PAP, PAP85-120, is present at a similar concentration in semen and was also found to enhance infection by HIV-19. Amyloidogenic fragments of the seminal proteins semenogelin-1 and -2 (SEM1 and SEM2)10, which are important components of the seminal coagulum, have also been found to enhance HIV-1 infection in vitro. The semenogelin proteins are cleaved by prostate specific antigen (PSA)10; the protease responsible for liberation of PAP85-120 and PAP248-286 fragments is unknown but may originate in the seminal vesicles10.
Section 2.2 – Mechanism of enhancement by seminal amyloids
All seminal amyloids described to date are highly basic. The PAP248-286 peptide which forms SEVI has a theoretical isoelectric point of 10.21 owing to its high proportion of positively charged residues (8 of 39 are lysine or arginine)6. This positive charge is essential for viral enhancement. Replacement of all positive residues on PAP248-286 with alanine does not impact its ability to form fibrils but abrogates its ability to enhance HIV-1 infection11. Further, treatment of the fibrils with anionic polymers such as dextran sulfate or molecular tweezers that mask the positively charged residues abrogates enhancement ability11,12. Combining these results with the observations that freshly dissolved peptide does not enhance viral infection, both positive charge and amyloid character are necessary for enhancement but neither is sufficient alone.
Live-imaging microscopy has revealed that SEVI fibrils are captured by cellular protrusions at the plasma membrane6. It is hypothesized that the positively charged PAP248-286 fibrils are liberated from PAP by proteolytic cleavage and assemble into SEVI fibrils (Figure 2.1). These fibrils bind virion membranes through interactions with negative charges on the virion membrane. The
27 Figure 2.1. Model of enhancement of HIV-1 infection by SEVI. 6.
SEVI fibrils then interact with target cells in a similar way, bringing the virion into close proximity with the cell membrane. In this way, attachment to the cell is enhanced, leading to increases in downstream entry processes including viral fusion6. The interaction between the seminal amyloids and virion membranes appears to be glycoprotein-independent to some degree. Similar fold- increases in cellular attachment have been observed with VLPs formed by the HIV-1 glycoprotein and matrix protein p24 and particles formed with p24 alone without a glycoprotein6. Seminal amyloids have been further characterized to enhance infection by HIV-1, HIV-2, simian immunodeficiency virus (SIV), cytomegalovirus (CMV), herpes simplex virus (HSV), and pseudoviruses bearing various glycoproteins on an HIV-1 core13–17. Enhancement may occur similarly for all enveloped viruses with similar membrane composition. Electron micrographs demonstrate internalization of SEVI fibrils into the cell in the absence of virus, suggesting that the fibrils may induce their own internalization6. However, inhibitors of phagocytosis were not sufficient to block the enhancement effect observed for HIV-1, which may fuse at the cell surface6.
28 Section 2.3 – Seminal amyloids and function
The physiological role of seminal amyloids has not been conclusively demonstrated. Sperm cells fluoresce when stained with Congo red, an amyloid-specific dye, suggesting that they may be coated in seminal amyloids18. SEM-1 and SEM-2 have been observed to bind to spermatozoa by immunofluorescence, though it is unclear whether the fraction of these proteins binding is formed into amyloid fibrils19. These proteins are under strong positive selection, suggesting these proteins may have an important role in evolutionary fitness20,21. Indeed, given the ability of these fibrils to enhance viral infection, one may posit that they may be evolutionarily selected against unless serving another function. Indeed, phylogenetic analysis suggests that these fibrils are conserved among primates, and the analogous peptide to SEVI in macaques enhances viral infection22,23. It has been hypothesized that seminal amyloids enhance fertilization through a similar mechanism by which they enhance viral infection. In analogous cases, a much smaller membrane-bound particle (sperm cell or enveloped virus) must overcome charge repulsion between its membrane and its target (egg cell or susceptible host cell). Both scenarios are often dependent upon lipid rafts and involve the action of glycoproteins to fuse membranes24. Interestingly, many compounds developed to inhibit fertilization will also inhibit viral infection to some degree, and vice versa24.
Recent experiments, however, have demonstrated that seminal amyloids (SEVI and
SEM1) actually inhibit fertilization of mouse gametes25. Subsequently, it was found that binding of amyloid fibrils restricted movement by spermatozoa25. In addition, treatment of spermatozoa with seminal amyloid fibrils was found to increase their internalization by macrophages, especially if the spermatozoa had been damaged by freeze-thaw cycles25. This led to a revised hypothesis that seminal amyloids may bind damaged or less fit spermatozoa and promote their clearance by phagocytic cells resident in the female genital tract. Interestingly, these same cell types are targeted for infection by several sexually transmitted viruses including EBOV, and the hypothesis that seminal amyloids may enhance their infection by a similar mechanism is untested.
29 Section 2.4 – Targeting amyloid fibrils
Heterosexual sexual transmission of HIV-1 and some other sexually transmitted viruses appears to be a relatively rare event. While HIV-1 transmission is highly dependent on viral load, studies suggest that transmission probabilities range from 0.01% to 0.23% per act26. Because these rates are very low, especially in the context of the huge burden of HIV-1 disease, even modest decreases in these rates may have large impacts on prevention of HIV-1 transmission. Because seminal amyloids are able to greatly enhance viral infection in vitro, the development of compounds that reduce this enhancement effect has been explored. Many of these strategies interfere with amyloid formation, stability, interaction with viral particles, or a combination of these27.
Many compounds have been explored to prevent the interaction of SEVI with viral particles, often by shielding the highly positive charge of the amyloid. Heparan sulfate and other polyanions have been shown to disrupt the interaction between SEVI and HIV-1 particles in addition to direct activity against HIV-111,28,29. Unfortunately, these molecules had detrimental effects in clinical settings due to induction of inflammatory responses that resulted in an increase in HIV acquisition30.
A similar strategy is to treat cells with molecules that antagonize the interaction between SEVI and the polyanions on the cell surface such as heparan sulfate proteoglycan. Surfen, such an antagonist, inhibits both interactions between the amyloid and cellular membrane as well as between the amyloid and viral membrane31. Another strategy has involved use of molecular tweezers that specifically bind arginine and lysine residues, shielding their positive charge from the virion. These molecules inhibit enhanced HIV-1 infection both through this mechanism and through direct antiviral effects on HIV-1 and other viral membranes12. This strategy is particularly attractive as it acts on both the virion and a host factor, making it unlikely that the virus will be able to evolve resistance to this mechanism.
Inhibition of the formation of precursor peptides into amyloid (e.g. PAP248-286 into SEVI fibrils) is a strategy that takes advantage of the observation that soluble peptides have no impact on viral infection. One such strategy is to design non-natural peptides that are able to interact with the amyloidogenic peptide but sterically block amyloid assembly. A computationally designed
30 peptide, WW61, was found to be able to prevent PAP248-286 from assembling into SEVI fibrils32.
However, because fibrils are present in their amyloid state in semen33, such a strategy may not be viable to prevent enhancement of viral infection in vivo. Epigallocatechin-3-gallate (EGCG), an antioxidant isolated from green tea, has been shown to disassemble SEVI fibrils slowly (over the span of 1-2 days) and prevent formation of PAP248-286 peptides into fibrils34. Again, it is unclear whether this strategy would be viable in vivo over such a timescale.
A final strategy is to remodel amyloid fibrils, either by disassembling the amyloid structure or by altering the conformation to one that does not enhance viral infection. In addition to EGCG described above, work has been done exploring the ability of disaggregase proteins to act upon amyloid substrates (Box 235). Hsp104, a yeast AAA+ protein, is able to both resolve and refold disordered protein aggregates and remodel amyloid fibrils36. It was found that Hsp104 is able to remodel SEVI, SEM1, and PAP85-120 amyloid fibrils in an ATP-dependent fashion to prevent enhancement of infection35. Interestingly, a mutant Hsp104 that was unable to hydrolyze ATP was still able to remodel the seminal amyloids into clusters that did not enhance HIV-1 infection35. This is particularly important because ATP is present at very low levels in the extracellular space. Thus, disaggregase proteins may represent a useful strategy for reducing the infection enhancement ability of amyloid fibrils.
31 Section 2.5 – Bibliography
1. Bouvet, J.-P., Grésenguet, G. & Bélec, L. Vaginal pH neutralization by semen as a
cofactor of HIV transmission. Clin. Microbiol. Infect. Off. Publ. Eur. Soc. Clin. Microbiol. Infect.
Dis. 3, 19–23 (1997).
2. Tevi-Bénissan, C. et al. In vivo semen-associated pH neutralization of cervicovaginal
secretions. Clin. Diagn. Lab. Immunol. 4, 367–374 (1997).
3. Bouhlal, H. et al. Opsonization of HIV-1 by semen complement enhances infection of
human epithelial cells. J. Immunol. Baltim. Md 1950 169, 3301–3306 (2002).
4. Pandya, I. J. & Cohen, J. The leukocytic reaction of the human uterine cervix to
spermatozoa. Fertil. Steril. 43, 417–421 (1985).
5. Thompson, L. A., Barratt, C. L., Bolton, A. E. & Cooke, I. D. The leukocytic reaction of the
human uterine cervix. Am. J. Reprod. Immunol. N. Y. N 1989 28, 85–89 (1992).
6. Münch, J. et al. Semen-derived amyloid fibrils drastically enhance HIV infection. 131,
1059–71 (2007).
7. Rusert, P. et al. Quantification of infectious HIV-1 plasma viral load using a boosted in
vitro infection protocol. Virology 326, 113–129 (2004).
8. Dimitrov, D. S. et al. Quantitation of human immunodeficiency virus type 1 infection
kinetics. J. Virol. 67, 2182–2190 (1993).
9. Arnold, F. et al. Naturally Occurring Fragments from Two Distinct Regions of the Prostatic
Acid Phosphatase Form Amyloidogenic Enhancers of HIV Infection. J Virol 86, 1244–1249
(2012).
10. Roan, N. R. et al. Peptides Released by Physiological Cleavage of Semen Coagulum
Proteins Form Amyloids that Enhance HIV Infection. Cell Host Microbe 10, 541–550 (2011).
11. Roan, N. R. et al. The cationic properties of SEVI underlie its ability to enhance human
immunodeficiency virus infection. J. Virol. 83, 73–80 (2009).
12. Lump, E. et al. A molecular tweezer antagonizes seminal amyloids and HIV infection.
eLife 4, (2015).
32 13. Kim, K.-A. et al. Semen-mediated enhancement of HIV infection is donor-dependent and
correlates with the levels of SEVI. Retrovirology 7, 1–12 (2010).
14. Münch, J. et al. Effect of semen and seminal amyloid on vaginal transmission of simian
immunodeficiency virus. Retrovirology 10, 148 (2013).
15. Tang, Q., Roan, N. R. & Yamamura, Y. Seminal plasma and semen amyloids enhance
cytomegalovirus infection in cell culture. J. Virol. 87, 12583–12591 (2013).
16. Torres, L., Ortiz, T. & Tang, Q. Enhancement of herpes simplex virus (HSV) infection by
seminal plasma and semen amyloids implicates a new target for the prevention of HSV
infection. Viruses 7, 2057–2073 (2015).
17. Wurm, M. et al. The influence of semen‐derived enhancer of virus infection on the
efficiency of retroviral gene transfer. J Gene Med. 12, 137–146 (2010).
18. Entwistle, K. W. Congo red--fast green FCF as a supra-vital stain for ram and bull
spermatozoa. Aust. Vet. J. 48, 515–519 (1972).
19. Bjartell, A. et al. Distribution and tissue expression of semenogelin I and II in man as
demonstrated by in situ hybridization and immunocytochemistry. J. Androl. 17, 17–26 (1996).
20. Ferreira, Z. et al. Reproduction and immunity-driven natural selection in the human
WFDC locus. Mol. Biol. Evol. 30, 938–950 (2013).
21. Hurle, B., Swanson, W., NISC Comparative Sequencing Program & Green, E. D.
Comparative sequence analyses reveal rapid and divergent evolutionary changes of the
WFDC locus in the primate lineage. Genome Res. 17, 276–286 (2007).
22. Zhou, R.-H. et al. Epigallocatechin Gallate Inhibits Macaque SEVI-Mediated
Enhancement of SIV or SHIV Infection. J. Acquir. Immune Defic. Syndr. 1999 75, 232–240
(2017).
23. Roan, N. R. et al. Liquefaction of semen generates and later degrades a conserved
semenogelin peptide that enhances HIV infection. J. Virol. 88, 7221–7234 (2014).
24. Doncel, G. F. Exploiting common targets in human fertilization and HIV infection:
development of novel contraceptive microbicides. Hum. Reprod. Update 12, 103–117 (2006).
33 25. Roan, N. et al. Semen amyloids participate in spermatozoa selection and clearance. eLife
6, e24888 (2017).
26. Gray, R. H. et al. Probability of HIV-1 transmission per coital act in monogamous,
heterosexual, HIV-1-discordant couples in Rakai, Uganda. Lancet Lond. Engl. 357, 1149–1153
(2001).
27. Castellano, L. M. & Shorter, J. The Surprising Role of Amyloid Fibrils in HIV Infection.
Biology 1, 58–80 (2012).
28. Rider, C. C. The potential for heparin and its derivatives in the therapy and prevention of
HIV-1 infection. Glycoconj. J. 14, 639–642 (1997).
29. Witvrouw, M. & De Clercq, E. Sulfated polysaccharides extracted from sea algae as
potential antiviral drugs. Gen. Pharmacol. 29, 497–511 (1997).
30. van de Wijgert, J. H. & Shattock, R. J. Vaginal microbicides: moving ahead after an
unexpected setback. AIDS Lond. Engl. 21, 2369–2376 (2007).
31. Roan, N. R., Sowinski, S., Münch, J., Kirchhoff, F. & Greene, W. C. Aminoquinoline
surfen inhibits the action of SEVI (semen-derived enhancer of viral infection). J. Biol. Chem.
285, 1861–1869 (2010).
32. Sievers, S. A. et al. Structure-based design of non-natural amino-acid inhibitors of
amyloid fibril formation. Nature 475, 96–100 (2011).
33. Usmani, S. et al. Direct visualization of HIV-enhancing endogenous amyloid fibrils in
human semen. Nat Commun 5, 3508 (2014).
34. Hauber, I., Hohenberg, H., Holstermann, B., Hunstein, W. & Hauber, J. The main green
tea polyphenol epigallocatechin-3-gallate counteracts semen-mediated enhancement of HIV
infection. Proc. Natl. Acad. Sci. U. S. A. 106, 9033–9038 (2009).
35. Castellano, L. M., Bart, S. M., Holmes, V. M., Weissman, D. & Shorter, J. Repurposing
Hsp104 to Antagonize Seminal Amyloid and Counter HIV Infection. Chem. Biol. 22, 1074–
1086 (2015).
36. Glover, J. R. & Lindquist, S. Hsp104, Hsp70, and Hsp40: a novel chaperone system that
rescues previously aggregated proteins. Cell 94, 73–82 (1998).
34 CHAPTER 3: ENHANCEMENT OF EBOLA VIRUS INFECTION BY SEMINAL
AMYLOID FIBRILS
Stephen M. Bart1, Courtney Cohen2, John Dye2, James Shorter3, Paul Bates1
1Department of Microbiology, Perelman School of Medicine, University of Pennsylvania,
Philadelphia, PA
2Virology Division, United States Army Medical Research Institute of Infectious Diseases,
Frederick, MD
3Department of Biochemistry and Biophysics, Perelman School of Medicine, University of
Pennsylvania, Philadelphia, PA
Manuscript in revision
This version contains unabridged Materials and Methods
The experiments presented in the manuscript were designed, executed, and analyzed by me under the supervision of Dr. Paul Bates. This includes the overarching hypothesis of SEVI-mediated enhancement of EBOV infection, execution of experiments to explore its mechanism, and identification of routes for further study. Dr. James Shorter provided reagents and insight into amyloid biology both during my rotation in his lab (in which I began work on seminal amyloids) and during this project. My infection protocol was amended for BSL-4 EBOV infections carried out by
Dr. Courtney Cohen under the supervision of Dr. John Dye at USAMRIID. I wrote the manuscript with critical reviews and edits from all authors.
35 Section 3.1 – Abstract
The 2014 Western Africa Ebola virus (EBOV) epidemic was unprecedented in magnitude, infecting over 28,000 and causing over 11,000 deaths. During this outbreak, multiple instances of
EBOV sexual transmission were reported, including cases where the infectious individual had recovered from EBOV disease months before transmission. Potential human host factors in EBOV sexual transmission remain unstudied. Several basic seminal amyloids, most notably semen- derived enhancer of viral infection (SEVI), enhance in vitro infection by HIV and several other viruses. To test the ability of these peptides to enhance EBOV infection, viruses bearing the EBOV glycoprotein (EboGP) were preincubated with physiological concentrations of SEVI before infection of physiologically relevant cell lines and primary cells. Pre-incubation with SEVI significantly increased EboGP-mediated infectivity and replication in epithelium- and monocyte-derived cells.
This enhancement was dependent upon amyloidogenesis and positive charge, and enhancement was observed with both viruses carrying EboGP and authentic EBOV. SEVI enhanced binding of virus to cells and markedly increased its subsequent internalization. SEVI also stimulated uptake of a fluid phase marker by macropinocytosis, a critical mechanism by which cells internalize EBOV.
We report a previously unrecognized ability of SEVI to significantly alter viral physical properties critical for transmissibility by increasing the stability of EboGP-bearing recombinant viruses during incubation at elevated temperature and providing resistance to desiccation. Given the potential for
EBOV sexual transmission to spark new transmission chains, these findings represent an important first interrogation of factors potentially important for this novel EBOV transmission route.
36 Section 3.2 – Significance Statement
During the 2014-2016 Ebola outbreak, multiple instances of male-to-female sexual transmission of
Ebola virus (EBOV) were reported. While relatively uncommon, EBOV sexual transmission presents a major public health concern, as these transmission events occurred months after recovery. Further, sexual transmission was linked to a resurgence of EBOV disease in Guinea, which had previously been declared Ebola-free. However, the role of host factors involved in sexual transmission remain unknown. We find that seminal amyloids are able to greatly enhance EBOV infection and alter the virion physical properties, stabilizing viral infectivity and protecting the virus from drying. These results suggest these seminal amyloids as possible targets for intervention to prevent EBOV sexual transmission and seeding new infection chains that reignite an outbreak.
37 Section 3.3 – Introduction
The 2014-2016 Western Africa Ebola outbreak was the largest Ebola outbreak on record, causing more than 28,652 cases and killing 11,3251. Ebola virus [EBOV] disease (EVD) is primarily spread by contact with bodily fluids of an infected individual or a person who has died from EVD.
Even after recovery from EVD, EBOV remains detectable in several anatomic locations and bodily fluids, including in semen2. Cohort studies of male EVD survivors demonstrated that infectious
EBOV could be isolated from semen up to 82 days after EBOV onset3,4. The maximum recorded persistence of EBOV RNA in semen is 965 days, although the concordance between persistent
RNA and infectious virus is unclear5. During the 2014-2016 EBOV outbreak, multiple cases of sexual transmission – some sparking new transmission chains – were reported in case reports supported by epidemiological and molecular evidence6–10. Transmission was reported from a male survivor 470 days after his EVD onset9. An epidemiological model of EBOV sexual transmission in
Sierra Leone predicted that if survivors experienced even a 3-month infectious period, the outbreak would be extended by an average of 83 days11.
EBOV tropism is broad12,13 and histological studies demonstrate a wide variety of cell types infected in vivo during infection12,14–16. It has been suggested that monocytes, macrophages, and dendritic cells in particular are important early targets for infection12,16–19. EBOV entry into cells is enhanced by interactions between the virion and cellular attachment factors that include C-type lectins (e.g. DC-SIGN and DC-SIGNR)20–24 and phosphatidylserine-binding molecules such as
Tyro3 family members (e.g. Axl)25 and TIM-126. EBOV requires macropinocytosis as an uptake mechanism27–29, resulting in its trafficking to acidified endosomes where the glycoprotein is processed by cathepsin B/L proteases30. After processing, the glycoprotein interacts with its receptor Niemann-Pick C1 (NPC1) to effect fusion between the viral and endosomal membranes31-33.
Screening of protein/peptide libraries derived from human semen identified peptides that dramatically enhance viral infection34. The most well-studied of these is PAP248-286, a highly basic
39-amino acid cleavage product of prostatic acid phosphatase (PAP)35. While having no impact on
38 HIV-1 infection as a soluble peptide, PAP248-286 assembles into amyloid fibrils termed semen- derived enhancer of viral infection (SEVI) which greatly increase HIV-1 infectivity35. Subsequent analyses have identified other amyloid fibrils in semen that enhance HIV-1 infection, including another cleavage product of PAP (PAP85-120)36 and fragments of semenogelin-1 and -2, components of the seminal coagulum37. Both positive charge and amyloid character are important for enhancement of HIV-1 infection by SEVI, as modified peptides without positive residues form amyloid fibrils with greatly diminished enhancement ability38. Anionic polymers such as dextran sulfate38 and molecular tweezers that bind positively charged amino acids39 inhibit SEVI’s enhancement effect, highlighting the importance of charge. Subsequent reports have identified enhancement roles for these fibrils for infection by SIV, CMV, and HSV-1, though the enhancement effects were generally lower for these viruses than those reported for HIV-140–42.
A leading model of SEVI enhancement of infection posits that the highly positive charge of the amyloid fibrils reduces electrostatic repulsion between the negative charges of the virion and cellular membranes. For HIV-1, an increase in virus attached to the cell surface was detected upon pretreatment of the virus with SEVI relative to virus alone35. SEVI fibrils are endocytosed by cellular protrusions, but the role this plays in enhancement of viral infection is not known35. It has also been reported that fusion of the viral and cellular membranes is enhanced upon pretreatment of HIV-1 with SEVI, though it is unclear whether SEVI enhances fusion per se or if increased attachment/binding results in an increase in downstream entry events35.
The ability of sexual transmission to reignite an outbreak warrants examination of factors affecting EBOV sexual transmission. In this study, we addressed whether seminal amyloids enhanced EBOV infection in vitro. Our results indicate that SEVI and other seminal amyloid fibrils greatly enhanced infection mediated by the EBOV glycoprotein using both non-pathogenic EBOV surrogates and authentic EBOV. This enhanced infection retained requirements for EBOV infection including macropinocytic uptake and cathepsin processing. In addition, we identify a new potential role for seminal amyloid fibrils in enhancing viral stability after extended incubation at elevated temperature or upon desiccation.
39 Section 3.4 – Materials and Methods
Viruses and cells. Recombinant VSV expressing the EBOV glycoprotein and mCherry (rVSV-
EboGP-mCherry) has been previously described43,44. To generate rVSV-EboGP-mCherry stocks,
Vero CCL81 cells (gift from Susan Weiss, U. Pennsylvania) were infected at an MOI of 0.001 for 3 days; clarified supernatant was buffered with 25 mM HEPES, aliquoted, frozen at -80°C, and titered by TCID50 on Vero CCL81 cells. EBOV/“Zaire 1995” (EBOV/H.sap-tc/COD/95/Kik-9510621) was used in authentic virus studies45. HeLa, A549, and THP1 cell lines and macrophages differentiated from purified human blood monocytes were used as target cells for infections. 293T cells were used for transfection. Samples obtained from the University of Pennsylvania Human Immunology Core are considered to be a secondary use of deidentified human specimens and are exempt via Title
55 Part 46, Subpart A of 46.101 (b) of the Code of Federal Regulations. 293T, HeLa, and A549 cells (ATCC) were maintained in DMEM with 4.5 g/L glucose and no sodium pyruvate supplemented with 10% FBS (Sigma). THP1 cells were maintained in RPMI-1640 supplemented with 10% FBS and 50 mM β-mercaptoethanol. Human monocyte-derived macrophages were maintained in RPMI-1640 with glutamine, 10% FBS, and penicillin/streptomycin, and were differentiated from peripheral blood monocytes by incubation in 20 μg/mL MCSF (Gemini) for 7 days.
Peptides and Fibrils. SEVI, SEVI-Ala, PAP85-120, SEM1, and SEM2 fibrils were generated by dissolving peptides (Keck Biotechnology Resource Laboratory, Yale University) in PBS, filtering through a 0.2 μm filter, seeding with 1% preformed amyloid, and incubating at 37°C with shaking at 1400 rpm35–37. Amyloid formation was confirmed by assessing thioflavin T fluorescence. Aliquots were stored at -80°C and working stocks kept at 4°C. Peptide sequences are available in
Supplementary Table 3.1.
Infection assays. rVSV-EboGP-mCherry was diluted into DMEM-10 alone or supplemented with amyloid fibrils or soluble peptides and incubated at 37°C for 15 minutes. 20 μL of the infection
40 mixture was added to each well of target cells in a 96-well plate (plated at 1.5e4 cells/well the previous day in 100 µL DMEM-10) and incubated at 37°C for 1 h, then the media was replaced with fresh DMEM-10 and incubated at 37°C for a total of 12 h. Cells infected in the presence of SEVI were harvested by trypsinization, fixed in 2% paraformaldehyde (PFA), and analyzed by flow cytometry for mCherry expression. For experiments containing amyloids other than SEVI, the cells were fixed, permeablized with 0.1% saponin in FACS buffer (1% BSA, 0.01% sodium azide in PBS), and stained for 1 h with a combination of 1:1000 mouse monoclonal anti-VSV(M) primary antibody
(gift from Robert Doms, U. Pennsylvania) and 1:5000 goat anti-mouse secondary antibody labeled with AF488 (Life Technologies) before analysis by flow cytometry. For each experiment, the average of triplicate technical replicates was log-transformed, and transformed percents infection of biological replicates were compared by repeated measures ANOVA with post hoc analysis using false discovery rate analysis to correct for multiple comparisons (GraphPad Prism). Monocyte- derived macrophages were treated with B18R (Abcam) for 24 h prior to as well as during infection to inhibit the interferon response.
For authentic virus infections, peptides were diluted to 5-50 µg/ml and pre-incubated with EBOV for 15 minutes. HeLa cells were exposed to peptide/virus inoculum at an MOI of 2.0 or 0.2 PFU/cell for 1 h, after which peptide/virus inoculum was removed and fresh culture media added. At 24-48 h post-infection, cells were formalin-fixed, removed from containment, and immunostained using the 13F6 antibody46 at 2 µg/ml. Infection was quantified using automated fluorescence microscopy as described47.
Binding/Internalization assays. rVSV-EboGP-mCherry was pretreated with or without SEVI then bound to HeLa cells on ice. Cells were either lysed in 1% Triton for 10 minutes on ice after 1 h
(binding) or warmed to 37°C for 1 h to allow viral internalization, washed 3x in PBS with Ca2+ and
Mg2+, trypsinized for 10 minutes at 37°C, and lysed with 1% Triton for 10 minutes on ice
(internalization). Lysates were separated on a 12% Criterion TGX gel, transferred to nitrocellulose
41 for 1 h, and blocked with TBS Odyssey Blocking Buffer (Li-Cor). Membranes were probed for VSV
M (1:1000, Ab as above) and GAPDH (1:2000, rabbit polyclonal, Santa Cruz Biotechnology) in TBS
Blocking Buffer/0.2% Tween simultaneously for 1 h, then with IRDye 800CW goat anti-mouse and
IRDye 680RD goat anti-rabbit (1:15,000, Li-Cor) in TBS blocking buffer/0.2% Tween. Membranes were then analyzed by quantitative Western blotting by comparing VSV M signal to GAPDH signal for each sample.
Virus-like particle generation. 293T cells were plated in 15 cm plates the day before transfection.
Cells were transfected with 7.5 μg each of pCAGGS-EboGP, pCAGGS-VP40, and either pCAGGS-
VP40(luc) or pCAGGS-VP40(GFP) with polyethylenimine48. Supernatants were collected at 24 and
48 h after transfection, concentrated through a 20% sucrose cushion by ultracentrifugation, resuspended in 1% BSA, 50 mM HEPES-buffered PBS, and frozen at -80°C until use.
VLP binding assay. HeLa cells were plated in a 96-well plate at 1.5e4 cells/well the day before the assay and incubated on ice for 30 min prior to the experiment. SEVI fibrils were diluted to 35 μg/mL in DMEM-10 and mixed with 3 μL concentrated EBOV VLP (VP40-luc) and incubated at 37°C for
10 min. 20 μL of the mixture was added to triplicate wells and spun at 1200g for 30 min at 4°C.
After spinning, the cells were washed 5X with cold DMEM-10 and lysed with Bright-Glo luciferase assay buffer (Promega). Luciferase activity was read on a Luminoskan Ascent (Thermo) 10 minutes after addition of assay buffer, and after background subtraction, readings were normalized to 0
μg/mL SEVI condition. Statistical significance was determined by paired t-test of log-transformed data (StataIC 14).
VLP internalization assay. The VLP internalization assay was done similarly to what has been previously described49. HeLa cells were plated in a 96-well plate at 1.5e4 cells/well the day before the assay. Cells treated with EIPA were pretreated with 100 μM EIPA for 1 h prior to the beginning of the experiment and incubated on ice for 30 minutes prior to the beginning of the experiment.
SEVI fibrils were diluted to 35 μg/mL in DMEM-10 and mixed with 2 μL EBOV VLP (VP40-GFP) and incubated at 37°C for 10 min. After incubation, 20 μL of this mixture was added to triplicate
42 wells and spun at 1200g for 30 min at 4°C. The plate was then shifted to 37°C for 1 h. The cells were then trypsinized, fixed in 2% PFA, and analyzed by flow cytometry for geometric mean fluorescence intensity in the GFP channel. Statistical significance was determined by repeated measured ANOVA with false discovery rate correction (GraphPad Prism).
Dextran uptake assay. HeLa cells were plated in a 96-well plate at 1.5e4 cells/well the day before the assay. Cells treated with EIPA were pretreated with 100 μM EIPA for 1 h. Culture medium was removed from each well and replaced with indicated concentrations of SEVI diluted in DMEM-10 with or without 100 μM EIPA in triplicate. The cells were incubated at 37°C for 20 minutes, then 2.5
μL of 20 mg/mL FITC-dextran (70 kDa in DMSO, Invitrogen) was added to each well for 10 minutes at 37°C. Afterward, the medium was removed and replaced with 100 μL of PBS pH 4.9 to bleach any uninternalized FITC. Cells were trypsinized and fixed in 2% PFA, washed 3X with FACS buffer
(1% BSA in PBS, 0.1% sodium azide) and analyzed by flow cytometry. Data were analyzed with
FlowJo to determine geometric mean fluorescence intensity in the FITC channel. Statistical significance was assessed by linear regression analysis or repeated measures ANOVA with false discovery rate correction (GraphPad Prism).
Inhibitor treatments. HeLa cells were plated in a 96-well plate at 1.5e4 cells/well the day before the assay. Cells treated with inhibitors [100 μM EIPA (Toronto Research Chemicals), 1 μM cytochalasin
D (Cayman Chemical Company), 0.5 μM 17-hydroxywortmannin (Cayman Chemical Company),
50 μM LY294002 (Cayman Chemical Company), 50 mM NH4Cl (Fisher), 10 μM Z-FF-FMK (EMD
Biosciences), 10 μM leupeptin (Sigma), 10 μM E64 (EMD Biosciences), or 10 μM MDL28170
(Calbiochem)] were pretreated for 1 h before infection. Cells were infected as described earlier.
After 1 h of infection, the virus- and inhibitor-containing medium was removed and replaced with medium without inhibitor for the remainder of the incubation. Infections were harvested and analyzed for percent infection as described above. To test for cytotoxic effects, HeLa cells were treated with inhibitors for 2 h before assessment for viability by the CellTiter 96 AQueous One
Solution Cell Proliferation assay kit (Promega) according to manufacturer instructions.
43 Virus stability analysis. rVSV-EboGP-mCherry was diluted from stock concentrations to concentrations of 1e7 TCID50/mL in artificial semen simulant, with or without SEVI fibrils or α- synuclein fibrils (35 μg/mL). For thermal stability experiments, samples were taken immediately (0 h timepoint) or after indicated times of incubation at 37°C in a thermocycler with heated lid to minimize evaporation. Samples were frozen at -80°C until titration by TCID50. For desiccation tolerance experiments, 10 μL of diluted virus in artificial semen simulant was spotted in the bottom of non-tissue-culture-treated 96-well plates and allowed to dry under laminar flow in a biosafety cabinet at room temperature for indicated lengths of time. For comparison, samples from the bulk liquid (maintained at room temperature in sealed tube) were taken at the initial timepoint and the last timepoint. Samples were immediately titered by TCID50 after addition of 200 μL of DMEM to recover virus from the dried samples.
TCID50. Vero cells were plated the previous day at 1.5e4 cells/well in 96-well plates. Viral samples were serially diluted in serum-free DMEM then added in 8-fold replicate to 96-well plates. After 48-
72 h, wells were scored by presence/absence of viral replication as marked by fluorescent protein expression and cytopathic effects. TCID50/mL was calculated by the Spearman & Kärber algorithm50. Data were log-transformed and analyzed for statistical significance by nonlinear regression (GraphPad Prism).
44 Section 3.5 – Results
Section 3.5.1 – Seminal amyloids enhance rVSV-EboGP-mCherry infection
The enhancement ability of seminal amyloids on EBOV glycoprotein-mediated infection was first assessed with a recombinant vesicular stomatitis virus expressing the EBOV glycoprotein
(rVSV-EboGP-mCherry, Fig 3.1A). Recombinant or pseudotyped VSV systems have been widely used to explore the interactions of the EBOV glycoprotein and viral membrane with cells during entry due to faithful mimicry of the EBOV infection process in a BSL-2 environment 30,31,51,52. SEVI is the best studied of the seminal amyloids, having been the first characterized and having variants available to explore mechanistic details35,38. Chemically synthesized PAP248-286 peptides were assembled into SEVI amyloid fibrils and preincubated with rVSV-EboGP-mCherry at indicated concentrations. These mixtures were used to infect cell lines representing potential target cell types during EBOV sexual transmission, including epithelium-derived HeLa and A549 cells and monocyte-derived THP1 cells. Percent infection was measured by flow cytometry for the mCherry reporter. Preincubation of rVSV-EboGP-mCherry with SEVI resulted in a striking increase in infectivity by 16.9- to 20.5-fold for the cell lines analyzed at 35 μg/mL, the concentration of SEVI reported in human semen35 (Fig 3.1B-C). A 4.3-fold increase in output virus was observed in primary monocyte-derived macrophages, indicating that the enhancement effect was not limited to cell lines (Fig 3.1D). These experiments were done at low multiplicity of infection, when enhancement by SEVI has been characterized as most effective35. However, due to concerns of reproducibility with such low initial percent infection, most subsequent studies were done at higher
MOI with a concomitant decrease in fold enhancement.
Other amyloidogenic peptides derived from the seminal proteins prostatic acid phosphatase (PAP85-120), semenogelin-1 (SEM1), and semenogelin-2 (SEM2) have been reported to enhance viral infection36,37,41. To determine whether these other amyloid fibrils enhance infection mediated by the EBOV glycoprotein, rVSV-EboGP-mCherry was preincubated with physiological concentrations of SEVI fibrils, PAP85-120 fibrils, representative SEM1 fibrils, or representative SEM2 fibrils and used to infect HeLa cells (MOI 3). Because of background for these
45 other peptides in the mCherry channel (Fig 3.S1), infection was quantified by flow cytometry after staining for VSV M protein. We observed that all four amyloid peptides significantly increased infection by rVSV-EboGP-mCherry, with the SEM1 amyloid fibrils exhibiting the highest fold increase of 20.3-fold (Fig 3.1E). The SEM2 and PAP85-120 peptides also significantly enhanced infection by the recombinant virus by 9.2- and 4.2-fold, respectively. These results demonstrate that rVSV-EboGP-mCherry infection is enhanced, to differing degrees, by four amyloid fibrils present in human semen.
SEVI-mediated enhancement of infection has been previously found to be dependent upon the charge and amyloid character of the peptides35,38. To determine whether SEVI-mediated enhancement of rVSV-EboGP-mCherry maintains similar specific requirements, rVSV-EboGP- mCherry was pretreated with SEVI fibrils, SEVI fibrils in which the positively-charged amino acids have been replaced with alanine (SEVI-Ala)38, or freshly dissolved PAP248-286. HeLa cells were infected with the virus-peptide mixtures (MOI 3) and infection was analyzed by flow cytometry for mCherry expression. Unlike SEVI, which effected a 7.5-fold enhancement of rVSV-EboGP- mCherry infection at this MOI, infection in the presence of SEVI-Ala or soluble PAP248-286-treated virus did not differ from rVSV-EboGP-mCherry alone (Fig 3.1F). These results indicate that much like enhancement of HIV-1 infection, the positive charge and amyloid character of the peptide are both critical for rVSV-EboGP-mCherry enhancement.
46
Figure 3.1. fibrils, SEVI fibrils, by ANOVA. μ SEVI; without n=3, ± mean SEM. monocyte human of titer output Normalized rVSV with infected cells THP1 and expression. mCherry for analyzed and SEVI of absence or presence the 1in MOI at mCherry A.
g/mL SEVIg/mL fibrils, 39
Schematic of the genomic organization of rVSV of organization genomic the of Schematic
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47 Section 3.5.2 – SEVI enhances EBOV VLP binding and internalization
To characterize the mechanism of SEVI-mediated enhancement of Ebola glycoprotein- mediated infection, binding and internalization experiments were performed. To assess impacts on binding, rVSV-EboGP-mCherry was preincubated with SEVI before binding to HeLa cells on ice for
1 h. Afterward, the cells were lysed on ice. Lysates were analyzed by quantitative Western blotting, probing for VSV M to assess quantity of virus present using GAPDH as a loading control. Signal in the presence of SEVI was compared to rVSV-EboGP-mCherry bound in the absence of SEVI. Only a modest increase in binding (1.5- to 2-fold) that saturated below the concentrations of SEVI tested was observed (Fig 3.2A). Binding was further quantified with a binding assay using EBOV virus- like particles (VLPs). These particles show filamentous morphology and replicate steps of the
EBOV entry process28,53–55. VLPs with a luciferase reporter were preincubated with SEVI and bound to HeLa cells at 4°C with centrifugation, then the cells were washed, lysed in luciferase assay buffer, and luminescence recorded. In agreement with previous results, an approximately 2-fold increase in binding was observed in the presence of SEVI (Fig 3.S2A).
Internalization by macropinocytosis is essential for EBOV entry, therefore the impact of
SEVI on internalization was also analyzed in a similar way. After binding to cells on ice, the cells were shifted to 37°C for an additional hour to permit internalization. The cells were then washed and trypsinized to remove bound but uninternalized virus, then lysed and analyzed by Western blotting. Strikingly, quantitative Western blotting analysis demonstrated a linear (R2=0.9894) dose- dependent relationship between SEVI preincubation concentration and relative internalization, with physiological concentrations of SEVI resulting in an approximately 10-fold increase in internalization of rVSV-EboGP-mCherry after background subtraction (Fig 3.2B). To further confirm this finding, EBOV VLPs labeled with GFP were preincubated with SEVI and bound to HeLa cells on ice then shifted to 37°C. After trypsinization, cells were analyzed by flow cytometry for GFP signal. While less quantitative than the blotting analysis, an increase in geometric mean fluorescence intensity of approximately 25% was observed in the presence of SEVI, suggesting that cells internalized a significantly higher quantity of VLPs when pretreated with SEVI than without
48 pretreatment (Fig 3.S2B). This effect was ablated by the macropinocytosis inhibitor N-(ethyl-N- isopropyl)-amiloride (EIPA), suggesting efficient removal of bound VLPs from the cell surface.
The ability of SEVI to enhance viral internalization has not been previously reported.
Previous reports have shown that SEVI is internalized into cells, raising the possibility it may induce endocytosis35. To explore the ability of SEVI to modulate macropinocytosis in a virus-free environment, HeLa cells were incubated at 37°C with different concentrations of SEVI for 20 minutes before the addition of a macropinocytic marker molecule FITC-dextran for 10 minutes. The cells were treated with PBS pH 4.9 to bleach any uninternalized FITC and trypsinized, and then geometric mean fluorescence intensity (gMFI) was determined by flow cytometry. A linear relationship (R2 = 0.9799) was observed between SEVI concentration and gMFI, with the highest concentrations of SEVI leading to a 3.7-fold increase in macropinocytic uptake (Fig 3.2C). This increase in fluorescent intensity was ablated in the presence of EIPA, indicating efficient bleaching of bound FITC signal and a specificity of macropinocytosis for uptake (Fig 3.2C). The enhanced
FITC-dextran internalization depended upon the charge and amyloid character of the SEVI peptide, as SEVI-Ala and soluble PAP248-286 peptide treatment showed no increase in dextran uptake
(Fig 3.2D). These results provide evidence for the ability of SEVI to promote macropinocytic uptake to increase internalization of EBOV particles.
49 2A 2B
2C 2D
2E 2F
Figure 3.2. SEVI enhances rVSV-EboGP-mCherry binding, internalization, and cellular macropinocytosis. 8 A. HeLa cells were prechilled and treated with rVSV-EboGP-mCherry pretreated with or without SEVI fibrils. Cells were lysed and the ratio of VSV M:GAPDH signal was compared to untreated control by quantitative Western blotting; n=3, mean ± SEM. B. HeLa cells were prechilled and treated with rVSV-EboGP-mCherry pretreated with or without SEVI fibrils. After 1 h, the cells were shifted to 37°C to allow internalization. Cells were washed, trypsinized, and lysed, and the ratio of VSV M:GAPDH signal was compared to untreated control by quantitative Western blotting; n=3, mean ± SEM. C. HeLa cells were treated with different concentrations of SEVI fibrils then treated with dextran-FITC in the presence or absence of EIPA. Geometric mean fluorescent intensity was measured by flow cytometry and normalized; n=3, mean ± SEM. **p<0.01 by linear regression analysis. D. HeLa cells were treated with or without 35 μg/mL SEVI fibrils, SEVI-Ala fibrils, or soluble PAP248-286 then 70 kDa dextran-FITC. n=3, mean ± SEM. **p≤0.01 by ANOVA of gMFI values. E. Normalized percents infection of HeLa cells pretreated with macropinocytosis inhibitors and infected with rVSV-EboGP-mCherry (MOI 5) with or without 35 μg/mL SEVI fibrils. F. Normalized percents infection of HeLa cells pretreated with DMEM-10 alone or supplemented with cathepsin inhibitors infected with rVSV-EboGP-mCherry (MOI 5) pretreated with or without 35 μg/mL SEVI fibrils. For both E and F: n=3, mean ± SEM, *p<0.05, **p<0.01, ***p<0.001 by two-factor repeated measures ANOVA.
50 Section 3.5.3 – SEVI-mediated enhancement maintains EBOV entry requirements
Following binding of the virus to the cell, EBOV particles are endocytosed by macropinocytosis and traffic through the endosomal system where cellular cathepsins process the glycoprotein, enabling an interaction of the glycoprotein with its receptor to initiate fusion of the viral and cellular membranes and infection56. To determine whether the fibril-enhanced infection diverges from the canonical EBOV entry pathway, cells were infected with rVSV-EboGP-mCherry in the presence or absence of inhibitors of macropinocytosis or cathepsin activity, with or without preincubation of the virus with SEVI. No cytotoxic effects were observed with any of these inhibitors
(Fig 3.S3). To inhibit macropinocytosis, HeLa cells were treated with EIPA, cytochalasin D, 17β- hydroxywortmannin, or LY294002 for 1 h before and 1 h during infection with the virus-peptide mixture (MOI 5). While infection by rVSV-EboGP-mCherry was enhanced by SEVI in this experiment in the absence of inhibitor, all macropinocytosis inhibitors equally reduced infection by
5- to 10-fold in both the absence and presence of SEVI. These results indicate that macropinocytic entry is not bypassed in the presence of SEVI and that infection maintains this cellular requirement for EBOV glycoprotein-dependent infection (Fig 3.2E). Similarly, inhibitors of cathepsin activity, including ammonium chloride, E64, Z-FF-FMK, leupeptin, and MDL28170, abrogated infection of
HeLa cells by 10- to 20-fold in both the presence and absence of physiological levels of SEVI (Fig
3.2F). These results imply that while SEVI enhances infection, it does not permit the virus to bypass critical cellular requirements for rVSV-EboGP-mCherry entry.
Section 3.5.4 – SEVI alters rVSV-EboGP-mCherry physical characteristics
We next hypothesized that seminal amyloid fibrils could affect the physical characteristics of the virus, which may impact transmissibility. To determine if the interaction of viral particles with amyloid fibrils alters the physical characteristics of the virus, we analyzed the effects of thermal and chemical stressors on viral infectivity. Whether SEVI pretreatment impacts rVSV-EboGP-mCherry stability over time was assessed by incubation of rVSV-EboGP-mCherry with or without physiologic
SEVI concentrations in artificial semen simulant57 for various lengths of time at 37°C. Artificial
51 semen simulant is designed to mimic the chemical composition of semen and contains 5 mg/mL
BSA, making any nonspecific effects of additional protein (e.g. amyloids or peptides) unlikely. At each timepoint, the titer of virus was determined by TCID50 on Vero cells. Values were normalized to the starting titer for each condition, log-transformed, and analyzed by nonlinear regression.
Strikingly, the presence of SEVI promoted increased viral viability relative to virus alone (p=0.0029).
The normalized titer of virus incubated in the presence of SEVI was approximately 17-fold higher than that of virus incubated in semen simulant alone after 36 h (Fig 3.3A). This enhancement in stability required the positive charge and amyloid character of SEVI, as SEVI-Ala and PAP248-286 had no effect on stability (Fig 3.3A). Further, addition of SEVI immediately before titration after incubation without SEVI was unable to rescue the effect, suggesting that SEVI exerts its stabilizing effects during the incubation itself and independently from its infection enhancement ability (Fig
3.3A, 0 μg/mL SEVI+35 μg/mL SEVI). To test whether SEVI promoted resistance to oxidative stress, we analyzed the ability of rVSV-EboGP-mCherry to resist exposure to dilute concentrations of chlorine bleach for one minute. Unlike the results found with viral infectivity during prolonged incubation, SEVI did not enhance the ability of rVSV-EboGP-mCherry to withstand oxidative stress by chlorine bleach, with equivalent decreases in titer observed both in the presence and absence of SEVI (Fig 3.S4).
52 3A
3B
Figure 3.3. SEVI alters rVSV-EboGP-mCherry physical characteristics.9 A. rVSV-EboGP-mCherry was incubated at 37°C in artificial semen simulant with or without 35 μg/mL SEVI fibrils, SEVI-Ala fibrils, or PAP248-286 peptide and titered by TCID50 on Vero cells. For the 0 μg/mL+35 μg/mL SEVI condition, SEVI was added immediately before titration after incubation without. Data were normalized to the 0 h timepoint independently for the conditions with or without SEVI and fit to one-step decay model; n=3, mean ± SEM. B. rVSV-EboGP- mCherry was diluted in artificial semen simulant with or without 35 µg/mL SEVI fibrils and dried under laminar flow before rehydration and titration by TCID50 on Vero cells. Data were normalized to the 0 h timepoint independently for the conditions with or without SEVI and fit to plateau followed by one-step decay model; n=3, mean ± SEM.
53 Lastly, we assessed whether the presence of SEVI fibrils could improve desiccation tolerance; as enveloped viruses, EBOV and rVSV-EboGP-mCherry are sensitive to drying. rVSV-
EboGP-mCherry was diluted in artificial semen simulant in the presence or absence of physiological concentrations of SEVI. Samples were maintained in bulk liquid or spotted into 96- well plates (10 μL) and allowed to air dry. Samples were taken immediately (0 h) or after varying lengths of time drying under laminar flow at room temperature. Dried samples were rehydrated with
200 μL of DMEM and infectivity was quantified by TCID50. Samples were also taken at 6 h from bulk liquid kept within a sealed tube to assess independently any effects of incubation at room temperature. TCID50/mL measurements were normalized to the initial timepoint for each and fitted to a plateau-one phase decay model, which reflects the lag in viral decay until after the liquid has evaporated. After 6 h of incubation, the normalized titer of virus desiccated in the presence of SEVI was approximately 10-fold lower than in its absence (Fig 3.3B). Notably, over this timescale at room temperature, there was no relative difference in viral titer between virus incubated in the presence or absence of SEVI in bulk liquid (p=0.10), suggesting the observed effects are actually due to desiccation and not temperature-induced decay (Fig 3.S5). Again, addition of SEVI immediately before titration after desiccation in its absence had no effect on desiccation kinetics (Fig 3.3B, 0
μg/mL SEVI+35 μg/mL SEVI). This result indicates that SEVI promotes viral viability after desiccation and rehydration. To better understand the mechanism of SEVI-mediated desiccation tolerance, rVSV-EboGP-mCherry was also incubated with SEVI-Ala and PAP248-286. While
PAP248-286 had no effect on desiccation tolerance, SEVI-Ala unexpectedly increased viral stability to the same extent as SEVI under these conditions. To determine if the presence of any amyloid could give this effect, rVSV-EboGP-mCherry was dried for 6 h in the presence of an equivalent mass of α-synuclein fibrils, which play a role in Parkinson’s disease pathogenesis58. No stabilizing effect was seen over incubation in semen simulant alone, suggesting that not all amyloids have this property (Fig 3.S6). Overall, these results indicate a previously unreported ability of seminal amyloid fibrils to stabilize viral infectivity, even after incubation at elevated temperatures over time or desiccation.
54 Section 3.5.5 – Seminal amyloids enhance infection by authentic EBOV
To confirm that the SEVI enhancement of rVSV-EboGP-mCherry faithfully mimics infection by authentic EBOV, HeLa cells were infected with EBOV after preincubation with various concentrations of SEVI fibrils. Preincubation of EBOV with SEVI led to a dose-dependent enhancement of infection similar to that observed with rVSV-EBOV-mCherry, resulting in a 28.9- fold increase in infection after 24 h at the physiologic concentration of SEVI (MOI 0.2) (Fig 3.4A).
As with EboGP-mediated VSV infection, the EBOV infection enhancement was dependent upon the charge and amyloid nature of the fibrils. In contrast to a 22.8-fold increase in infection observed in the presence of SEVI fibrils in this experiment, EBOV infection (MOI 2) was not enhanced by
SEVI-Ala fibrils or soluble PAP248-286 (Fig 3.4B). Preincubation of EBOV with the other seminal peptides also enhanced infection at 24 h by 37.2-fold for PAP85-120 fibrils, 34.3-fold for SEM1 fibrils, and 41.3-fold for SEM2 fibrils (all 35 μg/mL, MOI 2), in agreement with the results observed for rVSV-EBOV-mCherry (Fig 3.4C, 3.1E).
55
4A 4B
4C
Figure 3.4. Seminal amyloids enhance EBOV infection. 10 A. Normalized infection of HeLa cells infected with EBOV (MOI 0.2) preincubated with increasing concentrations of SEVI fibrils; n=2, mean ± SEM. B. Normalized infection of HeLa cells infected with EBOV (MOI 2) pretreated with or without 35 μg/mL SEVI fibrils, SEVI-Ala fibrils, or soluble PAP248-286; n=2, mean ± SEM. C. Normalized infection of HeLa cells infected with EBOV (MOI 2) pretreated with or without 35 μg/mL SEVI fibrils, PAP85-120 fibrils, SEM1 fibrils, or SEM2 fibrils; n=2, mean ± SEM.
56 Section 3.6 – Discussion
Reports of EBOV sexual transmission during the West Africa Ebola epidemic, though rare, are backed by strong epidemiological and/or molecular evidence6–10. Male-to-female transmission of persistent virus has been linked to resurgence of EBOV, but factors potentially involved in EBOV sexual transmission have not been characterized. This report provides evidence that seminal amyloid fibrils, a ubiquitous component of semen in healthy individuals, enhance in vitro infection by both an EBOV surrogate system and authentic EBOV. These fibrils act in a conformation- and charge-dependent manner to increase infection by increasing virion binding to host cells and enhancing macropinocytotic uptake. Further, we find that these fibrils act to protect virions from stresses potentially encountered during transmission, including thermal degradation and desiccation.
Ebola sexual transmission presented a significant and novel public health problem during and following the West Africa Ebola epidemic. Though rare and likely mitigated by public health agencies’ safe sex education initiatives, it has become apparent that semen of individuals with persistent EBOV is a potentially important vehicle to consider for EBOV transmission. For a successful male-to-female sexual transmission event, EBOV present in semen must either infect or cross the vaginal epithelium. Recent studies demonstrate that EBOV is able to infect the vaginal epithelium in guinea pig models59. Notably, the amount of infectious virus in semen has been difficult to determine, but at late time points is likely much lower than that in bodily fluids during acute illness. Prior studies on HIV indicate that the enhancement effect of SEVI is greatest with very low viral inoculums35; therefore, the effect of SEVI may be disproportionately important in
EBOV sexual transmission as seminal viral titers wane. Animal models have suggested that macrophages and dendritic cells are early targets of the virus12,16–19. Our results indicate that seminal amyloids enhance infection of epithelial and monocytic cells and subsequent viral replication and thus could impact early events in sexual transmission. These cells express numerous attachment factors that increase the efficiency of infection by EBOV, including DC-SIGN.
57 Whether SEVI permits EBOV to bypass this dependence upon attachment factors or synergistically enhances attachment is not known and worth further study.
In addition, our results demonstrate that amyloids impart resistance of the virus to potentially relevant environmental stresses, including extended incubations at physiological temperatures and desiccation. The ability of environmental factors to affect virion properties and infectivity is seen in other systems. As an example, recent studies have found that bacterial lipopolysaccharide (LPS) induces a conformational change in the poliovirus capsid, increasing virus binding to its cellular receptor and enhancing the stability of the virion60. These findings are similar to those we now report regarding the interaction of an enveloped virus and seminal amyloid fibrils.
After binding to SEVI, rVSV-EboGP-mCherry binding, internalization, and tolerance to environmental stresses increase. The mechanism for this enhanced environmental resistance is unclear, but could involve retention of water molecules by the large amyloid fibrils to create a microenvironment surrounding the virions that is relatively resistant to changes in the larger-scale environment. The exact characteristics necessary for desiccation tolerance are unclear given the unexpected ability of SEVI and SEVI-Ala to enhance tolerance while another amyloid did not.
Future experiments may involve exploration of the enhancement properties of a wider array of amyloids, as well as testing other viruses such as HIV-1. Resistance to environmental factors may be important to consider when assessing the ability of semen to remain infective over time.
Moreover, the seminal amyloids may represent prophylactic drug targets, as strategies to disassemble the amyloids have been investigated34.
Since the identification of seminal amyloid fibrils as enhancers of HIV-1 infection in 2007, infection by several viruses with sexual transmission routes have been found to also be enhanced by SEVI. While not all sexually transmitted viruses are enhanced – notably, Zika virus is not enhanced appreciably by SEVI despite reports of Zika virus sexual transmission, potentially because of the dense Zika virus glycoprotein structure (Fig 4.1) – enhancement of infection by these viruses may represent a common target for intervention. Mechanistically, enhancement by
58 Figure 3.5. Model of SEVI-mediated enhancement of EBOV infection.11 seminal amyloids has been proposed to enhance binding of the virus to the cell by alleviating repulsive interactions between the viral and cell membranes. Our results suggest that its effect on binding is modest, but SEVI stimulates macropinocytosis to increase viral internalization in addition to changing the physical properties of the virion (Fig 3.5). The enhancement of infection of primary monocyte-derived macrophages, as well as the striking increases in macropinocytosis observed in this study, are reminiscent of a recent report in which clearance of sperm cells by macrophages is enhanced by seminal amyloids61. Parallels between the clearance of spermatocytes by macrophages stimulated to engulf the cells by phagocytosis, and internalization of EBOV by phagocytic cells via a similar uptake mechanism are particularly intriguing.
An important limitation of the present study is the inability to study this phenomenon in an in vivo model, as no model for EBOV sexual transmission of persistent virus exists, and the challenges associated with developing one are considerable. However, seminal fibrils may represent an intriguing prophylactic target since agents that affect fibril stability or formation may target increased infectivity at a cellular level as well as enhanced viral stability ex vivo. Altogether, these findings represent the first analysis of molecular factors potentially involved in EBOV sexual transmission and may promote further study of this novel transmission route of an important human pathogen.
59 Section 3.8 – Supplemental Information
Supplementary Table 3.1. Peptide Sequences SEVI GIHKQKEKSRLQGGVLVNEILNHMKRATQIPSYKKLIMY SEVI-Ala GIHAQAEASALQGGVLVNEILNHMAAATQIPSYAALIMY PAP85-120 IRKRYRKFLNESYKHEQVYIRSTDVDRTLMSAMTNL SEM1 (45–107) GQHYSGQKGKQQTESKGSFSIQYTYHVDANDHDQSRKSQQYDLNALHKTTKSQRHLGGSQQLL SEM2 (49-107) GQKDQQHTKSKGSFSIQHTYHVDINDHDWTRKSQQYDLNALHKATKSKQHLGGSQQLL
60 Figure 3.S1. SEM1 and PAP85-120 exhibit autofluorescence in the mCherry channel. 13 HeLa cells were treated with seminal amyloids and autofluorescence was measured by flow cytometry. The percentage of cells in the 610_20 Green gate indicates background autofluorescence. S2A S2B
Figure 3.S2. SEVI enhances VLP binding and internalization. 12 A. HeLa cells were prechilled and treated with EBOV VLPs (VP40-luc) pretreated with or without 35 μg/mL SEVI fibrils. After spinfection, cell-associated luciferase signal was determined and normalized; n=3, mean ± SEM. **p<0.01 by paired t test. B. HeLa cells were prechilled and treated with EBOV VLPs (VP40-GFP) pretreated with or without 35 μg/mL SEVI fibrils. After allowing internalization, cells were analyzed for geometric mean fluorescence intensity of GFP. *p<0.05, ***p<0.001 by repeated measures ANOVA.
61 Figure 3.S3. Macropinocytosis and cathepsin inhibitors do not exhibit cytotoxicity. 14 HeLa cells were treated for 2 h with inhibitors in concentrations as described in Materials and Methods then cell viability was assessed by the CellTiter 96 AQueous One Solution Cell Proliferation assay. n=2, mean±SD.
Figure 3.S4. SEVI does not promote increased stability following treatment with dilute bleach 15 rVSV-EboGP-mCherry was diluted in DMEM-10 alone or supplemented with 35 μg/mL SEVI fibrils and treated with PBS or chlorine bleach diluted in PBS for a final concentration of 0.005% hypochlorite for 1 minute. The samples were then neutralized with sodium thiosulfate (10-fold excess of 0.01% solution) and titered by TCID50 on Vero cells. n=1, mean±SD.
62 Figure 3.S5. No differential thermal degradation occurs over six hours at room temperature in the presence or absence of SEVI.17 rVSV-EboGP-mCherry was diluted in artificial semen simulant alone or supplemented with 35 µg/mL SEVI fibrils and titered either immediately or after six hours of incubation in a sealed tube at room temperature by TCID50 on Vero cells. p=0.10 by paired t test.
Figure 3.S6. α-synuclein does not enhance rVSV-EboGP-mCherry desiccation tolerance.16 rVSV-EboGP-mCherry was incubated at 37°C in artificial semen simulant with or without 35 μg/mL SEVI fibrils and titered by TCID50 on Vero cells. n=1.
63 Section 3.9 – Acknowledgments
We thank the University of Pennsylvania Human Immunology Core (funding under P30-CA016520) for primary monocytes and Valeria Reyes-Ruiz for assistance in their differentiation. We acknowledge funding from T32-AI-007324 (SMB), Bill and Melinda Gates Foundation Grand
Challenges Explorations Award and R21-HD-074510 (JS), Defense Threat Reduction Agency,
Project #18855636 (CC & JD), and Department of Defense Peer Reviewed Medical Research
Program grant W81XWH-14-1-0204 (PB). Disclaimer: Opinions, interpretations, conclusions, and recommendations are those of the author and are not necessarily endorsed by the U.S. Army.
64 Section 3.10 – Bibliography
1. 2014-2016 Ebola Outbreak in West Africa | Ebola Hemorrhagic Fever | CDC. Available
at: https://www.cdc.gov/vhf/ebola/outbreaks/2014-west-africa/index.html. (Accessed: 6th
March 2018)
2. Vetter, P. et al. Ebola Virus Shedding and Transmission: Review of Current Evidence. J.
Infect. Dis. 214, S177–S184 (2016).
3. Rowe, A. K. et al. Clinical, Virologic, and Immunologic Follow‐Up of Convalescent Ebola
Hemorrhagic Fever Patients and Their Household Contacts, Kikwit, Democratic Republic of
the Congo. J Infect Dis 179, S28–S35 (1999).
4. Uyeki, T. et al. Ebola Virus Persistence in Semen of Male Survivors. Clin Infect Dis 62,
1552–1555 (2016).
5. Fischer, W. et al. Ebola Virus RNA Detection in Semen More than Two Years After
Resolution of Acute Ebola Virus Infection. Open Forum Infect Dis ofx155- (0).
doi:10.1093/ofid/ofx155
6. Mate, S. E. et al. Molecular Evidence of Sexual Transmission of Ebola Virus. N. Engl. J.
Med. 373, 2448–2454 (2015).
7. Blackley, D. J. et al. Reduced evolutionary rate in reemerged Ebola virus transmission
chains. Sci. Adv. 2, e1600378 (2016).
8. Thorson, A., Formenty, P., Lofthouse, C. & Broutet, N. Systematic review of the literature
on viral persistence and sexual transmission from recovered Ebola survivors: evidence and
recommendations. BMJ Open 6, e008859 (2016).
9. Diallo, B. et al. Resurgence of Ebola Virus Disease in Guinea Linked to a Survivor With
Virus Persistence in Seminal Fluid for More Than 500 Days. Clin Infect Dis 63, 1353–1356
(2016).
10. Keita, M. et al. Unusual Ebola Virus Chain of Transmission, Conakry, Guinea, 2014–
2015. Emerg Infect Dis 22, (2016).
65 11. Abbate, J., Murall, C., Richner, H. & Althaus, C. Potential Impact of Sexual Transmission
on Ebola Virus Epidemiology: Sierra Leone as a Case Study. PLoS Negl. Trop D 10,
e0004676 (2016).
12. Ryabchikova, E., Kolesnikova, L. & Luchko, S. An analysis of features of pathogenesis in
two animal models of Ebola virus infection. J Infect Dis 179 Suppl 1, S199-202 (1999).
13. Wool-Lewis, R. J. & Bates, P. Characterization of Ebola virus entry by using pseudotyped
viruses: identification of receptor-deficient cell lines. J. Virol. 72, 3155–3160 (1998).
14. Wyers, M. et al. Histopathological and immunohistochemical studies of lesions
associated with Ebola virus in a naturally infected chimpanzee. J. Infect. Dis. 179 Suppl 1,
S54-59 (1999).
15. Connolly, B. M. et al. Pathogenesis of experimental Ebola virus infection in guinea pigs.
J. Infect. Dis. 179 Suppl 1, S203-217 (1999).
16. Geisbert, T. W. et al. Pathogenesis of Ebola hemorrhagic fever in cynomolgus
macaques: evidence that dendritic cells are early and sustained targets of infection. Am. J.
Pathol. 163, 2347–2370 (2003).
17. Bray, M. & Geisbert, T. W. Ebola virus: the role of macrophages and dendritic cells in the
pathogenesis of Ebola hemorrhagic fever. Int J Biochem Cell Biol 37, 1560–6 (2005).
18. Feldmann, H. et al. Filovirus-induced endothelial leakage triggered by infected
monocytes/macrophages. J. Virol. 70, 2208–2214 (1996).
19. Gupta, M., Mahanty, S., Ahmed, R. & Rollin, P. Monocyte-derived human macrophages
and peripheral blood mononuclear cells infected with ebola virus secrete MIP-1alpha and TNF-
alpha and inhibit poly-IC-induced IFN-alpha in vitro. Virology 284, 20–5 (2001).
20. Alvarez, C. P. et al. C-type lectins DC-SIGN and L-SIGN mediate cellular entry by Ebola
virus in cis and in trans. J Virol 76, 6841–4 (2002).
21. Simmons, G. et al. DC-SIGN and DC-SIGNR bind ebola glycoproteins and enhance
infection of macrophages and endothelial cells. Virology 305, 115–23 (2003).
66 22. Matsuno, K. et al. C-type lectins do not act as functional receptors for filovirus entry into
cells. Biochem Bioph Res Co 403, 144–148 (2010).
23. Powlesland, A. et al. A Novel Mechanism for LSECtin Binding to Ebola Virus Surface
Glycoprotein through Truncated Glycans. J Biol Chem 283, 593–602 (2008).
24. Takada, A. et al. Human macrophage C-type lectin specific for galactose and N-
acetylgalactosamine promotes filovirus entry. J Virol 78, 2943–7 (2004).
25. Shimojima, M. et al. Tyro3 family-mediated cell entry of Ebola and Marburg viruses. J.
Virol. 80, 10109–10116 (2006).
26. Kondratowicz, A. S. et al. T-cell immunoglobulin and mucin domain 1 (TIM-1) is a
receptor for Zaire Ebolavirus and Lake Victoria Marburgvirus. Proc. Natl. Acad. Sci. U. S. A.
108, 8426–8431 (2011).
27. Mulherkar, N., Raaben, M., Torre, J., Whelan, S. & Chandran, K. The Ebola virus
glycoprotein mediates entry via a non-classical dynamin-dependent macropinocytic pathway.
Virology 419, 72–83 (2011).
28. Nanbo, A. et al. Ebolavirus is internalized into host cells via macropinocytosis in a viral
glycoprotein-dependent manner. PLoS Pathog. 6, e1001121 (2010).
29. Saeed, M., Kolokoltsov, A., Albrecht, T. & Davey, R. Cellular entry of ebola virus involves
uptake by a macropinocytosis-like mechanism and subsequent trafficking through early and
late endosomes. PLoS Pathog 6, e1001110 (2010).
30. Chandran, K., Sullivan, N. J., Felbor, U., Whelan, S. P. & Cunningham, J. M. Endosomal
proteolysis of the Ebola virus glycoprotein is necessary for infection. Science 308, 1643–1645
(2005).
31. Carette, J. E. et al. Ebola virus entry requires the cholesterol transporter Niemann-Pick
C1. Nature 477, 340–343 (2011).
32. Côté, M. et al. Small molecule inhibitors reveal Niemann-Pick C1 is essential for Ebola
virus infection. Nature 477, 344–8 (2011).
67 33. Spence, J. S., Krause, T. B., Mittler, E., Jangra, R. K. & Chandran, K. Direct Visualization
of Ebola Virus Fusion Triggering in the Endocytic Pathway. mBio 7, e01857-01815 (2016).
34. Castellano, L. M. & Shorter, J. The Surprising Role of Amyloid Fibrils in HIV Infection.
Biology 1, 58–80 (2012).
35. Münch, J. et al. Semen-derived amyloid fibrils drastically enhance HIV infection. Cell 131,
1059–1071 (2007).
36. Arnold, F. et al. Naturally Occurring Fragments from Two Distinct Regions of the Prostatic
Acid Phosphatase Form Amyloidogenic Enhancers of HIV Infection. J Virol 86, 1244–1249
(2012).
37. Roan, N. R. et al. Peptides Released by Physiological Cleavage of Semen Coagulum
Proteins Form Amyloids that Enhance HIV Infection. Cell Host Microbe 10, 541–550 (2011).
38. Roan, N. R. et al. The cationic properties of SEVI underlie its ability to enhance human
immunodeficiency virus infection. J. Virol. 83, 73–80 (2009).
39. Lump, E. et al. A molecular tweezer antagonizes seminal amyloids and HIV infection.
eLife 4, (2015).
40. Münch, J. et al. Effect of semen and seminal amyloid on vaginal transmission of simian
immunodeficiency virus. Retrovirology 10, 148 (2013).
41. Tang, Q., Roan, N. R. & Yamamura, Y. Seminal plasma and semen amyloids enhance
cytomegalovirus infection in cell culture. J. Virol. 87, 12583–12591 (2013).
42. Torres, L., Ortiz, T. & Tang, Q. Enhancement of herpes simplex virus (HSV) infection by
seminal plasma and semen amyloids implicates a new target for the prevention of HSV
infection. Viruses 7, 2057–2073 (2015).
43. Jones, S. M. et al. Live attenuated recombinant vaccine protects nonhuman primates
against Ebola and Marburg viruses. Nat. Med. 11, 786–790 (2005).
44. Haines, K., Burgt, N., Francica, J., Kaletsky, R. & Bates, P. Chinese hamster ovary cell
lines selected for resistance to ebolavirus glycoprotein mediated infection are defective for
NPC1 expression. Virology 432, 20–8 (2012).
68 45. Jahrling, P. B. et al. Evaluation of immune globulin and recombinant interferon-alpha2b
for treatment of experimental Ebola virus infections. J. Infect. Dis. 179 Suppl 1, S224-234
(1999).
46. Wilson, J. A. et al. Epitopes involved in antibody-mediated protection from Ebola virus.
Science 287, 1664–1666 (2000).
47. Wec, A. Z. et al. A ‘Trojan horse’ bispecific-antibody strategy for broad protection against
ebolaviruses. Science 354, 350–354 (2016).
48. Boussif, O. et al. A versatile vector for gene and oligonucleotide transfer into cells in
culture and in vivo: polyethylenimine. Proc. Natl. Acad. Sci. U. S. A. 92, 7297–7301 (1995).
49. Johansen, L. et al. FDA-approved selective estrogen receptor modulators inhibit Ebola
virus infection. Sci Transl Med. 5, 190ra79 (2013).
50. Hierholzer, J. C. & Killington, R. A. Virus isolation and quantitation. in Virology methods
manual 25–46 (Elsevier, 1996).
51. Takada, A. et al. A system for functional analysis of Ebola virus glycoprotein. Proc Natl
Acad Sci 94, 14764–14769 (1997).
52. Lee, J. E. et al. Structure of the Ebola virus glycoprotein bound to an antibody from a
human survivor. Nature 454, 177–182 (2008).
53. Noda, T. et al. Ebola virus VP40 drives the formation of virus-like filamentous particles
along with GP. J. Virol. 76, 4855–4865 (2002).
54. Kallstrom, G. et al. Analysis of Ebola virus and VLP release using an immunocapture
assay. J. Virol. Methods 127, 1–9 (2005).
55. Shoemaker, C. J. et al. Multiple cationic amphiphiles induce a Niemann-Pick C
phenotype and inhibit Ebola virus entry and infection. PloS One 8, e56265 (2013).
56. Moller-Tank, S. & Maury, W. Ebola virus entry: a curious and complex series of events.
PLoS Pathog. 11, e1004731 (2015).
57. Owen, D. & Katz, D. A Review of the Physical and Chemical Properties of Human Semen
and the Formulation of a Semen Simulant. J Androl 26, 459–469 (2005).
69 58. Serpell, L. C., Berriman, J., Jakes, R., Goedert, M. & Crowther, R. A. Fiber diffraction of
synthetic alpha-synuclein filaments shows amyloid-like cross-beta conformation. Proc. Natl.
Acad. Sci. U. S. A. 97, 4897–4902 (2000).
59. Cooper, T. K. et al. Histology, immunohistochemistry, and in situ hybridization reveal
overlooked Ebola virus target tissues in the Ebola virus disease guinea pig model. Sci. Rep. 8,
1250 (2018).
60. Robinson, C. M., Jesudhasan, P. R. & Pfeiffer, J. K. Bacterial Lipopolysaccharide Binding
Enhances Virion Stability and Promotes Environmental Fitness of an Enteric Virus. Cell Host
Microbe 15, 36–46 (2014).
61. Roan, N. R. et al. Semen amyloids participate in spermatozoa selection and clearance.
eLife 6, (2017).
70 CHAPTER 4: DISCUSSION AND FUTURE DIRECTIONS
Section 4.1 – Summary
Sexual transmission of persistent EBOV during the unprecedented 2014 Ebola epidemic represents an avenue by which outbreak control can be compromised. Despite the major public health problem that this presents, very little is known about the mechanisms involved in EBOV sexual transmission. This work is the first to study the role of host factors involved in EBOV sexual transmission at a molecular level.
Seminal amyloids, typified by SEVI, have been shown to greatly enhance the ability of
HIV-1 and other viruses to infect cells in vitro. This work demonstrates that SEVI and other seminal amyloids are able to greatly enhance infection not only by a recombinant virus vector bearing the
EBOV glycoprotein, but also by authentic EBOV in experiments conducted under Biosafety Level
4 conditions. This enhancement occurs in multiple cell types, including epithelium-derived cell lines, a monocyte-derived cell line, and primary monocyte-derived macrophages.
At a molecular level, SEVI appears to enhance infection by rVSV-EboGP-mCherry by enhancement of entry. Similar to effects on other viruses including HIV-1, SEVI induces a modest increase in binding of the virus to the cell. However, this work shows a novel function of SEVI to greatly increase internalization of the virus into the cell. SEVI accomplishes this function by enhancing macropinocytosis in a dose-dependent manner. After the virus is internalized by this critical pathway, it appears to follow the typical EBOV entry process and maintains requirements for cathepsin processing of its glycoprotein.
This work demonstrates further novel functions for SEVI in viral stability. As an enveloped virus, EBOV (and the recombinant VSV) are sensitive to prolonged incubations at physiologic temperature as well as desiccation. Incubation of virus with SEVI instills additional stability in both situations, suggesting that seminal amyloid fibrils may increase viral infectivity under adverse environmental conditions.
The mechanistic details for SEVI-mediated enhancement of infection and stability were also explored. Much like the results observed for HIV-1 enhancement, enhancement of rVSV-
71 EboGP-mCherry and EBOV infection required that SEVI maintain its positive charge as well as its amyloid character. Uncharged and soluble forms of SEVI were unable to increase macropinocytic uptake in cells. Similarly, only SEVI fibrils were able to enhance stability at physiologic temperature, with uncharged and unpolymerized forms showing no benefit over virus alone. In contrast, fibrils with positive residues replaced by alanine were able to instill protection from desiccation to the same degree as wild type SEVI fibrils, while soluble peptide had no effect.
Altogether, these findings indicate that amyloid fibrils are potentially able to enhance EBOV by two distinct mechanisms. At a molecular level, SEVI enhances infection by increasing binding and internalization. In an environmental context, SEVI enhances the ability of the virus to withstand environmental stresses, which may permit increased transmissibility.
72 Section 4.2 – Future directions
The identification of seminal amyloids as a pro-viral component of semen presents numerous avenues for future research. While this work has given insight into some mechanistic details regarding the SEVI-EBOV interaction, further study would elucidate a number of factors that remain unclear.
Section 4.2.1 – Ex vivo infections
The first and perhaps most straightforward next step would be to determine if amyloid fibrils endogenously present in semen enhance infection and stability of rVSV-EboGP-mCherry.
Protocols for infection as well as for removing seminal amyloids from seminal plasma exist1,2. Thus, the ability of semen to enhance infection and stability can be compared to semen devoid of amyloid fibrils. Collaborating with the Penn Medicine Division of Reproductive Endocrinology and Infertility for seminal samples, preliminary results suggest that semen does in fact enhance rVSV-EboGP- mCherry infection. To further explore the enhancement effect in a model that may better reflect
EBOV sexual transmission, vaginal explant models can be infected with either rVSV-EboGP- mCherry or EBOV in the presence and absence of SEVI. The microenvironment in tissue may be significantly different from that in a culture plate, especially given the diversity of cells in tissue compared to clonal cell lines. These studies may give insight into the ability of seminal amyloids to enhance infection of a specific subset of cells, which would perhaps mirror the results of experimental animal infections that showed early and sustained infection of cells including DCs and macrophages. Alternatively, the enhanced infection seen in epithelium-derived cell lines lends credence to a hypothesis that the virus may be able to replicate in vaginal epithelial cells that form a barrier, establish local infection, then spread to other cell types. Notably, vaginal epithelial cells are infected in an acute EBOV model in guinea pigs3. No experiments have been done with explant models and no animal EBOV sexual transmission models exist, leaving the expected results for these experiments up for conjecture.
73 Section 4.2.2 – Enhancement in the context of other viruses
While infection by numerous sexually transmitted viruses is enhanced by SEVI, it does not appear to be universal. Zika virus (ZIKV), which was responsible for a pandemic across the
Americas marked by an uptick in birth defects including microcephaly, has been demonstrated to be transmitted sexually in addition to its much more common mosquito transmission route. ZIKV
MR766 (Figure 4.1) and ZIKV Mex 2-81 (data not shown) infection do not appear to be enhanced by SEVI nearly to the same degree as rVSV-EboGP-mCherry, EBOV, or HIV-1. Because the mechanism of SEVI-mediated enhancement is hypothesized to be glycoprotein-independent, this result would seem to be contrary to current models. However, cryo-electron microscopy has been used to generate models of ZIKV virions that show very little plasma membrane area not occluded by the E glycoprotein4. Other viruses (including EBOV) have a lower density of glycoprotein on the virion surface; HIV-1 in particular is notable for its very low density of glycoprotein, with as few as
7-14 spikes being present per virion5–7. Thus, enhancement may not occur with ZIKV due to the inability of amyloid fibrils to effectively contact the virion membrane. It is unclear, and would be worth testing, whether ZIKV would exhibit increased stability and desiccation tolerance as does rVSV-EboGP-mCherry since infection is not enhanced. The ability of SEVI-Ala to promote desiccation tolerance (Fig 3.3B) suggests that an electrostatic interaction may not be required. It would also be interesting to assess whether SEVI enhances stability of other viruses whose infection is enhanced by the fibrils, especially HIV-1. Comparing the Figure 4.1. SEVI does not dramatically increase ZIKV effects on stability may give clues infection.18 ZIKV was incubated with SEVI at different concentrations for to the mechanism of action of the 15 minutes before infection of A549 cells. Infection was quantified by immunostaining and flow cytometry. n=1. fibrils for stability promotion.
74 Section 4.2.3 – Stabilization against other environmental factors
The ability of SEVI to promote resistance to some forms of environmental stress raises the possibility that it may enhance resistance to others. Semen contains proteases and antibodies that may inhibit viral infection. It is possible that amyloids could bind to viral particles and physically occlude proteases and antibodies from respectively cleaving or neutralizing the virus. To test the hypothesis that SEVI could protect virus from proteolysis, rVSV-EboGP-mCherry was treated with tryspin or proteinase K for 30 minutes at 37°C with the intention of determining whether SEVI would reduce the sensitivity of the virus to proteolytic inactivation. However, under the conditions tested, no loss in infectivity was observed after protease treatment. It is unclear whether EboGP is sensitive to digestion by these enzymes. To more directly test the hypothesis, protocols assessing seminal protease activity could be adapted for use in a viral setting8.
SEVI has been previously described to reduce the sensitivity of cytomegalovirus (CMV) to neutralizing antibodies, though extensive characterization of the effect was not completed9. To assess whether SEVI impacts neutralization sensitivity of EboGP, rVSV-EboGP-mCherry was incubated with two different neutralizing anti-EboGP antibodies with or without SEVI for 1 h at 37°C then used to infect cells. Normalized infection values were used to calculate IC50 values in the presence and absence of SEVI (Figure 4.2). There was no difference between IC50 values in the presence or absence of SEVI, suggesting no protective effect. It is unclear what difference there is between the CMV and rVSV-EboGP-mCherry results except that the EboGP neutralization capacity was tested at numerous concentrations, while the CMV data were a single point and may not have been representative. It is also possible that the CMV glycoprotein targeted (gH) interacts with SEVI in some way that renders it less sensitive to neutralization. Studies testing a wider array of anti-gH antibodies targeting different domains of the glycoprotein could help to clarify the ability of SEVI to enhance neutralization sensitivity. Further, both anti-EboGP antibodies targeted the base region of the glycoprotein, and a wider panel of antibodies could assess specific changes in neutralization sensitivity at other sites.
75 Figure 4.2. SEVI does not reduce neutralization sensitivity to anti-EboGP antibodies.19 rVSV-EboGP-mCherry was pretreated or not with SEVI and incubated with different concentrations of anti-EboGP neutralizing antibody for 30 min at 37°C, then used to infect Vero cells. Infection was quantified by flow cytometry. n=1.
The finding that both SEVI and SEVI-Ala enhance desiccation tolerance was unexpected.
This finding supports a hypothesis that this stabilization effect occurs through a different mechanism than enhancement of infection or stability at physiological temperature. It may be that the large fibrillar structure is able to retain water, creating a local microenvironment in which the virion is protected from desiccation. The exact requirements for this effect remain unclear, as incubation with the amyloid α-synuclein had no effect on desiccation tolerance. To better define the mechanism, future studies could test desiccation tolerance in the presence of a panel of amyloid fiibrils to determine if this effect is specific to SEVI (and related peptides containing the same amyloidogenic domains, like SEVI-Ala). Specifically, the other seminal amyloids as well as their uncharged variants could be used to better understand this phenomenon. Lastly, several species of bacteria including E. coli have been identified to form extracellular amyloid fibrils using curli proteins10. Given that EVD is primarily a diarrheal disease, it would be highly clinically relevant if amyloid secretions by gut bacteria enhance either infection or environmental stability of EBOV.
This could be tested by incubating rVSV-EboGP-mCherry with purified curli then testing for
76 enhancement of infection and of stability enhancement. The finding that microbiota could play a role in EBOV transmission would be novel.
Section 4.2.4 – Mechanisms of enhancement at the cellular and molecular level
It is unclear how SEVI-mediated enhancement interacts with cellular attachment factors.
Expression of attachment factors including DC-SIGN on the surface of cells increases their susceptibility to infection by EBOV and EboGP-expressing particles. It is unknown how enhancement of binding and especially internalization by SEVI is modulated by the presence of these attachment factors. It is possible that seminal amyloids and cellular attachment factors could synergize to result in an even greater increase in infection, or the presence of seminal amyloids could physically occlude binding sites on the virion to antagonize the effects of attachment factors.
To assess these possibilities, HEK-293 cells that have a DC-SIGN gene under expression of a doxycycline-inducible promoter were infected in the presence or absence of physiologic levels of
SEVI, in the presence or absence of doxycycline. No additional increases in rVSV-EboGP-mCherry infection were seen when SEVI was added to infection of cells expressing DC-SIGN by fluorescence microscopy, and quantification was not performed. However, for this experiment relatively high concentrations of SEVI (35 μg/mL) and doxycycline (2 μg/mL) were used, and each condition alone greatly increased infection. Each factor alone may enhance the entry process of rVSV-EboGP-mCherry to biologically maximal levels. To address this concern, future experiments are planned in which lower levels of SEVI (10 μg/mL) and doxycycline (0.2 μg/ml) are used in order to see any potential synergistic effects. These preliminary results do not support a model of antagonism between the fibrils and cellular attachment factors, at least as far as attachment factors that bind EboGP are concerned. PS-binding attachment factors may be more sensitive to occlusion of membrane phospholipids, and this can be tested in a similar fashion.
The enhancement of EBOV entry via increased macropinocytic uptake is an important new finding of this work, but the mechanism by which SEVI induces macropinocytosis is unknown. It is likely that the large, positively charged fibrils will engage multiple cell surface receptors in a
77 nonspecific manner to induce uptake. Macropinocytosis requires activation of Rho GTPases which regulate actin polymerization that forms membrane ruffles11,12. A number of kinases are also activated depending upon the upstream signal. Interestingly, while both antibody-dependent enhancement (ADE) of EBOV infection and antibody-independent infection require macropinocytosis as an entry mechanism, distinct intracellular signaling pathways are activated.
While inhibition of the non-receptor tyrosine kinases Src and Syk have little impact on infection by rVSV-EboGP pseudotypes, antibody-enhanced infection is blocked in their presence13. These results indicate that internalization signals downstream of the Fc receptor are distinct from those normally activated during rVSV-EboGP pseudotype entry. To determine whether a similar disparity exists during SEVI-enhanced rVSV-EboGP-mCherry infection, HeLa cells were pretreated for 1 h with the Src inhibitor PP2 then infected with rVSV-EboGP-mCherry. Qualitative observation by fluorescence microscopy showed no differences in infection between treated and untreated samples in the presence or absence of SEVI (data not shown). These data do not support the hypothesis that SEVI-enhanced infection specifically requires a Src-dependent uptake mechanism and thus is dissimilar to Fc-mediated uptake of rVSV-EboGP. Further candidates, however, could be tested in the same way to determine if there is differential activation of intracellular signaling molecules during SEVI enhancement.
It is notable that sexual transmission of EBOV had not been previously described before the 2014 outbreak. While it is possible that EBOV sexual transmission is simply a rare event that has not occurred before this outbreak, it is also possible that mutations that arose during the outbreak contributed to sexual transmission. As previously described, the A82V mutation in EboGP became prominent during the outbreak14. This mutation results in an increase in particle infectivity but detracts from glycoprotein stability during incubations at physiological temperatures15. Notably, published sequences of sexually transmitted EBOV contain the V82 allele, though correlations are difficult to draw due to the small number of sequences available16. This work describes a novel function of SEVI to enhance viral stability under those same conditions. It is possible that SEVI increases stability of the virion to mitigate this decrease in stability. This would result in viral stability
78 being perhaps unchanged while the particle is more infective during the transmission event. To test this hypothesis, the rVSV-EboGP-mCherry vector is being mutagenized to replace the ancestral
A82 allele with V82. After rescue of infectious virus by previously described mechanisms, the A82 and V82 viruses will be compared in their relative stabilities in the presence and absence of SEVI.
If the stability of the V82 virus increases disproportionately in the presence of SEVI, this would provide support to a hypothesis that this interaction may be important for sexual transmission.
Section 4.2.5 – Implications for antiviral development
The identification of a pro-viral component of semen presents an exciting opportunity for the development of antivirals that can target the interaction. In this manner, both the molecular enhancement mechanism as well as the enhancement of environmental stability could be ameliorated. There has been significant interest in developing compounds that disassemble amyloid fibrils, both in the context of SEVI and otherwise. Recently, it has been demonstrated that
CLR01, a molecule designed to mask the positive charge of amyloids but that was subsequently found to disrupt viral membranes, has activity against EBOV pseudoviruses17,18. It will be interesting to see the extent to which existing anti-amyloid compounds affect whether these compounds have effects on the environmental stabilization properties of SEVI in addition to its infection- enhancement activity. In particular, the finding that SEVI-Ala enhances desiccation tolerance may require development of broader-acting anti-amyloid agents. While SEVI-Ala is not naturally occurring and thus not per se a concern, its activity raises the possibility that other amyloids or aggregates may demonstrate similar actions that should also be targeted for maximal impact.
79 Section 4.3 – Concluding remarks
In conclusion, this work identifies for the first time that EBOV infection can be enhanced by amyloid fibrils present in human semen. These fibrils act to enhance infection at a molecular level by increasing particle binding and internalization by increasing macropinocytosis in target cells.
Further, a novel function of SEVI to enhance stability in the environment is described, suggesting a potential role for these fibrils in transmission dynamics. This represents the first pro-viral host factor identified in the context of EBOV sexual transmission and may represent a viable target for therapeutic options moving forward.
80 Section 4.4 – Materials and methods for preliminary work
This section provides materials and methods information for experiments not described in Section
3.4.
ZIKV infection assays. A549 cells were plated at 1.5e4 cells/well one day prior to infection. ZIKV
MR766 or ZIKV Mex 2-81 (Susan Weiss, U. Pennsylvania) were diluted in DMEM-10 in the presence or absence of SEVI and used to infect cells at MOI 1 as calculated by plaque assay on
Vero cells. After 1 h, infection medium was replaced with fresh DMEM-10. After 16-20 h, cells were trypsinized and fixed in 2% paraformaldehyde for >20 min. Cells were permeablized in 0.1% saponin in FACS buffer (1% BSA in PBS) for 25 minutes, then incubated in FACS buffer with saponin with the “pan-flavivirus” 4G2 antibody for 1 h, then anti-mouse AF647-conjugated secondary antibody for 1 h. Infection was quantified by flow cytometry.
Protease treatment. rVSV-EboGP-mCherry was diluted in serum-free DMEM. TPCK trypsin or proteinase K were added to final concentrations of 50 μg/mL or 20 μg/mL, respectively. The virus was then incubated for 30 minutes at 37°C. Digestion was halted with DMEM-10 containing soybean trypsin inhibitor or DMEM-10 alone. Afterward, virus was titered by TCID50 on Vero cells.
Antibody neutralization. rVSV-EboGP-mCherry was incubated with SEVI, then added to serial dilutions of anti-EboGP antibodies (gift from Erica Ollmann-Saphire, Scripps Research Institute) for
1 h at 37°C. Mixtures were then used to infect Vero cells for 1 h. Afterward, infection medium was replaced with fresh DMEM-10 and the infection was permitted to proceed for 12 h total. Cells were trypsinized and analyzed by flow cytometry for percent infection, then relative percents infection were used to calculate IC50 values (GraphPad Prism).
DC-SIGN cells. 293 T-Rex DC-SIGN cells were maintained in DMEM-10 without doxycycline. To upregulate DC-SIGN, the cells were treated for 24 h prior to infection with 2 μg/mL doxycycline.
Inhibitor treatments. HeLa cells were treated with the Src inhibitor PP2 (10 μM) for 1 h prior to and
1 h during infection with rVSV-EboGP-mCherry in the presence or absence of SEVI.
81 Section 4.4 – Bibliography
1. Münch, J. et al. Semen-derived amyloid fibrils drastically enhance HIV infection. Cell 131,
1059–1071 (2007).
2. Kim, K.-A. et al. Semen-mediated enhancement of HIV infection is donor-dependent and
correlates with the levels of SEVI. Retrovirology 7, 1–12 (2010).
3. Cooper, T. K. et al. Histology, immunohistochemistry, and in situ hybridization reveal
overlooked Ebola virus target tissues in the Ebola virus disease guinea pig model. Sci. Rep. 8,
1250 (2018).
4. Sirohi, D. et al. The 3.8 Å resolution cryo-EM structure of Zika virus. Science 352, 467–
470 (2016).
5. Beniac, D. R. & Booth, T. F. Structure of the Ebola virus glycoprotein spike within the
virion envelope at 11 Å resolution. Sci. Rep. 7, 46374 (2017).
6. Chertova, E. et al. Envelope glycoprotein incorporation, not shedding of surface envelope
glycoprotein (gp120/SU), Is the primary determinant of SU content of purified human
immunodeficiency virus type 1 and simian immunodeficiency virus. J. Virol. 76, 5315–5325
(2002).
7. Zhu, P. et al. Electron tomography analysis of envelope glycoprotein trimers on HIV and
simian immunodeficiency virus virions. Proc. Natl. Acad. Sci. U. S. A. 100, 15812–15817
(2003).
8. Olsen, J. S. et al. Seminal plasma accelerates semen-derived enhancer of viral infection
(SEVI) fibril formation by the prostatic acid phosphatase (PAP248-286) peptide. J. Biol. Chem.
287, 11842–11849 (2012).
9. Tang, Q., Roan, N. R. & Yamamura, Y. Seminal plasma and semen amyloids enhance
cytomegalovirus infection in cell culture. J. Virol. 87, 12583–12591 (2013).
10. Barnhart, M. M. & Chapman, M. R. Curli biogenesis and function. Annu. Rev.
Microbiol. 60, 131–147 (2006).
82 11. Mercer, J. & Helenius, A. Vaccinia Virus Uses Macropinocytosis and Apoptotic Mimicry to
Enter Host Cells. 320, 531–535 (2008).
12. Ridley, A. J., Paterson, H. F., Johnston, C. L., Diekmann, D. & Hall, A. The small GTP-
binding protein rac regulates growth factor-induced membrane ruffling. Cell 70, 401–410
(1992).
13. Furuyama, W. et al. Fcγ-receptor IIa-mediated Src Signaling Pathway Is Essential for the
Antibody-Dependent Enhancement of Ebola Virus Infection. PLoS Pathog. 12, e1006139
(2016).
14. Diehl, W. E. et al. Ebola Virus Glycoprotein with Increased Infectivity Dominated the
2013–2016 Epidemic. Cell 167, 1088–1098.e6 (2016).
15. Wang, M. K., Lim, S.-Y., Lee, S. M. & Cunningham, J. M. Biochemical Basis for
Increased Activity of Ebola Glycoprotein in the 2013-16 Epidemic. Cell Host Microbe 21, 367–
375 (2017).
16. Mate, S. E. et al. Molecular Evidence of Sexual Transmission of Ebola Virus. N. Engl. J.
Med. 373, 2448–2454 (2015).
17. Lump, E. et al. A molecular tweezer antagonizes seminal amyloids and HIV infection.
eLife 4, (2015).
18. Röcker, A. E. et al. The molecular tweezer CLR01 inhibits Ebola and Zika virus infection.
Antiviral Res. 152, 26–35 (2018).
83 APPENDIX – IDENTIFICATION OF HOST FACTORS THAT RESTRICT ZIKA VIRUS
INFECTION
Stephen M. Bart1, Kangjian Zhang2, Angelíca Ortiz2, Serge Y. Fuchs2, Paul Bates1
1Department of Microbiology, Perelman School of Medicine, University of Pennsylvania
2Department of Biomedical Sciences, School of Veterinary Medicine, University of Pennsylvania
In addition to identification of seminal amyloids as a host factor that enhanced EBOV infection, I have conducted work to identify factors that restrict viral infection. Specifically, I have developed a system in which factors that promote cell survival after viral challenge can be enriched from a genome-wide pool. This ongoing work seeks to identify factors that restrict Zika virus replication. K. Zhang and A. Ortiz in the Fuchs lab assisted in the generation of IFNAR1 KO cells using reagents developed and produced by me. I conceived and conducted the remainder of the experiments and wrote this appendix under the supervision of Paul Bates.
84 Section 1.1 – Introduction
Zika virus (ZIKV) was discovered in Uganda in 1947 and identified as a member of the
Flaviviridae family. The virus, which was traditionally restricted to Africa and Asia, emerged in several Pacific islands before spreading to the Americas during the 2015-2016 Zika epidemic. The epidemic was marked by a sharp uptick in the number of cases of microcephaly, other birth defects, and Guillain-Barré syndrome, prompting the World Health Organization to declare a Public Health
Emergency of International Concern1–5.
The interactions of ZIKV with host cells are poorly characterized. Several loss-of-function screens have been performed to identify factors required for ZIKV replication6–8. Several factors that inhibit ZIKV replication have also been described; these mainly focus upon the interferon response. ZIKV replication is antagonized by the action of types I and III interferon9,10, which induce a signaling cascade leading to the expression of numerous interferon-stimulated genes (ISGs).
These genes, among other functions, encode factors with antiviral activity to inhibit viral replication.
Screens employing cDNAs of various ISGs have been used to identify which factors out of the hundreds of ISGs act upon specific viruses to limit their replication. The breadth of factors that affect ZIKV replication is unknown, but IFITM1, IFITM3 and viperin have each been described to inhibit ZIKV replication11–13. It is further probable that there are cellular factors that are not necessarily interferon-stimulated that also have antiviral activity. The zinc metallopeptidase
ZMPSTE24 has recently been described to have anti-ZIKV activity in a mechanism involving
IFITMs14. A systematic exploration of factors involved in ZIKV restriction, whether interferon- dependent or not, has not been conducted.
Recently, CRISPR/Cas9 technology has been adapted to allow for targeting gene activation. One system, synergistic activation mediators (SAM), uses transcriptional coactivators derived from NF-κB (p65), HSF1, and VP64 along with a modified guide RNA in order to target an activating Cas9 to a specific locus and upregulate gene expression. When a pool of cells is transduced with a library of guide RNAs then challenged with some selective pressure, factors that promote cellular tolerance to that stress are enriched. This library targets 23,430 genes in triplicate
85 for a total of 70,290 guide RNAs. These factors can be identified by comparing the abundance of each guide RNA in the selected population against unselected cells15. This technology has identified that sialic acid-modifying enzymes have anti-influenza activity by reducing receptor binding16.
This report describes the generation and characterization of cells to perform a SAM screen to identify factors with antiviral activity against ZIKV.
Section 1.2 – Materials and Methods
Cells and viruses. A549 cells (ATCC) were maintained in F12K media supplemented with
10% FBS (Sigma), 293T (ATCC) and Vero CCL81 cells (gift from Susan Weiss, U. Pennsylvania) were maintained in DMEM with 4.5 g/L glucose and no sodium pyruvate supplemented with 10%
FBS (Sigma). ZIKV MR766 (gift from Susan Weiss, U. Pennsylvania) and ZIKV Mex 2-81 (gift from
Scott Hensley, U. Pennsylvania) were propogated in Vero cells. Newcastle disease virus-GFP was a gift from Susan Weiss, U. Pennsylvania.
Plasmids. plentiCRISPR (AddGene) was modified to contain a loxP site within the 5’ long terminal repeat (lentiCRISPRloxP) by restriction enzyme cloning and insertion of a gBlock (IDT).
Components of the SAM system (lentiviral plasmids encoding dCas9-VP64, MS2-p65-HSF1, guide
RNA library) were acquired from AddGene and the SAM library was propagated according to
AddGene instructions. The gRNA2.0 plasmid was also modified to express GFP in place of the drug resistance marker.
Lentivirus production and transduction. Lentiviral pseudotypes were generated by transfecting 15 μg genome plasmid, 10.5 μg pSPAX2, and 3.75 μg VSV G with polyethylenimine into a 15 cm plate of 293T cells. Supernatants were harvested 24-48 h post-transfection, clarified by centrifugation, and frozen at -80°C.
qPCR. DNA was extracted from half of a 24-well plate well according to manufacturer’s protocols (Qiagen Blood and Tissue Kit). 5 μL of DNA was used per reaction along with 2 μL of 5
μM combined F and R plasmids (Cas9, puromycin, or GAPDH), 3 μL water, and 10 μL SYBER 2X master mix. After qPCR, data were analyzed by ddCt method.
86 Flow cytometry. Cells were trypsinized, fixed in 2% PFA, then permeablized for >20 minutes with saponin. Cells were incubated with 4G2 antibody (gift from Sara Cherry, U.
Pennsylvania) for 1 h in FACS buffer (1% BSA in PBS) with saponin (4G2 1:4000) then incubated with AF647-anti-mouse secondary antibody (1:5000) for 1 h. After resuspending cells in FACS buffer without saponin, cells were ran on LSRII and data analyzed with FlowJo.
Section 1.3 – Results
A549 cells were selected for the screen because of the cells’ intact intrinsic immune system as well as the ability of ZIKV to induce cytopathic effect (data not shown). To reduce interferon signaling, which may result in nonspecific survival of cells and increase screen background, a modified lentiCRISPR construct was first used to knock out the IFNAR1 gene, a component of the type I interferon receptor. To avoid downstream “crosstalk” between the nucleolytic Cas9 used for knockout and the catalytically inactive Cas9 used in SAM, the lentiCRISPR vector was modified such that the lentiviral genome contains loxP sites within the LTRs (Fig A.1A). After treatment of cells with Cre recombinase, the genome (including Cas9) is excised, leaving only a single LTR element integrated into the genome. A549 cells were transduced with lentiCRISPRloxP containing a guide against IFNAR1, then single cell cloned. Clones were screened for knockout by treatment with interferon-α and γ; a putative IFNAR1 KO clone (1-4) exhibited phosphorylation of STAT1 in response to IFN- γ but not α (Fig A.1B). Inactivation of IFNAR1 was further confirmed by cell- surface staining for IFNAR and analyzing cells by flow cytometry (data not shown) and genomic sequencing of the IFNAR1 gene (Fig A.1C).
IFNAR1 KO clones also exhibited a functional inability to respond to exogenous IFN-α.
Cells were pretreated for 24 h with 500 U/mL IFN-α then infected with Newcastle disease virus that expresses GFP (NDV-GFP). This virus is exquisitely sensitive to IFN and is used as a biosensor for IFN activity17. As opposed to wild type cells that become resistant to NDV-GFP infection after
IFN treatment, knockout cells show no difference in infectivity (Fig A.1D). Lastly, knockout cells were infected with ZIKV MR766 at a multiplicity of infection (MOI) of 0.1 for 3 days. At that point, the supernatants were harvested, cleared of cell debris by centrifugation, and UV-treated to
87 inactivate virus. This supernatant was applied to WT or KO cells for 24 h prior to infection with ZIKV
MR766. While the supernatant led to a decrease in infection in WT cells, no difference was seen in infection among treated or untreated KO cells (Fig A.1E). This indicates that the cytokines produced during ZIKV infection of IFNAR KO A549 cells do not induce an antiviral state in other
IFNAR KO cells, a key requirement for screening.
IFNAR KO cells were then transduced with lentiviral vectors containing the dCas9-VP64 and MS2-p65-HSF1 expression vectors as well as Ad-Cre to remove the integrated provirus genome containing Cas9. After selection with blasticidin and hygromycin, cells were single cell cloned. Individual clones were then harvested for genomic DNA that was subsequently subjected to qPCR to identify clones negative for the integrated provirus, as marked by the puromycin resistance gene. While the parent IFNAR KO cells exhibited high Cas9 and puromycin signal compared to the GAPDH control, several clones showed ablation of the puromycin signal (Fig
A.2A). These puromycin-negative clones were then screened for the clone that had the highest activity for upregulation of target genes. Cells were transduced with a lentiviral vector containing a sgRNA for CD4 as well as a GFP marker and analyzed by flow cytometry. CD4 was chosen because of the abundance of reagents to detect the molecule as well as its lack of expression on
A549 cells. Cells exhibited a range of expression profiles, including no upregulation, upregulation in a subset of cells, or full upregulation (Fig A.2B). Two clones exhibited full upregulation, and these cells were independently transduced with three different guides targeting IRF1, an antiviral gene with wide activity, again with a GFP marker. ZIKV infection (assessed by immunostaining for ZIKV
E) was compared among GFP- (untransduced) cells as well as GFP+ (IRF1 guide transduced cells). Clone 1.9 was found to have lower relative levels of infection among GFP+ cells in 2/3 guides compared to clone 1.6, and thus was selected to proceed for screening (Fig A.2C).
Cells of the Clone 1.9 lineage were renamed SAM cells, were expanded, and transduced with the guide RNA library containing 70,290 guides at an MOI of 0.2. Library was titered by counting number of cells following treatment with Zeocin following several rounds of replating at low densities. Cells were transduced at a level designed to give at least 500 transduced cells per
88 each guide. After 7 days to permit expansion and gene upregulation, cells were infected with ZIKV at MOI 1 or passaged in parallel without infection. After 4 days, the cells were washed vigorously and harvested for DNA to be used in sequencing (Fig A.3).
Section 1.4 – Bibliography
1. WHO | WHO Director-General summarizes the outcome of the Emergency Committee
regarding clusters of microcephaly and Guillain-Barré syndrome. WHO Available at:
http://www.who.int/mediacentre/news/statements/2016/emergency-committee-zika-
microcephaly/en/. (Accessed: 4th April 2018)
2. Cauchemez, S. et al. Association between Zika virus and microcephaly in French Polynesia,
2013-15: a retrospective study. Lancet Lond. Engl. 387, 2125–2132 (2016).
3. Schuler-Faccini, L. et al. Possible Association Between Zika Virus Infection and Microcephaly -
Brazil, 2015. MMWR Morb. Mortal. Wkly. Rep. 65, 59–62 (2016).
4. Dirlikov, E. et al. Guillain-Barré Syndrome During Ongoing Zika Virus Transmission - Puerto
Rico, January 1-July 31, 2016. MMWR Morb. Mortal. Wkly. Rep. 65, 910–914 (2016).
5. Cao-Lormeau, V. M. et al. Guillain-Barré Syndrome outbreak associated with Zika virus
infection in French Polynesia: a case-control study. Lancet Lond. Engl. 387, 1531–1539
(2016).
6. Zhang, R. et al. A CRISPR screen defines a signal peptide processing pathway required by
flaviviruses. Nature 535, 164–168 (2016).
7. Savidis, G. et al. Identification of Zika Virus and Dengue Virus Dependency Factors using
Functional Genomics. 16, 232–246 (2016).
8. Marceau, C. D. et al. Genetic dissection of Flaviviridae host factors through genome-scale
CRISPR screens. Nature 535, 159–163 (2016).
9. Bayer, A. et al. Type III Interferons Produced by Human Placental Trophoblasts Confer
Protection against Zika Virus Infection. Cell Host Microbe 19, 705–712 (2016).
10. Lazear, H. M. et al. A Mouse Model of Zika Virus Pathogenesis. Cell Host Microbe 19,
720–730 (2016).
89 11. Savidis, G. et al. The IFITMs Inhibit Zika Virus Replication. Cell Rep. 15, 2323–2330
(2016).
12. Van der Hoek, K. H. et al. Viperin is an important host restriction factor in control of Zika
virus infection. Sci. Rep. 7, 4475 (2017).
13. Panayiotou, C. et al. Viperin Restricts Zika Virus and Tick-Borne Encephalitis Virus
Replication by Targeting NS3 for Proteasomal Degradation. J. Virol. 92, (2018).
14. Fu, B., Wang, L., Li, S. & Dorf, M. E. ZMPSTE24 defends against influenza and other
pathogenic viruses. J. Exp. Med. 214, 919–929 (2017).
15. Konermann, S. et al. Genome-scale transcriptional activation by an engineered CRISPR-
Cas9 complex. Nature 517, 583–588 (2015).
16. Heaton, B. E. et al. A CRISPR Activation Screen Identifies a Pan-avian Influenza Virus
Inhibitory Host Factor. Cell Rep. 20, 1503–1512 (2017).
17. Park, M.-S. et al. Newcastle disease virus (NDV)-based assay demonstrates interferon-
antagonist activity for the NDV V protein and the Nipah virus V, W, and C proteins. J. Virol. 77,
1501–1511 (2003).
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D. absence of conditioned, UV conditioned, of absence byre selection. puromycin after clones integrated. copy repeat terminal long single a only leaving excised is provirus the A. A.1 Figure
Relative NDV
Schematic of integrated provirus construct within A549 IFNAR KO cells. Upon expression of Cre recombinase, recombinase, Cre of expression Upon cells. KO IFNAR A549 within construct provirus integrated of Schematic
peated measures ANOVA log of measures peated
.
Generation
-
GFPinfection in the presence or absence of interferon
of IFNAR1 knockout cells. IFNAR1 knockout of
-
inactivated supernatant. n=2; n=2; mean±SD. inactivatedsupernatant.
C. C.
Sequencing from the IFNAR1 locus in parental cells or cloned CRISPR cells. CRISPR cloned or cells parental in locus IFNAR1 the from Sequencing
-
transformed percents infection. percents transformed
20
E.
-
α pretreatment. α n=3;pretreatment. mean±SEM, *p<0.05
Relative ZIKV infection in the presence or the ZIKV in presence infection Relative
B.
Western blot used to screen screen to used blot Western
91
sequence (quantified by qPCR) after selection with ZIKV MR766 for 3 days. days. 3 for MR766 ZIKV with selection after qPCR) by (quantified sequence infection (quantified by flow cytometry) of n=1. SAM transduced; clones guide transduced with CD4 are dCas9 IRF1 thus guides; and n=1. GFP express SAM that cells indicate peaks Blue or guide. CD4 the have gene Cas9 lentiCRISPRloxP either from Red lentisgRNA_CD4_GFP. with transduction after clones SAM of plots flow Representative be can signal Cas9 cassette; cells. lentiCRISPRloxP parental KO IFNAR clonal the to normalized and GAPDH A. A.2. Figure
qPCR qPCR screening of clones after transduction with SAM components and Ad
Generation of SAM cells for screening. screening. for cells SAM of Generation
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Lack of puromycin signal indicates successful excision of of excision successful indicates signal puromycin of Lack
-
Cre. Cre. Puro and Cas9 signal is relative to genomic
D. D.
Relative Relative
peaks are GFP are peaks
enrichment enrichment for lentisgRNA
-
C. C.
-
cells that do not not do that cells
VP64 gene. gene. VP64
Relative ZIKV ZIKV Relative B. B.
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Figure
A.3.
Schematic for screening A549 SAM cells. SAM A549 for screening Schematic
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