CHAPTER0 4 © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC NOT Forvirus SALE OR Distributionreplication Cyclesnot for SALE OR DISTRIBUTION

CHAPTER0 4 © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC NOT FORVirus SALE OR DISTRIBUTIONReplication CyclesNOT FOR SALE OR DISTRIBUTION

© Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC Some human and animal virusesNOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION are grown in tissue culture cells in vitro. This researcher is seeding dishes of tissue culture cell monolayers.© Jones The monolayers & Bartlett are Learning, LLC © Jones & Bartlett Learning, LLC grownNOT in a CO2 FOR incubator SALE before OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION the cells are infected with a laboratory strain of virus. Viruses that can be grown in cell cultures © Jones &can Bartlett be well characterized Learning, in the LLC © Jones & Bartlett Learning, LLC NOT FORresearch SALE laboratory. OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION

o u t l i n e

© Jones & BartlettIn the struggleLearning, LLC4.1 One-Step Growth© Curves Jones & Bartlett4.3 Learning, The Error-Prone LLC RNA “ Polymerases: Genetic Diversity NOT FORfor SALE survival, OR DISTRIBUTION the 4.2 Key Steps of theNOT Viral FOR SALE OR DISTRIBUTION Replication Cycle 4.4 Targets for Antiviral Therapies fittest win out at 1. Attachment (Adsorption) RNA Virus Mutagens: A New Class the expense of 2. Penetration (Entry) of Antiviral Drugs? their rivals © Jones & Bartlett 3. Uncoating Learning, (Disassembly LLC Virus© Fil Jonese 4-1: How& Bartlett Are Cellular Learning, LLC NOT FOR SALEand OR Localization) DISTRIBUTION ReceptorsNOT Used FOR for ViralSALE OR DISTRIBUTION because they Attachment Discovered? 4. Types of Viral Genomes and succeed in adapt- Their Replication Virus File 4-2: Unraveling the Life Cycle of Mimivirus ing themselves 5. Assembly © Jones & Bartlett Learning, LLC © Jones & Vi rusBartlett File 4-3 Learning,: Stamping DownLLC 6. Maturation Flu Viruses bestNOT to theirFOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION 7. Release Refresher: Molecular Biology environment.” Charles Darwin © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION

67 2ND REVISE © Jones & Bartlett Learning, LLC. NOT FOR SALE OR DISTRIBUTION. CASE STUDY: HUMAN METAPNEUMOVIRUS

© Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION The campus day care was recently closed during the samples are sent to diagnostic laboratories, and a peak of the winter flu season because many of the lesser percentage in adults. young children were sick with a lower respiratory Both hMPV and RSV cause upper and lower tract infection. An email announcement was sent respiratory tract infections associated with serious to all students,© Jones faculty, &and Bartlett staff at the Learning, college that LLC illness in the young, immunosuppressed,© Jones & Bartlett elderly, Learning, LLC stated the closureNOT wasFOR due SALE to a metapneumovirus OR DISTRIBUTION and chronically ill. CommonNOT FOR symptoms SALE includeOR DISTRIBUTION outbreak. The announcement briefed the campus cough, fever, wheezing or exacerbation of asthma, community with information about human meta- and rhinorrhea (runny nose). It can produce se- pneumonoviruses (hMPVs). vere enough symptoms to cause intensive care The announcement stated that hMPV was a admission and ventilator support. Healthy adults © Jonesnewly & identified Bartlett respiratory Learning, tract LLC pathogen discov- can get© aJones mild form & Bartlettof the disease Learning, that is character- LLC NOTered FOR in theSALE Netherlands OR DISTRIBUTION in 2001. New tests confirm ized byNOT a cough, FOR hoarseness,SALE OR congestion,DISTRIBUTION runny that it is one of the most significant and common nose, and sore throat. viral infections in humans. It is clinically indistin- Members of the campus day care staff were guishable from a viral relative known as respiratory doing their best to limit the epidemic spread of the syncytial virus (RSV). Both RSV and hMPV infec- hMPV outbreak at the center. Primary care physi- © Jones & Bartletttions Learning, occur during LLC the winter. hMPV may account© Jones cians& Bartlett need up-to-date Learning, knowledge LLC and heightened NOT FOR SALE ORfor 2% DISTRIBUTION to 12% or more of previously unexplainedNOT FORawareness SALE OR to recognizeDISTRIBUTION this new viral disease in pediatric lower respiratory infections for which patients.

n Chapter© Jones2, you learned & Bartlett that viruses Learning, are de- LLC animals were difficult© to Jones work with & Bartlettand expensive Learning, LLC pendentNOT upon hostFOR cells SALE for their OR reproduction, DISTRIBUTION to maintain. AnotherNOT drawback FOR was SALE that animals OR DISTRIBUTION Iyet to do so viruses must overcome certain cel- were not very permissive to infection with human lular constraints. Only those viruses that have viruses due to the species barrier and the animal been able to adapt to their hosts have been able to immune response. exist in nature. The previous chapter focused on Enders, Weller, and Robbins won the 1954 © Jonestaxonomy & Bartlett and structure Learning, of viruses LLC and in sum- Nobel© Prize Jones in Physiology & Bartlett or Medicine Learning, for theLLC cul- NOTmary FOR stated SALE that OR several DISTRIBUTION different virus families tivationNOT of FORpoliovirus SALE in nonnervousOR DISTRIBUTION tissue cul- cause similar viral disease syndromes. tures (human embryonic skin and muscle cells). This chapter focuses on experiments such as Their observations and procedures used to grow one-step growth curves, which are used to viruses in vitro contributed to the refinement of study virus–host interactions. These studies have tissue culture techniques and played a monumental © Jones & Bartlettpro Learning,vided information LLC about the events that ©occur Jones role& Bartlett in the development Learning, of LLC vaccines against polio NOT FOR SALE ORat each DISTRIBUTION step of the infection cycle (attachmentNOT, FORvir SALEus in the OR 1950s DISTRIBUTION (Salk vaccine) and 1960s (Sabin penetration, uncoating, replication, assembly, vaccine). maturation, and release), including the intricate One-step or single-step growth curve ex- details of the strategies that animal and human periments are used to study a single replication viruses use to express and replicate their diverse cycle of viruses. Max Delbruck developed the one- genomes. © Jones & Bartlett Learning, LLC step growth curve experiment© Jones &while Bartlett using Learning, an LLC NOT FOR SALE OR DISTRIBUTIONEscherichia coli-T4 bacterialNOT system, FOR which SALE gave OR faster DISTRIBUTION experimental results than did traditional methods. 4.1 One-Step Growth With the advent of cell culture systems, these experiments were carried out with viruses that infect Curves tissue culture cells. These experiments are per- © Jones & Bartlett Learning, LLC formed© Jonesin special & cell Bartlett culture facilities.Learning, LLC NOTVirologists FOR SALE could OR not DISTRIBUTION study animal and human Briefly,NOT FOR monolayers SALE OR(or cellDISTRIBUTION suspensions in viruses well in the laboratory before tissue cul- liquid medium) of tissue culture cells such as mon- ture methods were developed by John F. Enders, key kidney cells are allowed to adhere and form Thomas H. Weller, and Frederic C. Robbins in the monolayers on the bottom of plastic dishes. The late 1940s. Before their work, viruses were injected monolayers of cells are subsequently infected with © Jones & Bartlettinto Learning, animals and LLC tissues were analyzed for© theJones the& Bartlett virus of choice.Learning, They LLCare infected at a high NOT FOR SALE ORpathological DISTRIBUTION signs of viral infection. ExperimentalNOT FORmultiplicity SALE OR ofDISTRIBUTION infection (MOI) to ensure that

68 Chapter 4 Virus Replication Cycles 2ND REVISE 2ND REVISE © Jones & Bartlett Learning, LLC. NOT FOR SALE OR DISTRIBUTION. General Procedure: One-Step Growth Curves Figure 4-1 The diagram briefly outlines the steps involved © Jones & Bartlett Learning, LLC © Jones & inBartlett performing Learning,one-step growth LLCexperiments. Step 5 includes NOT FOR SALE OR DISTRIBUTIONStep 1: NOT FOR SALEa photograph OR of DISTRIBUTION viral plaques (clearings where the virus Infect monolayers of tissue destroyed the cell monolayer). The plaque assay is a culture cells (using a vertical quantitative assay used to determine the number of viruses laminar flow biosafety hood) present in a given sample. The results of these assays can be and allow the infection to used to generate a one-step growth curve for a particular virus. proceed in a CO2 incubator. For more details about virological methods see Chapter 5, Laboratory Diagnosis of Viral Diseases and Working with © Jones & Bartlett Learning, LLC Viruses in the Research Laboratory.© Jones & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION Step 2: Monitor experiments via CO2 incubator. inverted microscope.

Step 3:© Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC Collect infected cell lysates at variousNOT time pointsFOR after SALE infection. OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION

Step 4: Perform serial dilutions on infected cell lysates and do plaque assays. © Jones & Bartlett Learning, LLC One-Step Growth Experiment© Jones & Bartlett Learning, LLC release NOT FOR SALE OR DISTRIBUTION maturationNOT FOR SALE OR DISTRIBUTION synthesis eclipse Step 5: Stain and analyze plaque assays. attachment extracellular virions Record results. intracellular virions © Jones & Bartlettproteins Learning, LLC © Jones & Bartlett Learning, LLC NOT FOR SALEnucleic acids OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION time

every cell of the monolayer is infected simultane- during the life cycle of a typical bacterium. The ously.© The Jones MOI is& the Bartlett average Learning, number of viruses/LLC viral attachment,© eclipse,Jones and & productiveBartlett Learning, (matura- LLC cell. NOTHence, FOR classic SALE one-step OR growth DISTRIBUTION experiments tion and release)NOT stages FOR will SALEbe discussed OR DISTRIBUTIONin detail usually use an MOI of 10 (10 viruses/cell). The as these key steps of viral replication are dissected infected cells are maintained in CO2 incubators in the following section. and monitored throughout the course of infection. At various times during the infection, infected cells © Jones &and/or Bartlett tissue Learning, culture fluid LLC are harvested and ©4.2 Jones &Key Bartlett Steps Learning, of the LLC Viral NOT FORplaque SALE assays OR areDISTRIBUTION performed. Plaque assays are NOT FOR SALE OR DISTRIBUTION used to quantitate the number of intracellular or Replication Cycle extracellular virus particles present during that point of infection. All viruses should be going 1. Attachment (Adsorption) through the same step in the viral replication cycle at the same time (Figure© Jones4-1). & Bartlett Learning,The LLCfirst step in the life cycle of a ©virus Jones is attachment. & Bartlett Learning, LLC From these experiments,NOT FOR virologists SALE have OR de- DISTRIBUTIONThe virus must be able to attachNOT to its FOR host SALE and OR DISTRIBUTION termined that there is a general pattern observed enter the “correct” or “target” cell. The attachment during the life cycle of a virus that distinguishes it event is electrostatic and does not require any from the life cycle of a bacterium. Shortly after the cellular energy. This step is a critical step in the infection, the input or inoculated virus disappears. viral replication cycle and a great target for anti- No virus© Jones particles & areBartlett detected Learning, at this time. LLC This is viral therapies ©developed Jones &to Bartlettprevent viral Learning, infec- LLC termedNOT the FOReclipse SALE period OR (Fig ureDISTRIBUTION 4-2b). This con- tions. If virus attachmentNOT FOR is blocked,SALE OR the infectionDISTRIBUTION tinues until progeny viruses are detectable (any- is prevented. A virus is said to exhibit a tropism where from one to several hours or even days for a particular cell type when it targets and in- depending on the virus), which is termed the pro- fects that cell type. In many cases these cell types ductive stage. Figure 4-2b illustrates that there is are a specific population of cells within organs. © Jones &a lag Bartlett phase in Learning, which few bacteria LLC are detected but ©Table Jones 4-1 lists & Bartlettexamples ofLearning, viruses and LLC their cellular NOT FORthere SALE is never OR aDISTRIBUTION disappearance of bacteria observed NOTtropism. FOR Sometimes SALE OR viruses DISTRIBUTION also display species

4.2 Key Steps of the Viral Replication Cycle 69 2ND REVISE 2ND REVISE © Jones & Bartlett Learning, LLC. NOT FOR SALE OR DISTRIBUTION. 1000 © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC Stationary phase NOT FOR SALE OR DISTRIBUTIONCell-associated NOT FOR SALE OR DISTRIBUTION 100 virus Yield

Death 10 Cell-free virus

NOT FOR SALE OR DISTRIBUTION units per cell Infectious NOT FOR SALE OR DISTRIBUTION 0.1

Log numbers (CFU/ml) 0 5 10 15 Lag phase Hours after addition of virus

Figure 4-2 Typical bacterial growth versus a one-step growth curve of a naked virus. (a) Bacterial growth generally proceeds in a series of phases: lag, log (exponential growth in which the rate of multiplication is most rapid and constant), stationary, and death. © Jones & BartlettVir usesLearning, require hos t LLCcells for growth and reproduction. CFU© Jones/ml = colony & for Bartlettming units perLearning, milliliter. Mod LLCified from an illustration by H. Douglas Goff, Ph.D., University of Guelph. (b) Viruses are assembled from preformed “parts” when enough of the preformed parts NOT FOR SALE ORhave beenDISTRIBUTION made. Adapted from White, D. E., and Fenner,NOT F. J. Medical FOR Virology, SALE Fourth OR Edition. DISTRIBUTION Academic Press, 1994.

tropism. For example, poliovirus infects only pri- tory tract). Routes of entry and mechanisms for mate cells. viral spread in the body are covered in Chapter 6. Host range© Jones is a term & Bartlett that refers Learning, to the differ- LLC In order to infect© cells, Jones the attachment& Bartlett pro- Learning, LLC ent types ofNOT tissue FOR culture SALE cells or OR organisms DISTRIBUTION (spe- teins located on the outsideNOT FOR of the SALE virus must OR beDISTRIBUTION cies) that the virus can infect. The host range may able to bind to cellular surface receptors. be broad (infecting many different animals or cell Cellular receptors are usually proteins, glycopro- lines of different species) or narrow. An example teins, carbohydrates, or lipids. Table 4-2 provides of a broad-range virus is rabies, for which all mam- examples of viruses and their cellular receptor(s). © Jonesmals have & Bartlett varying susceptibility.Learning, LLCHuman immuno- Viruses© haveJones evolved & Bartlett to use these Learning, receptors for LLCattach- NOTdeficiency FOR SALE virus OR (HIV), DISTRIBUTION which infects humans and ment NOTand entry FOR to theirSALE hosts. OR You DISTRIBUTION may ask, “Why monkeys but causes disease only in humans, falls haven’t cells evolved to keep up with the evolu- into the narrow range. Human viruses and animal tion of viruses?” The answer is that viruses have viruses also have preferred routes of entry (e.g., evolved to use essential components of the cells as influenza and rhinoviruses enter via the respira- receptors. Without these essential components, the © Jones & Bartlett Learning, LLC © Jones cell& Bartlett can’t exist. Learning, Cell surface receptorsLLC play important NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION Table 4-1 Viral Cell Tropism Table 4-2 Cell Surface Receptors Used by Virus(es) Cell Type HIV CD4+ T lymphocytes, macrophages Viruses to Attach and Enter Cells © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC Rabies Muscle, neurons NOT FOR SALE OR DISTRIBUTIONVirus Cell SurfaceNOT Receptor FOR SALE OR DISTRIBUTION Human papilloma Differentiating keratinocytes Influenza A Sialic acid Hepatitis A, B, C Liver (hepatocytes) HIV-1 CD4 and chemokine co-receptors (CXCR5, CCR4) Human herpes Mucoepithelium Hepatitis C Low-density lipoprotein receptor simplex 1 and 2 Rabies Acetylcholine receptor, neural cell adhesion Influenza A Respiratory epithelium © Jones & Bartlett Learning, LLC © Jonesmolecule, & Bartlett nerve growth Learning, factor, gangliosides, LLC NOT FORRotavirus SALE OR DISTRIBUTIONIntestinal epithelium NOT FORphospholipids SALE OR DISTRIBUTION Norovirus Intestinal epithelium Rhinovirus Intracellular adhesion molecule 1 (ICAM-1) Cytomegalovirus Epithelium, monocytes, lymphocytes Hepatitis B IgA receptor Rhinovirus Nasal epithelium Adenovirus Integrins avb3 and avb5 © Jones & BartlettPoliovirus Learning, LLCIntestinal epithelium © Jones &Type Bartlett 2 Learning, LLC NOT FOR SALE OREpstein-Barr DISTRIBUTION B cell NOT FOR PoliovirusSALE ORImmunoglobulin DISTRIBUTION superfamily protein (CD155)

70 Chapter 4 Virus Replication Cycles 2ND REVISE 2ND REVISE © Jones & Bartlett Learning, LLC. NOT FOR SALE OR DISTRIBUTION. VIRUS FILE 4-1 How Are Cellular Receptors Used for Viral Attachment Discovered? © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION

Scientists have developed several techniques to identify cell surface receptors and co-receptors to which viruses attach in order to initiate infection. These approaches may be viral receptor- interference© Jones studies & Bartlett or genetic techniques.Learning, Parts LLC (a)–(c) of Figure VF 4-1 illustrate© Jones the general & Bartlett Learning, LLC schemeNOT of FORthe various SALE methods OR employed. DISTRIBUTION NOT FOR SALE OR DISTRIBUTION

Sialic acid Figure VF 4-1 Identification of host cell receptors. (a) Removal of cell surface © Jones & Bartlett Learning, LLC © Jones & recBartletteptors. Ada Learning,pted from LLC Host cell Treat cells with Paulson, J. C., and Nucleus NOT FOR SALE OR DISTRIBUTIONneuraminidase NOT FOR SALERogers, G. OR N. Met DISTRIBUTIONhods (Removes Enzymol (1987):162–168. sialic acid) (b) Monoclonal antibodies block cell surface receptors. Adapted from Staunton, D. E., et al. Influenza © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning,Cell 56 (19 LLC89):849–853. virus NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION(c) Gene-transfer experiments. Adapted Influenza attaches to Influenza cannot from Mendelsohn, C., a sialic acid cell receptors attach to host cell et al. PNAS USA 20 (1986):7845–7849. Rhinovirus© Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC Monoclonal NOT FOR SALEantibody OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION

Host cell

Nucleus © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC NOT FOR SALEICAM-1 OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION

PVR gene PVR gene Poliovirus © Jones & Bartlett Learning,Poliovirus LLC © Jones & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTIONreceptor (PVR) NOT FOR SALE OR DISTRIBUTION

Nucleus

Transfer PVR gene into mouse cells © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC Human cells NOT FOR SALE OR DISTRIBUTIONMouse cells NOT FOR SALE OR DISTRIBUTION (susceptible expressing PVR to poliovirus are susceptible to infection) poliovirus infection

Mouse cells lacking © Jones & Bartlett Learning, LLCPVR gene are resistant © Jones & Bartlett Learning, LLC c to poliovirus infection NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION

4.2 Key Steps of the Viral Replication Cycle 71 2ND REVISE 2ND REVISE © Jones & Bartlett Learning, LLC. NOT FOR SALE OR DISTRIBUTION. roles in normal cellular activities. We do not know ligand-mediated fusion it is the viral ligand rather © Jones & Bartlettall Learning,of the cellular LLC receptors for every virus; ©how- Jones than& Bartlett the host Learning, receptor that LLC mediates the fusion NOT FOR SALE ORever, DISTRIBUTION research continues in this area of host–virusNOT FORevent.) SALE OR DISTRIBUTION interactions. In ligand-receptor-mediated fusion, the virus There are several factors that may influence attaches to the plasma membrane of the cell and the efficiency of viral attachment, such as the den- fusion takes place between the viral and cellular sity of receptors present on the host cell surface, membranes. The nucleocapsid of the virus is the density© of Jones the ligands & Bartlett on the Learning,viral surface, LLC released inside of the© cell.Jones The &remaining Bartlett viral Learning, LLC and the concentrationsNOT FOR SALE of virus OR and DISTRIBUTION host cells. envelope remains asNOT a “patch” FOR onSALE the cellular OR DISTRIBUTION Temperature, pH, and the presence or absence of plasma membrane (Figure 4-3a). The fusion at specific ions may also play a role in the efficiency the plasma membrane mode of penetration is of attachment. For some viruses, such as polio pH independent. In receptor-mediated endocytotis (engulfment), © Jonesvirus, rhinovirus,& Bartlett and Learning, influenza LLCvirus, a single type © Jones & Bartlett Learning, LLC of cellular receptor is sufficient for virus attach- the enveloped virus attaches to a receptor on the NOTment. FOR In SALE other cases, OR DISTRIBUTIONincluding HIV Type 1 (HIV‑1) plasmaNOT membrane FOR SALE of the cell OR and DISTRIBUTION the cell is stimu- and adenoviruses, one type of cellular receptor is lated to engulf the entire virus, thus forming an required for the initial attachment and attachment endocytotic vesicle (Figure 4-3b). This endocytotic to a co-receptor is necessary for viral entry into vesicle may fuse with the lysosomes, which possess © Jones & Bartlettthe Learning, cell. LLC © Jones an& Bartlettinternal acidic Learning, pH. In the LLC acidic pH of the endocytic vesicles, conformational changes in the viral NOT FOR SALE OR 2.DISTRIBUTION Penetration (Entry) NOT FORenvelope SALE proteinsOR DISTRIBUTION facilitate the fusion of the viral After the animal or human virus attaches to a cel- membrane with the endocytic membrane and the lular receptor, it must cross the lipid bilayer plasma subsequent release of the viral nucleocapsid into membrane (or in some cases the nuclear mem- the cytoplasm. This mode of viral penetration is pH brane) of the© Joneshost cell. & Activity Bartlett at Learning,the surface of LLC dependent because it© is Jones only at an& Bartlettacidic pH thatLearning, LLC the fusion between the viral envelope and the host cellular membranesNOT FOR is dynamic SALE ORand DISTRIBUTIONthese mem- NOT FOR SALE OR DISTRIBUTION branes are constantly being recycled. Clathrin, cell membrane occurs (Figures 4-3b and 4-3c). which is a large, fibrous protein, is instrumental in the formation of specialized regions of the cell Naked Virus Entry membrane called clathrin-coated pits. These It is more difficult to envision how naked or non- © Jonespits appear & Bartlett as invaginations Learning, that LLC are coated with enveloped© Jones viruses & cross Bartlett the cellular Learning, membrane, LLC and NOTdark FOR material, SALE and OR are DISTRIBUTION located on the cytoplasmic muchNOT remains FOR to beSALE understood OR DISTRIBUTION about how these side of the membrane. The pits are short lived and viruses enter cells. Studies suggest that the majority soon bud off to form clathrin-coated vesicles. of naked viruses enter via receptor-mediated endo- These vesicles are for transport and are coated cytosis. The virus ligand–cell surface receptor inter- with a latticelike network of clathrin. Shortly after action causes a clathrin-coated pit formation/ © Jones & Bartlettformation, Learning, the clathrinLLC coat is removed and© theJones invagination& Bartlett atLearning, the cell surface. LLC The clathrin-coated NOT FOR SALE ORresultant DISTRIBUTION vesicles are referred to as endosomesNOT. FORpits SALE encase OR the DISTRIBUTIONvirus and bud off to form a clathrin- Sometimes these vesicles contain viruses, which coated vesicle. Within seconds this clathrin coat is can penetrate directly at the plasma membrane or shed and the vesicle containing the virus fuses with via endosomes. The virus particle must then disas- lysosomes. The low pH, along with proteases in this semble to make the viral genome available in the endocytic vesicle, disassociate the capsid, releasing cytoplasm, ©where Jones it is targeted& Bartlett to the Learning, correct loca- LLC the nucleic acid genome© Jonesof the virus & Bartlettinto the cyto- Learning, LLC tion in the cellNOT for FOR genome SALE replication. OR DISTRIBUTIONplasm. Figure 4-4 demonstratesNOT FOR this SALEtype of entry.OR DISTRIBUTION Enveloped Virus Entry 3. Uncoating (Disassembly and Localization) Enveloped viruses contain a lipid bilayer, or en- This step refers to the removal or degradation of velope, that surrounds the nucleocapsid. These the capsid (uncoating), thereby releasing the ge- © Jonesviruses & enterBartlett cells Learning,via fusion of LLCthe viral and cel- nome© intoJones the &host Bartlett cell. The Learning, genome is LLC trans- NOTlular FOR membranes. SALE OR This DISTRIBUTION process is driven by the portedNOT to the FOR site whereSALE transcription/replication OR DISTRIBUTION viral glycoproteins located on the viral surface. can begin. In some viruses there is no degrada- The two basic modes of entry of an enveloped tion of the capsid because the capsid proteins play human/animal virus are by ligand-mediated a role in viral transcription and replication. For fusion of the virus and the cellular plasma mem- these viruses uncoating refers to changes in the © Jones & Bartlettbrane Learning, or by receptor-mediated LLC endocytotic© Jones nucleocapsid& Bartlett Learning, that make it LLCready for transcription NOT FOR SALE ORentry DISTRIBUTION of an enveloped virus (Figure 4-3aNOT). (In FORand/or SALE replication. OR DISTRIBUTION

72 Chapter 4 Virus Replication Cycles 2ND REVISE 2ND REVISE © Jones & Bartlett Learning, LLC. NOT FOR SALE OR DISTRIBUTION. Receptor-mediated fusion of an enveloped virus with the plasma membrane © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC NOT FORNucleocapsid SALE OR DISTRIBUTIONEnvelope NOT FOR SALE OR DISTRIBUTION

Viral envelope forms patch on plasma membrane © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION

Ligand

Attachment Fusion of viral and Nucleocapsid released © Jones & Bartlett Learning,cellular envelopesLLC inside cell© Jones & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION

a Receptor-mediated endocytotic entry of an enveloped virus © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTION Formation of an NOT FOR SALE OR DISTRIBUTIONRelease of nucleocapsid endocytotic vesicle into cell’s interior

NOT FOR SALE OR DISTRIBUTION Hϩ NOT FOR SALE OR DISTRIBUTION Hϩ

Attachment Hϩ Hϩ © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTION AcidificationNOT FOR SALE OR DISTRIBUTION

Figure 4-3 (a) Viral entry steps in receptor-mediated fusion. Adapted from Wagner, © Jones & Bartlett Learning, LLC E. K., and© JonesHewlett, M. & J. BasBartlettic Virology, Learning, Second Edition. BlaLLCckwell Publishing, 2003. (b) Viral entry steps in a receptor-mediated endocytotic entry of an enveloped virus. NOT FOR SALE OR DISTRIBUTION AdaptedNOT from WagFORner, SALEE. K., and OR Hew lett,DISTRIBUTION M. J. Basic Virology, Second Edition. Blackwell Publishing, 2003. (c) Electron micrograph of mouse hepatitis viruses (family Coronaviridae) are absorbed into mouse intestinal cells via receptor-mediated endocytosis. The plasma membrane is invaginated and will release the viruses inside the cell. Magnification 40,000×. © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION

4.2 Key Steps of the Viral Replication Cycle 73 2ND REVISE 2ND REVISE © Jones & Bartlett Learning, LLC. NOT FOR SALE OR DISTRIBUTION. Figure 4-4 Steps that naked viruses use to enter © Jones cells.& Bartlett Adapted from Learning, Wagner, E. K., LLC and Hewlett, M. J. © Jones & Bartlett Learning, LLC Basic Virology, Second Edition. Blackwell Publishing, Clathrin-coated pit forms— triggered by virion-ligand cell NOT FOR200 SALE3. OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION Virion surface receptor interaction.

Endocytotic vesicle forms and becomes acidified © Jones & Bartlett Learning, LLC © Jones & BartlettPartial degradationLearning, LLC NOT FOR SALE OR DISTRIBUTION NOT FOR SALEof virionOR andDISTRIBUTION ATP potential expression of processed antigen

H+

The uncoating step may occur simultane- dsDNA Viruses © Jonesously &with Bartlett penetration Learning, or it may LLC immediately © Jones & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTION The genomeNOT FOR replication SALE ORof most DISTRIBUTION RNA viruses follow penetration of the virus into the host cell. occurs in the cytoplasm of the host. Presumably, It is a necessary step before replication of the ge- this is because their replication is associated with nome can occur. When the nucleic acid genome RNA-dependent RNA polymerases that the host is uncoated, infectious particles are no longer cell nucleus cannot provide. In contrast, most detected in one-step growth experiments. This is © Jones & Bartlett Learning, LLC © Jones DNA& Bartlett viruses Learning, replicate theirLLC genomes in the the start of the eclipse phase, which continues nucleus and utilize the host’s DNA and RNA syn- NOT FOR SALE ORuntil DISTRIBUTION new infectious virus particles are madeNOT (see FORthesizing SALE ORmachinery, DISTRIBUTION along with the host’s RNA Figure 4-2b). processing machinery. This means the viral genome must traverse the nuclear membrane to 4. Types of Viral Genomes and Their Replication utilize the aforementioned cellular machinery When viruses© Jonesinfect cells, & twoBartlett important Learning, and sepa- LLC (Figure 4-6). © Jones & Bartlett Learning, LLC rate events NOTmust occur:FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION • the production of virus structural proteins and Viral genomes enzymes, and Single stranded • replication of the viral genome. DNA Double stranded

© JonesThe genome & Bartlett of a Learning,virus may consist LLC of DNA or Nucleic© Jones & Bartlett Learning, LLC NOTRNA, FOR which SALE may OR be DISTRIBUTIONsingle stranded (ss) or double acid NOT FOR SALE OR DISTRIBUTION stranded (ds) and linear or circular (Figure 4-5). Double stranded The entire genome may occupy either one nucleic RNA Positive (ϩ) sense Single stranded acid molecule (monopartite or linear genome) Negative (Ϫ) sense or several nucleic acid molecules (multipartite or RNA DNA © Jones & Bartlettsegmented Learning, genome). LLC The different types of genome© Jones & Bartlett Learning, LLC NOT FOR SALE ORnecessitate DISTRIBUTION different replication strategies. NOT FORFi gSALEure 4-5 OR Types DISTRIBUTION of viral nucleic acid genomes.

74 Chapter 4 Virus Replication Cycles 2ND REVISE 2ND REVISE © Jones & Bartlett Learning, LLC. NOT FOR SALE OR DISTRIBUTION. Figure 4-6 Example of a life cycle of a dsDNA © Jones & Bartlett Learning,1 Attachment LLC © Jones & Bartlett Learning,virus (cytomegalovirus). LLC Cytomegalovirus is a NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTIONmember of the Herpesviridae family. The gH and Virion gB glycoproteins present on the outside of the mRNA cytomegalovirus particle bind to the cellular 2 Penetration receptors. The attachment event triggers cellular Tegument 7 Release 3 Uncoating proteins Translation of transcription factors SP1 and NF-κB to migrate to viral proteins the host cell nucleus. After the penetration and Viral SP1 uncoating step, the viral cytomegalovirus dsDNA NFkB © Jones & BartlettDNA Learning, LLC © Jones & Bartlett Learning, LLC activation activation is released and enters the host cell nucleus NOT FOR SALE OR DISTRIBUTION where theNOT DNA isFOR replicated SALE and tra ORnscribed DISTRIBUTION 6 Maturation with the help of host cell transcription factors SP1 and NF-κB. Viral mRNAs are exported into the cytoplasm where they are translated by the Transcription cellular machinery. Viral dsDNA, and viral and of viral genes Cytoplasm cellular proteins are packaged into the virion. The virion buds from the cell, gaining an envelope © Jones & NucleusBartlett Learning, LLC © Jones & Bartlett Learning, LLC Cellular 5 Virion from the plasma membrane as the particle is NOT FOR SALE ORtranscription DISTRIBUTIONassembly NOT FORreleased. SALE OR DISTRIBUTION

4 Replication

Replication of many DNA viruses involves host’s nuclear enzymes and machinery. The mon- strategies that are familiar in cell biology: DNA ocistronic mRNAs are transcribed directly from the replication and mRNA© transcription Jones & Bartlett from dsDNA. Learning, viral LLC dsDNA (Figure 4-7a). Refer© to Jones Chapter & 14 Bartlett for Learning, LLC Viral proteins are translatedNOT FOR from SALE the monocis- OR DISTRIBUTIONmore details about poxvirus replication.NOT FOR SALE OR DISTRIBUTION tronic mRNAs generated via transcription of viral Six families of viruses comprise a group of vi- mRNAs, as shown in Figure 4-7a, which also lists ruses collectively designated Nucleo-Cytoplasmic examples of dsDNA viruses, the diseases they cause, Large DNA Viruses (NCLDV). These families of vi- and the families to which they belong. Many DNA ruses include major pathogens of humans and viruses© Joneshave evolved & Bartlett ways to Learning,evade host defenses LLC other mammals© as Jones well as viruses& Bartlett that infect Learning, cold- LLC and can cause tumors in animals. NOT FOR SALE OR DISTRIBUTION blooded vertebrates,NOT insects, FOR SALEand algal OR hosts. DISTRIBUTION All of Papovaviruses and herpesviruses are dsDNA the viruses contain dsDNA genomes and replicate viruses and have the most straightforward replica- either exclusively in the cytoplasm of the host cells tion strategy. These viruses utilize the cellular or possess both cytoplasmic and nuclear stages in DNA-dependent RNA polymerase II located in the their life cycle (Table 4-3). host’s nucleus to transcribe the viral mRNAs from Phycodnaviruses are members of the Phycod- © Jones &the Bartlett dsDNA viral Learning, genome. LLC The host cell must be © Jones & Bartlett Learning, LLC naviridae family. The family name is derived from NOT FORcycling SALE through OR DISTRIBUTION the cell cycle for its DNA poly- NOT FOR SALE OR DISTRIBUTION two distinguishing features: “phyco” from their algal merase to be available for use by these DNA vi- hosts and “dna” because all of these viruses have ruses. The viral RNA transcripts are spliced and cleaved via cellular machinery to produce mono- dsDNA genomes. The phycodnaviruses are recog- cistronic mRNAs that are exported into the cyto- nized as important ecological virioplankton in plasm and translated© accordinglyJones & Bartlett by the cell’s Learning, aquatic LLC ecosystems. Virioplankton© now Jones represent & Bartlettthe Learning, LLC translation machinery.NOT The FORviral dsDNASALE is OR pack- DISTRIBUTIONmost abundant viruses in natural NOTwaters, FOR surpassing SALE OR DISTRIBUTION aged, along with the necessary structural proteins the number of bacteria by an order of magnitude. and enzymes, resulting in the generation of the The phycodnaviruses, along with other viruses, in- newly assembled progeny viruses. cluding bacteriophages, play roles in nutrient re Poxviruses differ from the other dsDNA virus cycling (refer to Chapter 1, Viruses and Aquatic families© Jones listed in & Figure Bartlett 4-7a inLearning, that they replicate LLC Ecosystems) and© algal Jones blooms. & Bartlett There are Learning, currently LLC solelyNOT in the FOR cytoplasm. SALE These OR DISTRIBUTIONviruses carry their extensive metagenomicsNOT FOR SALEstudies ORunderway DISTRIBUTION to own DNA-dependent DNA polymerase (to repli- determine aquatic viromes. Metagenomics is the cate the viral dsDNA genome) within the virus genomic analysis of all DNA applied to entire com- particle. The genomes of poxviruses are large munities of microbes and/or viruses, bypassing the (ranging from 130–230 kbp, or roughly 100–200 need to isolate and culture individual microbes or © Jones &genes), Bartlett allowing Learning, these viruses LLC to be fully equipped ©viruses Jones in the& Bartlett laboratory. Learning, A virome is LLCthe genomes NOT FORwith SALE the genesOR DISTRIBUTION to make them independent of the NOTof all virusesFOR SALEthat inhabit OR aDISTRIBUTION particular environment.

4.2 Key Steps of the Viral Replication Cycle 75 2ND REVISE 2ND REVISE © Jones & Bartlett Learning, LLC. NOT FOR SALE OR DISTRIBUTION. Figure 4-7 (a) List of dsDNA viruses and their dsDNA Viruses © Jones rep& licationBartlett strategy. Learning, Adapted from LLCHarper, D. R. © Jones & Bartlett Learning, LLC Molecular Virology, Second Edition. BIOS Scientific Virus Disease Family NOT FORPub SALElishers, 199OR9. (b)DISTRIBUTION List of ssDNA viruses. Adapted HerpesNOT simplex FOR SALE OR DISTRIBUTION Herpesviridae from Harper, D. R. Molecular Viorology, Second Edition. Type 1 Cold sores BIOS Scientific Publsihers, 1999. Type 2 Genital herpes Adenovirus Respiratory infections Adenoviridae © Jones & Bartlett Learning,Cytomegalovirus LLC Infectious mononucleosis© Jones & BartlettHerpesviridae Learning, LLC NOT FOR SALE OR DISTRIBUTIONVariola Smallpox NOT FOR SALEPoxviridae OR DISTRIBUTION Human Papillomavirus Cervical cancer Papovaviridae *Types 16 and 18 Genital warts *Types 6 and 11 Plantar warts *Types 1, 2, and 4 *common types © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTION mRNANOT FOR ProteinsSALE OR DISTRIBUTION dsDNA Assembly dsDNA

a © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC ssDNA Viruses NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION Virus Disease Family Human parvovirus B19 Fifth disease Parvoviridae (slapped-cheek syndrome) Transfusion transmitted Hepatitis? Circoviridae © Jones & Bartlett Learning,virus (TTV) LLC © Jones & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTION mRNANOT Proteins FOR SALE OR DISTRIBUTION ssDNA dsDNA Assembly ssDNA

© Jones & Bartlett Learning, LLCb © Jones & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION ssDNA Viruses the viral dsDNA into viral mRNA in the nucleus. The Parvoviruses are the smallest of the human viruses cellular splicing machinery is also used in the pro- (only 20–25 nm in diameter). In contrast to the duction of the viral mRNAs. The general outline of dsDNA virus genomes, these ssDNA viruses con- their replication strategy is illustrated in Figure 4-7b. tain very small linear genomes (the genome of the © Jones & Bartlett Learning, LLC © Jones ss/dsDNA& Bartlett Viruses Learning, (Using an LLC RNA Intermediate) NOT FOR SALE ORhuman DISTRIBUTION parvovirus B19 is 5 kb). ParvovirusesNOT do FOR SALE OR DISTRIBUTION not carry any enzymes in the virus particle. These Hepadnaviruses replicate via a very unique and viruses infect cells that are in the cell cycle because somewhat complicated mechanism. This textbook they are dependent upon the host’s DNA poly- focuses on hepatitis B virus (HBV) because it spe- merase to synthesize the viral ssDNA and the cell’s cifically infects humans. Other members of the DNA-dependent© Jones RNA &polymerase Bartlett II Learning, to transcribe LLC Hepadnaviridae family© infect Jones woodchucks, & Bartlett ground Learning, LLC NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION Table 4-3 Host Range and Replication Sites of Selected Families of the Eukaryotic Nucleo-Cytoplasmic Large DNA Viruses

© JonesVirus Family& Bartlett Learning,Replication SiteLLC Host Range © Jones & Bartlett Learning, LLC NOT FORAscorviridae SALE OR DISTRIBUTIONNucleus and Cytoplasm Insects, mainlyNOT noctuids FOR (night-flying SALE moths) OR DISTRIBUTION Asfarviridae Cytoplasm Mammals Iridoviridae Nucleus and Cytoplasm Insects, cold-blooded vertebrates (reptiles, amphibians, and fish) Mimiviridae Cytoplasm Acanthamoeba © Jones & BartlettPhycodnaviridae Learning, LLC Nucleus and Cytoplasm © JonesChlorella-like & Bartlett green algae, Learning,algal symbionts of LLC paramecia and hydras NOT FOR SALE ORPoxviridae DISTRIBUTION Cytoplasm NOTInsects, FOR mammals, SALE birds, OR reptiles DISTRIBUTION

76 Chapter 4 Virus Replication Cycles 2ND REVISE 2ND REVISE © Jones & Bartlett Learning, LLC. NOT FOR SALE OR DISTRIBUTION. squirrels, chipmunks, ducks, geese, chimps, gib- ssDNA. As a result, the gapped regions contain only © Jones bo& ns,Bartlett and orangutans. Learning, Interestingly, LLC HBV-infected ©(–) Jones sense ssDNA. & Bartlett After the Learning, HBV has entered LLC its host NOT FORcells SALE produce OR different DISTRIBUTION forms of virus-related par- NOTcell and FOR the SALEvirus is partiallyOR DISTRIBUTION uncoated, the partial ticles. Electron microscopy of partially purified dsDNA genome of the Dane particle migrates to the virus particle preparations reveal three types of nucleus, where it is completed or repaired by a viral particles: a 42- to 47-nm mature spherical virus reverse transcriptase. The dsDNA enters the nu- particle (known as Dane particles, named after cleus and the ends are ligated by cellular enzymes, their discoverer); 22-nm© Jones spherical & particles, Bartlett which Learning, forming LLC a circular episome. (The© term Jones episome & Bartlett ap- Learning, LLC are found in 10,000- NOTto 100,000-fold FOR SALE excess OR over DISTRIBUTION plies to a viral genome that is maintainedNOT FOR in cells SALE by OR DISTRIBUTION the Dane particle; and filamentous particles that autonomous replication.) Next, the repaired viral are 22 nm in diameter and of varying lengths. All dsDNA associates with cellular histones and is tran- three forms contain the same surface protein, scribed into separate viral mRNA transcripts and a called the hepatitis B surface antigen (HbsAg). The full-length ssRNA pre-genome (Figure 4-9). Dane© particle Jones is the & Bartlettonly infectious Learning, particle ofLLC HBV. The viral mRNAs© Jones are translated& Bartlett to Learning,yield the LLC The 22-nmNOT FORspheres SALE and filaments OR DISTRIBUTION do not contain hepatitis B coreNOT antigens FOR and SALE the viralOR DISTRIBUTIONreverse nucleic acid (Figure 4-8). transcriptase. The RNA pre-genome associates The genome of the Dane particles consists of a with the viral reverse transcriptase and is pack- 3.2-kb linear DNA that is arranged in a relaxed aged with the core proteins to form an immature circle. Some parts of the genome are dsDNA, virus particle in the cytoplasm of the cell. The viral © Jones &whereas Bartlett others Learning, consist of LLCssDNA regions or gaps. ©reverse Jones transcriptase & Bartlett Learning, synthesizes theLLC (–) sense NOT FORThis SALE partially OR duplexed DISTRIBUTION DNA consists of a full-length NOTssDNA FOR strand SALE using ORthe ssRNADISTRIBUTION intermediate as a (–) sense ssDNA and a shorter length (+) sense template (see Refresher: Molecular Biology, which

Figure 4-8 (a) HBV infection results in the formation of three different types of virus particles: 42- to 47-nm intact © Jones & Bartlett Learning, LLC infectious Dane particles,© Jones22-nm spheres, & Bartlett and 22-nm Learning, LLC filaments of varying lengths. (b) Illustration depicting the NOT FOR SALE OR DISTRIBUTION different forms of HBVNOT particles. FOR Mat ureSALE hepatitis OR B DISTRIBUTION Dane particles contain dsDNA with associated protein, but their mode of replication is different from the other dsDNA viruses and their replication strategy. Adapted from University of South Carolina, School of Medicine. “Virology: Hepatitis Viruses.” Microbiology and © Jones & Bartlett Learning, LLC Imm© Jonesunology. Uni &versity Bartlett of Sou thLearning, Carolina, 2008. LLC NOT FOR SALE OR DISTRIBUTION http://pathmicro.med.sc.edu/virol/hepatitis-virus.htm.NOT FOR SALE OR DISTRIBUTION

HBsAg Pol protein DNA© Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION

Core Membrane

Virus Filamentous particle Spherical particle Dane particle up to 200 nm long ~20 nm diameter © Jones & Bartlett40 nm diameter Learning, LLC © Jones & Bartlett Learning, LLC NOT FORb SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION

4.2 Key Steps of the Viral Replication Cycle 77 2ND REVISE 2ND REVISE © Jones & Bartlett Learning, LLC. NOT FOR SALE OR DISTRIBUTION. © Jones Fi&g ureBartlett 4-9 ssDNA/dsDNA Learning, virus (thLLCat uses ssRNA Virus © Jones &Disease Bartlett Learning, LLCFamily as an intermediate) and its replication strategy. NOT FORAdapted SALE from OR Harper, DISTRIBUTION D. R. Molecular Virology, Hepatitis BNOT FOR SALEHepatitis ORassociated DISTRIBUTIONHepadnaviridae with liver cancer Second Edition. BIOS Scientific Publishers, 1999.

mRNA Proteins Assembly

© Jones & Bartlett Learning, LLC © Jones &genome Bartlett Learning, LLC NOT FOR SALEss/dsDNA OR DISTRIBUTIONdsDNA ssRNA NOT FOR maturesSALE OR DISTRIBUTION Reverse intermediate in particle transcriptase (pre-genome) extends linear Reverse regions of the transcription DNA genome of ssRNA into © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning,viral (Ϫ) ssDNA LLC NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION(ssRNA intermediate degraded)

DNA polymerase synthesizes (ϩ) ssDNA strand © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTIONmature particle

Refresher Molecular Biology © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTIONPolymerase NOT FOR SALE OR DISTRIBUTION

What is reverse transcriptase? Reverse transcriptase (RT) has three distinct enzymatic activities: © Jones & Bartlett Learning, LLC 1. RNA-dependent© DNA Jones polymerase & Bartlett Learning, LLC 2. RNase H activity (cleaves/degrades RNA from NOT FOR SALE OR DISTRIBUTION RNA/DNA hybrids)NOT FOR SALE OR DISTRIBUTION 3. DNA-dependent DNA polymerase 5´ 3´ + ssRNA

RT (RNA dep. DNA pol. activity) © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC 5´ 3´ NOT FOR SALE OR3´ DISTRIBUTION5´ cDNA (–ssDNA) NOT FOR SALE OR DISTRIBUTION

RT (RNase H activity)

5´ 3´ 3´ 5´ cDNA (–ssDNA) © Jones & Bartlett Learning, LLC RT second strand synthesis© Jones & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTION (DNA-dependent polymeraseNOT FOR activity) SALE OR DISTRIBUTION 3´ 5´ cDNA (–ssDNA) 5´ 3´ second strand (+ ssDNA) Retroviruses and hepadnaviruses utilize RT in their life cycles. © Jones & Bartlett Learning,Figure RB 4-1 LLC Reverse Transcriptase Courtesy of ©Dav Jonesid S. Goodsell, & BartlettScripps Research Learning, Institute. Pro LLCtein Data Bank: 2hmi. Reproduced from J. Ding, et al., J. Mol. Biol. 284 (1998): 1095–1111. NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION

78 Chapter 4 Virus Replication Cycles 2ND REVISE 2ND REVISE © Jones & Bartlett Learning, LLC. NOT FOR SALE OR DISTRIBUTION. is about reverse transcriptase functions). The pre- ϩ sense ssRNA genome: AUG GCA CGA met ala arg © Jones genome& Bartlett is degraded Learning, by the LLC RNase H activity of the © Jones & Bartlett Learning, LLC NOT FORreverse SALE transcriptase OR DISTRIBUTION enzyme, but it leaves a short NOTϪ sense FOR ssRNA SALE genome: OR UAC DISTRIBUTION CGU GCU sequence of RNA at its 5′ end that acts as a primer for DNA polymerase to synthesize a complemen- Figure 4-10 Differences between positive (+) and negative (–) tary (+) DNA strand in the mature particle. sense ssRNA viral genomes. Hepatitis B is one of a few known nonretroviral © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC viruses that uses reverse transcription as part of its RNA-dependent RNA polymerase that will synthe- replication process. OtherNOT viruses FOR that SALE utilize ORreverse DISTRIBUTION size the viral +ssRNA, mRNAs, NOTand –ssRNA FOR SALEviral OR DISTRIBUTION transcriptase are retroviruses such as human T-cell genomes into the host cell with them. leukemia virus (HTLV) and HIV, which possess an RNA genome. For these retroviruses reverse tran- dsRNA Viruses scription is one of the first steps in viral replication, whereas© Jones for hepatitis & Bartlett B reverse Learning, transcription LLC occurs Rotaviruses have© Jonesemerged & as Bartlett the main Learning, agent of LLC duringNOT maturation FOR SALE (the latter OR steps) DISTRIBUTION in making new acute gastroenteritisNOT in FOR infants SALE and children OR DISTRIBUTION world- virus particles. In addition, in contrast to retrovi- wide. These viruses have dsRNA segmented ge- ruses, HBV does not have integrase activity. The nomes. The rotavirus particle contains 11 segments DNA of hepatitis B is usually not integrated into cel- or pieces of the viral dsRNA. The host does not lular DNA; it is found as an independent episome. produce RNA-dependent RNA polymerases; thus, © Jones &Inte Bartlettgrated parts Learning, of the hepatitis LLC B genome, however, ©the Jones virus carries& Bartlett its own Learning, RNA-dependent LLC RNA NOT FORare SALE found ORin the DISTRIBUTION chromosomes of hepatocellular tu- NOTpolymerase FOR SALEand the ORreplication DISTRIBUTION cycle occurs solely mors from cancer patients. Retroviruses have inte- in the cytoplasm. grase activity (see Chapter 10). A rotavirus particle is nonenveloped and icosahedral with a double-capsid. One of its two layers RNA Viruses is removed but the other is not; the transcription RNA viruses are unique© Jones because &their Bartlett genetic Learning, in- takes LLC place inside of this single© capsidJones and & Bartlettthe Learning, LLC formation is encoded inNOT RNA. FOR The genomes SALE ORof RNA DISTRIBUTION mRNAs are released in the cytoplasmNOT FORfor transla- SALE OR DISTRIBUTION viruses are diverse [ss or ds, (+) or (–) sense, linear tion. After attachment, entry, and uncoating, the or segmented]. The type of RNA genome deter- virus synthesizes a +ssRNA from each of the 11 mines if the first step after uncoating will be trans- dsRNA segments (using the –ssRNA strands of the lation, transcription, or RNA replication. dsRNA genome as a template) via a viral RNA- Viruses© Jones that & contain Bartlett +ssRNA Learning, genomes LLC have dependent RNA© polymerase. Jones & Bartlett These viral Learning, ssRNAs LLC genomesNOT that FOR can SALE be directly OR translated DISTRIBUTION using the are also cappedNOT via a FORviral cappingSALE enzyme.OR DISTRIBUTION The host cell machinery because the +ssRNA acts like an RNAs are not polyadenylated. Half of the newly mRNA (Figure 4-10). These +ssRNA viruses, how- synthesized capped +ssRNAs strands (mRNAs) are ever, do need to carry the gene that encodes the translated by the cellular machinery in the cyto- replicase that produces the viral genomic RNA. plasm. The remaining strands are packaged into a © Jones & BartlettAll other typesLearning, of RNA virusesLLC (–ssRNA, dsRNA, ©viral Jones capsid & during Bartlett assembly Learning, (Figure 4-11LLC). At this stage of the life cycle, the RNAs inside NOT FORlinear, SALE segmented) OR DISTRIBUTION must be transcribed into mRNA NOT FOR SALE OR DISTRIBUTION before translation can occur. Eukaryotic host cells the particles are sensitive to RNase treatment. do not contain RNA-dependent RNA polymerases During maturation of the virus particle, the com- (see Section 2.3, Molecular Constraints of the Host plementary –ssRNA strands are synthesized using Cell), and as a result these viruses must carry an the capped +ssRNAs as a template within the virus © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC Virus NOTDisease FOR SALE OR DISTRIBUTIONFamily Figure 4-11 List ofNOT dsRNA FORviruses andSALE their replication OR DISTRIBUTION strategies. Adapted from Harper, D. R. Molecular Virology, Rotavirus Gastroenteritis Reoviridae Second Edition. BIOS Scientific Publishers, 1999. Reovirus Mild respiratory and Reoviridae gastrointestinal symptoms © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC NOT FOR SALE OR ProteinsDISTRIBUTION NOT FOR SALE OR DISTRIBUTION dsRNA mRNA Assembly dsRNA

4.2 Key Steps of the Viral Replication Cycle 79 2ND REVISE 2ND REVISE © Jones & Bartlett Learning, LLC. NOT FOR SALE OR DISTRIBUTION. particle to form the remaining dsRNA genomic seg- turn serves as a template for the genomic +ssRNA © Jones & Bartlettments Learning, (Figure 4-11). LLC These final dsRNA segments© Jones (Figure& Bartlett 4-12). Learning, Note that there LLC are exceptions to this NOT FOR SALE ORare resistant DISTRIBUTION to RNase treatment. There are stillNOT sev- FORreplication SALE OR strategy. DISTRIBUTION Not all ssRNA viruses produce eral remaining questions about the replication a single polyprotein that is cleaved by proteases into cycle of rotaviruses and other viruses of the individual proteins. Some produce more than one Reoviridae family; for example, how does the virus mRNA, allowing greater control of the production particle manage to contain only one of copy of of individual proteins; for example, early replica- each of the© 11 Jones mRNAs? & Bartlett Learning, LLC tion proteins and later© structural Jones &proteins Bartlett are pro-Learning, LLC NOT FOR SALE OR DISTRIBUTIONduced at different timesNOT during FOR the viralSALE replication OR DISTRIBUTION +ssRNA Viruses cycle (Figure 4-13). The +ssRNA viruses include several families of viruses. Members of the Picornaviridae, Flaviviridae, –ssRNA Viruses © Jonesand Caliciviridae & Bartlett families, Learning, in particular, LLC are ubiqui- Viruses© Jonesin the Paramyxoviridae, & Bartlett Learning, Rhabdoviridae, LLC tous in nature and cause a wide range of diseases. and Filoviridae families contain –ssRNA nonseg- NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION Their success and widespread distribution suggest mented genomes. All of these viruses encode their that their replication strategy is very effective. The own RNA-dependent RNA polymerases that tran- RNA in the virus particle itself functions as mRNA. scribe the –ssRNA genome into several different This genomic RNA is a polycistronic mRNA that is viral monocistronic +ssmRNAs that can be recog- © Jones & Bartlettrecognized Learning, by cellular LLC machinery and translated© Jones as nized& Bartlett by the Learning,host cell machinery. LLC The different NOT FOR SALE ORone DISTRIBUTIONopen reading frame into a single polyproteinNOT FOR+ss SALERNAs ORare madeDISTRIBUTION by a complicated start–stop precursor that is subsequently cleaved into indi- type of mechanism. In other words, a range of vi- vidual viral proteins by viral and cellular proteases ral mRNAs are each translated to make different (Figure 4-12). One of the viral encoded proteins is viral proteins rather than a polyprotein. All of the an RNA-dependent RNA polymerase that replicates proteins are not produced to the same level, and a the viral genome.© Jones It transcribes & Bartlett the Learning, viral +ssRNA LLC number of control mechanisms© Jones are & used.Bartlett The sec-Learning, LLC into a –ssRNANOT replicative FOR SALE intermediate, OR DISTRIBUTION which in ond function of the NOTviral RNA-dependent FOR SALE OR RNA DISTRIBUTION

Figure 4-12 List of +ssRNA viruses and their replication Virus Disease Family strategies. Adapted from Harper, D. R. Molecular Virology, Second Edition. BIOS Scientific Publishers, 1999. Poliovirus Poliomyelitis Picornaviridae © Jones & Bartlett Learning, LLC © JonesPostpolio & Bartlett syndrome Learning, LLC Rhinovirus (many types) Common cold Picornaviridae NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION Hepatitis A Hepatitis Picornaviridae Cocksackie Picornaviridae Group A Types 21, 24 Common cold Group A Types 4, 5, 9, 10, 16 Hand, foot, and mouth disease © Jones & Bartlett Learning, LLC Group© JonesB Types 1–5 & Bartlett Learning,Myocarditis LLC Group B Types 2, 5 Hand, foot, and NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTIONmouth disease Echoviruses Picornaviridae Various Types Diarrhea Types 1–7, 9, 11, 13–23, 25, 27 Aseptic meningitis Rubivirus Rubella Togaviridae © Jones & Bartlett Learning,Yellow fever LLC Hemorrhagic© Jones fever & BartlettFlaviviridae Learning, LLC NOT FOR SALE OR DISTRIBUTIONHepatitis C HepatitisNOT FOR SALEFlaviviridae OR DISTRIBUTION liver cancer Dengue Dengue fever Flaviviridae West Nile Fever, rash, myalgia Flaviviridae encephalitis © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC Norovirus Gastroenteritis Caliciviridae NOT FOR SALE OR DISTRIBUTIONSapovirus NOT FORGastroenteritis SALE OR DISTRIBUTIONCaliciviridae

(Cleavage) Polyprotein ϩssRNA ϩssRNA Assembly © Jones & Bartlett Learning, LLC © Jones & BartlettϪssRNA Learning, LLC NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION

80 Chapter 4 Virus Replication Cycles 2ND REVISE 2ND REVISE © Jones & Bartlett Learning, LLC. NOT FOR SALE OR DISTRIBUTION. Envelope protein Virus © Jones & Bartlett Learning, LLCNucleocapsid © Jones & Bartlett Learning, LLC Lipid NOT FOR SALE OR DISTRIBUTION NOT FOR SALE ORbilayer DISTRIBUTION 1 Attachment

Nucleocapsid assembles 5 Capsid envelope proteins Coated assembly pit © Jones & Bartlett Learning,Capsid LLC © Jones & Bartlett Learning, LLC 2 Entry/penetration protein Progeny NOT FOR SALE OR DISTRIBUTIONcapsid NOT FOR SALE OR DISTRIBUTION containing Budding Coated viral RNA 7 Release vesicle Capsid disassembly 3 Uncoating RNA 4 Replication © Jones & Bartlett Learning, LLC © Jones & ProgenyBartlett virus Learning, LLC Synthesis and glycosolation 6 Maturation NOT FOR SALE OR DISTRIBUTION of envelope proteins NOT FOR SALE OR DISTRIBUTION Insertion of Endosome envelope proteins into plasma membrane Nucleus

ER © Jones & Bartlett Learning, LLC © JonesGolgi & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION Glycosolation of envelope proteins is completed

Figure 4-13 Example of a life cycle of a +ssRNA enveloped virus. All steps of the life cycle take place in the cytoplasm. Like a typical eukaryotic mRNA, the viral +ssRNA© Jones genome & isBartlett directly translated Learning, by the cellular LLC ribosomal machinery in the cytoplasm.© Jones Viral & Bartlett Learning, LLC glycoproteins are synthesized on and inserted into the rough endoplasmic reticulum (ER) membrane, where they are subsequently transported to the trans GolgiNOT network FOR and then SALE inserted OR into theDISTRIBUTION plasma membrane. A viral polymerase synthesizesNOT the viral FOR genome. SALE OR DISTRIBUTION Newly synthesized capsid proteins bind to the replicated genome, forming a nucleocapsid that buds out of the plasma membrane to form the final enveloped virion.

polymerase© Jones is to & synthesize Bartlett the Learning, viral/progeny LLC ge- The main focus© Jones on retroviruses & Bartlett has Learning, been on LLC nomeNOT using FOR the +ssRNA SALE as OR a template. DISTRIBUTION Hence, the the avian (chicken)NOT or FOR human SALE retroviruses: OR DISTRIBUTION Rous RNA-dependent RNA polymerase is sometimes sarcoma virus (RSV, discovered in 1911, see Chap referred to as having a transcriptase and repli- ter 10), HIV (discovered in 1983), and HTLVs (dis- case function (Figure 4-14a). covered in 1981). Retrovirus infections cause a wide The –ssRNA viruses containing segmented spectrum of diseases including cancer, immune © Jones &genomes Bartlett also Learning, encode their LLC own RNA-dependent ©deficiencies, Jones & Bartlettand neurological Learning, disorders. LLC Most ret- NOT FORRNA SALE polymerase OR DISTRIBUTION that functions as a transcriptase NOTroviral FOR infections, SALE however, OR DISTRIBUTION occur without having and replicase. Each segment produces a mono- any detectable, deleterious damage to the host. cistronic mRNA or an RNA that is differentially The replication cycle of retroviruses includes spliced to make monocistronic mRNAs. The ge- the integration of the viral complementary DNA nomes of the viruses in the Arenaviridae and (cDNA) into the chromosomal DNA of the host Bunyaviridae families© areJones more & complicated Bartlett Learning, in cell. LLC The result of this integration© event Jones is that & Bartlettthe Learning, LLC that at least one of theNOT viral FOR ssRNA SALE genomic OR seg- DISTRIBUTIONretroviral DNA is inherited fromNOT parent FOR to SALE off- OR DISTRIBUTION ments are ambisense [the ssRNA is both (+) and spring of the infected host if germline cells (sperm (–) sense on the same ssRNA segment]. The pro- and egg) contain the integrated viral genome. cess of replication (Figure 4-14b) and transla- These are termed endogenous retroviruses or tions of these RNAs is not completely understood. proviruses, and their biologic properties and func- © Jones & Bartlett Learning, LLC tions are still under© Jones investigation. & Bartlett Approximately Learning, LLC VirusesNOT with FOR ssRNA SALE Genomes OR That DISTRIBUTION Use 0 8% to 12% of theNOT human FOR genome SALE consistsOR DISTRIBUTION of se- a dsDNA Intermediate to Replicate quences of human endogenous retroviruses The Retroviridae family contains viruses that have (HERVs). Retroviruses that are not integrated in been identified in virtually all organisms including germline cells of their hosts are called exogenous invertebrates. This suggests that these viruses have retroviruses (or external viruses). © Jones &an Bartlettevolutionarily Learning, successful LLC design. Their biology © JonesThe genome & Bartlett of retroviruses Learning, contains LLC two copies NOT FORis SALEquite unique. OR DISTRIBUTION NOTof a +ssRNA FOR moleculeSALE OR that DISTRIBUTION is reverse transcribed into

4.2 Key Steps of the Viral Replication Cycle 81 2ND REVISE 2ND REVISE © Jones & Bartlett Learning, LLC. NOT FOR SALE OR DISTRIBUTION. :Figure 4-14 (a) List of –ssRNA viruses (nonsegmented ؊ssRNA Viruses with Non-segmented Genomes © Jones and& Bartlett segmented) andLearning, their replication LLC strategies. Adapted © Jones & Bartlett Learning, LLC from Harper, D. R. Molecular Virology, Second Edition. Virus Disease Family NOT FORBIOS SALE Scientific OR Publishers, DISTRIBUTION 1999. (b) Ambisense RNA RabiesNOT FOR SALERabies OR DISTRIBUTIONRhabdoviridae viruses: Strategies for replication and mRNA synthesis of Ebola Hemorrhagic fever Filoviridae RNA genome. Adapted from Harper, D. R. Molecular Virology, Second Edition. BIOS Scientific Publishers, Marburg Hemorrhagic fever Filoviridae 1999. Nipah Encephalitis and Paramyxoviridae © Jones & Bartlett Learning, LLC respiratory infections© Jones & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTIONMeasles Measles NOT FORParamyxoviridae SALE OR DISTRIBUTION Mumps Mumps Paramyxoviridae Metapneumovirus Respiratory tract infections Paramyxoviridae Borna Psychiatric disorders? Bornaviridae

Jones & Bartlett Learning, LLC ؊ssRNA Viruses© Jones with Segmented & Bartlett Genomes: Learning, LLC © NOT FOR SALE OR DISTRIBUTIONVirus DiseaseNOT FOR SALE OR DISTRIBUTIONFamily Influenza A, B, C Influenza Orthomyxoviridae Crimean-Congo Hemorrhagic fever Bunyaviridae Sin nombre Hantavirus pulmonary Bunyaviridae © Jones & Bartlett Learning, LLC © Jones & Bartlettsyndrome Learning, LLC NOT FOR SALE OR DISTRIBUTION HantaanNOT FOR SALEHemorrhagic OR DISTRIBUTION fever Bunyaviridae Rift Valley fever Hemorrhagic fever Bunyaviridae Lassa Hemorrhagic fever Arenaviridae

Proteins © Jones & Bartlett Learning,Ϫ LLCssRNA ϩssRNA © JonesAssembly & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTION ssRNANOT FOR SALE OR DISTRIBUTION a (Ambisense RNA) ؉ ؉ ؉ ؉ ؉ ؉ ؉ ؉ ؉ ؉ ؉ ؉ ؉ ؉ ؉ ؉ ؊ ؊ ؊ ؊ ؊ ؊ ؊ ؊ ؊ ؊ ؊ ؊3´ ؊Viral ؊ ؊ ؊ ؊ ´5 © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning,genome RNA LLC NOT FOR SALE OR DISTRIBUTION NOT FORmRNA SALE synthesis OR DISTRIBUTION

3´ 5´ mRNA (ϩssRNA) © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION 3´ 5´ Antigenome RNA (ϪssRNA) mRNA synthesis

dsDNA by a viral RNA-dependent DNA polymerase by the cellular protein synthesis machinery. Some © Jones(reverse & Bartletttranscriptase) Learning, to produce LLC an RNA:DNA full-length© Jones +ssRNA & Bartlett transcripts Learning, will be packaged LLC NOThybrid, FOR SALEwhich in OR turn DISTRIBUTION is converted to dsDNA. The into NOTthe new FOR retrovirus SALE ORparticles DISTRIBUTION (Figure 4-16). viral dsDNA is inserted into the host chromosomal Refer to Chapter 10 (Sections 10.3 and 10.4) for dsDNA (Figure 4-15). The integrated DNA (provirus) more information. is subsequently transcribed by the host’s DNA- 5. Assembly dependent RNA polymerase II. The mRNA tran- © Jones & Bartlettscripts Learning, are then LLC spliced and exported into© theJones It& isBartlett not always Learning, possible to LLC identify the assembly, NOT FOR SALE ORcytoplasm DISTRIBUTION of the cell, where they will be translatedNOT FORmaturation, SALE OR and DISTRIBUTION release of virus particles as distinct

82 Chapter 4 Virus Replication Cycles 2ND REVISE 2ND REVISE © Jones & Bartlett Learning, LLC. NOT FOR SALE OR DISTRIBUTION. Virus Disease Family Figure 4-15 List of ssRNA viruses (that use a DNA © Jones & Bartlett Learning, LLC © Jones & Bartlettintermediate) Learning, and their rep LLClication strategies. Adapted HIV-1 and 2 AIDS Retroviridae from Harper, D. R. Molecular Virology, Second Edition. NOT FOR HTLVSALE I OR DISTRIBUTIONT-Lymphocyte Leukemia NOTRetroviridae FOR SALEBIOS OR Scientific DISTRIBUTION Publishers, 1999. HTLV II ? Retroviridae

Proteins ssRNA dsDNA Integration mRNA reverse © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC Assembly transcriptase NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION

and separate© Jones stages & Bartlett of the viral Learning, life cycle. Virus LLC as- specific sites within© Jones the infected & Bartlett cell. The Learning, genomic LLC semblyNOT is a FOR key step SALE in the OR replication DISTRIBUTION cycles of nucleic acids areNOT packaged FOR into SALE preexisting OR DISTRIBUTION shells viruses. It involves the process in which the im- that form via self-assembly (spontaneous as- mature virus particle is formed. Despite the struc- sembly, also refer to Chapter 3) of viral capsid tural diversity of virus particles, the repertoire of proteins, or are coated with capsid proteins, or assembly mechanisms is limited. All of the compo- are co-assembled with capsid proteins. Assembly © Jones &nents Bartlett of the Learning,virus must be LLC assembled to create a ©sites Jones (for example,& Bartlett the Learning, cytoplasm, LLCnucleus, on NOT FORstable SALE structure. OR DISTRIBUTION At the same time, the newly as- NOTthe inner FOR surface SALE ofOR the DISTRIBUTION plasma membrane of sembled virus must accomplish disassembly to cells) differ according to the virus and have some start a new infectious life cycle. influence on how the virus particle is released. The assembly event occurs when an appro- Historically, research directed toward virus priate concentration of virus proteins and ge- assembly mechanisms has received less attention nomic nucleic acids ©is reachedJones &and Bartlett localized Learning, at because LLC of more interest in the mechanisms© Jones of& viralBartlett Learning, LLC NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION

1 Attachment

7 Release

3 Uncoating Reverse 6 Maturation transcription Trafficking

5 Integration 6 Transcription Budding © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC NOT FOR SALE ORProvirus DISTRIBUTION NOT FOR SALE OR DISTRIBUTION

Early Late © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC FigureNOT 4-16 FORBasic life SALE cycle of aOR typical DISTRIBUTION retrovirus. Retroviruses undergo either a latent (early)NOT phase FOR in which SALE the virus OR begins DISTRIBUTION the steps of a typical virus life cycle: virus attaches to a host cell receptor(s) followed by uncoating of the virus particle in which the genome is translocated (also referred to as trafficking) to the nucleus where the genome is reverse transcribed into dsDNA that is integrated into the host’s chromosome. At this point, the retroviral DNA is replicated along with the host chromosome. This early stage is latent in that no infectious particles are made while the retroviral DNA has integrated into the chromosomal DNA. After an “activation” event, the integrated retroviral DNA is transcribed. Viral mRNAs are exported to the cytoplasm where the cellular translational machinery produces © Jones &the Bartlettviral proteins. Learning, The capsid proteins LLC and retroviral RNA is assembled/packaged© Jones into & a nucleocapsidBartlett thatLearning, buds from the LLC plasma membrane, forming the final particle that undergoes a maturation step before the virus is fully infectious. Modified from an illustration NOT FORby SALE Kate Bishop, OR MRC DISTRIBUTION National Institute for Medical Research, UK. NOT FOR SALE OR DISTRIBUTION

4.2 Key Steps of the Viral Replication Cycle 83 2ND REVISE 2ND REVISE © Jones & Bartlett Learning, LLC. NOT FOR SALE OR DISTRIBUTION. gag core precursor Cell membrane Budding through Protease cuts © Jones polyproteins& Bartlett [p55] Learning, LLC the cell membrane© Jones & Bartlettlong length Learning, LLC NOT FOR SALE OR DISTRIBUTION NOT FOR SALEprotein OR chainsDISTRIBUTION gp120 p17

gp41 © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC

RNA NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION

p24 gag-pol core and enzymes precursor © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC

NOT FOR SALE OR DISTRIBUTION RNA NOT FOR SALE OR DISTRIBUTION Reverse Structural proteins Finalized proteins transcriptase, and enzymes still now form HIV core Integrase Envelope protein linked to each other © Jones & BartlettFig ureLearning, 4-17 The structural LLC proteins within the immature© HIV Jones virus particle & Bartlett must be cleaved Learning, by a viral protease LLC inside of the particle in NOT FOR SALE ORorder DISTRIBUTIONfor the virus to be infectious. Adapted from Vella, S.,NOT et al., AIDSFOR Soc. SALE 4 (1996): OR 15–18. DISTRIBUTION

gene expression and replication. There is now re- orthomyxoviruses, paramyxoviruses, bunyaviruses, newed interest in understanding virus assembly coronaviruses, rhabdoviruses, and hepadnaviruses). because of© the Jones development & Bartlett of new Learning, molecular LLC Viruses that are released© Jonesvia budding & Bartlett may damage Learning, LLC technologiesNOT and theFOR success SALE of therapeutic OR DISTRIBUTION agents the cell (as is the case withNOT paramyxoviruses, FOR SALE OR rhab- DISTRIBUTION designed to inhibit virus-specific reactions involved doviruses, and togaviruses) or they may not (as is in the production of infectious virus particles. the case with retroviruses). Some viruses bud from These advances in understanding continue at an other membranes and are released from the cell via accelerated pace. a secretory-like mechanism. © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC 6. Maturation NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION This is the stage of the virus life cycle in which the virus becomes infectious. Viral or cellular proteases are often involved in maturation. One or more capsid or envelope proteins may undergo © Jones & Bartlettspecific Learning, proteolytic LLC cleavage within the particle.© Jones & Bartlett Learning, LLC NOT FOR SALE ORThe DISTRIBUTIONcleavage event results in a subtle structuralNOT FOR SALE OR DISTRIBUTION change of the virus particle, which may give it increased stability. Virus-encoded proteases are attractive targets for antiviral therapies; for example, the protease inhibitors© Saquinavir Jones & Bartlett mesylate Learning, (Invirase), LLC © Jones & Bartlett Learning, LLC SaquinavirNOT (Fortovase), FOR SALE Ritonavir OR DISTRIBUTION (Norvir), NOT FOR SALE OR DISTRIBUTION Indinavir (Crixivan), Nelfinavir (Viracept), Amprenavir (Agenerase), and ABT-378 (Kaletra) target the HIV-encoded protease by preventing the maturation of virions capable of infecting other © Jonescells ( &Fig ureBartlett 4-17). Learning, LLC © Jones & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION 7. Release Newly formed viruses are either released to the outside environment upon lysis, escaping the cell as it disintegrates (lytic viruses), or are released by bud- © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC ding (Figure 4-18) through the plasma membrane of Figure 4-18 Transmission electron micrograph of measles NOT FOR SALE ORthe cellDISTRIBUTION (as is the case with retroviruses, togaviruses,NOT FORvirus SALE released OR by budding. DISTRIBUTION

84 Chapter 4 Virus Replication Cycles 2ND REVISE 2ND REVISE © Jones & Bartlett Learning, LLC. NOT FOR SALE OR DISTRIBUTION. Lytic Viruses viral DNA remains integrated and does not excise © Jones & Bartlett Learning, LLC © Jones Most& Bartlett naked virusesLearning, are released LLC when infected itself from the chromosome as is done in bacterio- NOT FORcells SALE break OR open DISTRIBUTION (cell lysis/destruction) due to the NOTphages FOR (see ChapterSALE OR21). LatencyDISTRIBUTION is considered to activity of viral enzymes, rather than distention. be one of the biggest reasons why drug therapy Poliovirus is an example of a lytic virus. A lytic life fails to eradicate HIV from patients. cycle is one that kills the host cell. Many envel- Herpesviruses enter and migrate down neu- oped viruses also do this. rons, where they become latent in the body of neu- © Jones & Bartlett Learning,rons. LLC They do not integrate their DNA;© Jones it remains & Bartlett as Learning, LLC Latent Eukaryotic VirusesNOT FOR SALE OR DISTRIBUTIONan episome. Subsequent activationNOT of theFOR latently SALE OR DISTRIBUTION Retroviruses such as HIV-1 undergo a latent (non- infected neurons by a variety of factors (such as ex- lytic) cycle in which the viral DNA (provirus) treme temperatures, physical trauma, emotional becomes inserted into the host’s DNA. In certain stress, and immune suppression) enables the viruses cell types, the proviral DNA replicates “silently” to migrate back up the nerve cell and replicate again along© with Jones the cellular & Bartlett DNA, Learning,and the virus LLC is un- in the epithelial© cells. Jones Two common& Bartlett herpesviruses Learning, LLC detectedNOT for FOR many SALE years. TheOR provirus,DISTRIBUTION however, are herpes simplexNOT virus FOR type 1SALE (HSV-1), OR which DISTRIBUTION usu- can be activated at any time, allowing a productive ally causes fever blisters or oral herpes, and herpes infection or lytic cycle (one that produces infec- simplex virus type 2 (HSV-2), which usually causes tious exogenous particles) to occur. Note that the genital herpes (see Chapter 15). © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION VIRUS FILE 4-2 Unraveling the Life Cycle of Mimivirus

The© discovery Jones of & Mim Bartlettivirus in 200 Learning,3 sent a sho LLCckwave through the community© of Jones virologists & Bartlett Learning, LLC andNOT evolutionists. FOR SALEThe virus OR was 750DISTRIBUTION nm in size—gargantuan compared to typNOTical vir FORuses— SALE OR DISTRIBUTION and was termed a Girus (see Chapter 3, Viruses That Challenge the Definition of a Virus). It contained the largest known viral genome at a whopping 1,181,404 base pairs of dsDNA, coding for 911 gene products. It contained many genes not found before in other viruses such as aminoacyl-tRNA synthetases that are central to components of cellular © Jones & traBartlettnslation mac Learning,hinery, gen esLLC that are components of cellular© repJoneslication & and Bartlett transcriptional Learning, LLC NOT FOR SALEmachinery, OR and DISTRIBUTION genes associated with metabolic pathways,NOT and fou FORr of its SALE genes con ORtained DISTRIBUTION introns. But Mimivirus still appears to be absolutely dependent on its host cells for synthesis of its proteins. Following the deciphering of the genome sequence of Mimivirus, efforts began to elucidate the life cycle of the virus. Research teams led by Didier Raoult or Abraham Minsky resulted © Jones & Bartlett Learning,in extensive LLC ultrastructural studies to det©ermine Jones the lif &e cyc Bartlettle of Mim Learning,ivirus. In their stuLLCdies, NOT FOR SALE OR DISTRIBUTIONMimiviruses were “fed” and allowed to infNOTect Aca FORnthamoeba SALE pol yphagaOR DISTRIBUTION at a cell–virus ratio of 1:10. At various times post-infection within a 24-hour period, the infected cells were collected and prepared for transmission electron microscopy. Electron microscopy demonstrated that within 30 minutes, the Mimiviruses appeared to enter the amoeba cells by phagocytosis. Upon eng©ulfment, Jones the Mim& Bartlettiviruses und Learning,erwent uncoating LLC steps by fusing with the hos©t phaJonesgosomes. & Bartlett Learning, LLC DuringNOT the fusionFOR event, SALE the ORviral capsidsDISTRIBUTION morphed into 5-fold star-shaped structuresNOT that FOR acted SALE OR DISTRIBUTION like a “stargate,” or portal, through which viral DNA was released into the cytoplasm of the host cell. Subsequently, the viral DNA was imported into the host nucleus where its first round of replication began. Like poxviruses, Mimiviruses possess their own transcriptional apparatus (refer to Chapter 14). © Jones & BartlettBy 3 hou rsLearning, post-infection, LLC Mimivirus DNA exited the hos©t nucJonesleus to & for Bartlettm cytoplasmic Learning, LLC NOT FOR SALEviral rep licationOR DISTRIBUTION factories surrounded by mitochondria. By NOT5 to 8 houFORrs pos SALEt-infection, OR the DISTRIBUTIONse viral factories increased 50% in size and viral proteins were detected. In addition to electron microscopy, direct fluorescent staining was performed on the infected cells in order to study the Mimivirus factories. The viral factories appeared to have three zones: an electron- © Jones & Bartlett Learning,dense inn LLCer replication center, an intermediate© Jones assembly & Bartlett zone, and Learning,the peripheral LLCzone NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION(continued)

4.2 Key Steps of the Viral Replication Cycle 85 2ND REVISE 2ND REVISE © Jones & Bartlett Learning, LLC. NOT FOR SALE OR DISTRIBUTION. © Jones & BartlettVIRUS Learning, FILE 4-2 LLCUnraveling the Life Cycle of ©Mimivirus Jones (continued) & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION

where particles matured and acquired fibrils. At 8 hours after the infection, the viral factories contained empty, fiberless capsids that were partially assembled as well as some icosahedral © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC capsids undergoing DNA packaging through a transient “stargate” or aperture. By 10 to NOT FOR12 hours SALE post-infection, OR DISTRIBUTION mature fibril-coated particles budded from NOTthe viral FOR factories SALE and were OR DISTRIBUTION released through cell lysis (Figure VF 4-2). Overall, the takeover of the cellular machinery by Mimivirus was rapid and efficient. Many of the viral events in the life cycle took place in a giant volcano-like viral factory. © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC

NOT FOR SALE OR DISTRIBUTION Mature particles released NOT FOR SALE OR DISTRIBUTION

Cell lysis 1 Phagocytosis (0-30 minutes)

Phagosome Amoeba 1 2 2 Fusion with © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC phagosome NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION

Nucleus

3 Uncoating Fibers coat 12 hours later Mitochondria particles Stargate— © Jones & Bartlett Learning, LLC © Jones & BartlettDNA genome Learning, LLC released NOT FOR SALE OR AssemblyDISTRIBUTION & maturation NOT FOR SALE OR DISTRIBUTION of particles within viral Viral factories DNA

3 Viral DNA 4 enters © Jones & Bartlett Learning, LLC © Jonesnucleus & Bartlett Learning, LLC NOT FOR SALEFiberless OR DISTRIBUTION Viral factory NOT FOR SALE OR DISTRIBUTION empty capsid expands

Viral factory

NOT FOR SALE OR DISTRIBUTIONMitochondria NOT FOR SALE OR DISTRIBUTION

Viral DNA 8 hours later exits nucleus 3 hours later © FiJonesgure VF &4-2 Bartlett The Mimivirus Learning, life cycle bas LLCed on observations of infected amo©eba Jones cells at &var Bartlettious times Learning, LLC after infection. Adapted from Suzan-Monti, M., et al. 2007. “Ultrastructural characterization of the giant volcano- NOTlike virus FOR factory SALE of Acanthamoeba OR DISTRIBUTION polyphaga Mimivirus.” PLoS ONE 3:e328. FigureNOT 8. FOR SALE OR DISTRIBUTION

References Claverie, J. M., et al. 2009. “Mimivirus and Mimiviridae: Giant viruses with an increasing number of potential hosts, including corals and sponges.” J Invertebr Pathol 101:172–180. © Jones & Bartlett Claverie,Learning, J. M., and LLCAbergel, C., 2009. “Mimivirus and its virophage.”© Jones Ann Rev &Gen Bartlett 43:49–66. Learning, LLC NOT FOR SALE ORSuz DISTRIBUTIONan-Monti, M., et al. 2007. “Ultrastructural characterization ofNOT the giant FOR volcano-like SALE virus fac toryOR of AcaDISTRIBUTIONnthamoeba polyphaga Mimivirus.” PLoS ONE 3:e328. Zauberman, N., et al. 2008. “Distinct DNA exit and packaging portals in the virus Acanthamoeba polyphaga Mimivirus.” PLoS Biol 6:1104–1114.

86 Chapter 4 Virus Replication Cycles 2ND REVISE 2ND REVISE © Jones & Bartlett Learning, LLC. NOT FOR SALE OR DISTRIBUTION. Why Don’t the Viruses Get Stuck on the Cellular Figure 4-19 A © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC Vietnamese woman Receptors as They Are Released? NOT FOR SALE OR DISTRIBUTIONweighs a chicken at a NOT FORThe SALE release OR of DISTRIBUTIONvirus particles poses an interesting market in Hanoi, dilemma. Viruses are designed to enter rather than Vietnam (January 2004). At the time, leave cells. Some viruses, such as influenza A, pro- the avian influenza duce a protein during their life cycle to destroy the virus infected several cellular receptors as they© Jones exit the &cell. Bartlett The protein, Learning, LLC © Jones humans& Bartlett who had Learning, LLC neuraminidase, cleaves the sialic-acid receptors direct contact with NOT FOR SALE OR DISTRIBUTION NOT FORchickens. SALE The OR virus DISTRIBUTION on the outside of cells as the infectious particles are infected millions of released (see Chapter 12). As a result, the viruses chickens, raising fears do not aggregate at the cell surface. that it might mutate to a strain that could readily pass from © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning,human to human, LLC 4.3 The Error-Prone leading to the next NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTIONhuman influenza RNA Polymerases: pandemic. Genetic Diversity

© Jones &Viruses Bartlett replicate Learning, rapidly. LLCDuring the process of © Jones & Bartlett Learning, LLC NOT FORreplication, SALE OR an DISTRIBUTIONerror, or point mutation, may occur. NOT FOR SALE OR DISTRIBUTION The mutation rate of DNA viruses is usually similar to those of their cellular hosts because most of these polymerases used to replicate DNA genomes resistant strains of HIV), changes in virulence (for possess proofreading ability; for example, the mu- example, the 1997 Hong Kong avian influenza A tation rate of herpesviruses© Jones (with & proofreadingBartlett Learning, was LLChighly virulent), changes that© allow Jones the virus & Bartlett to Learning, LLC ability) is one error inNOT every FOR 108 to SALE 1011 bases. OR The DISTRIBUTION evade the host’s immune system NOT(such asFOR the influ- SALE OR DISTRIBUTION genome sizes of herpesviruses range from 1.3 × 105 enza viruses and antigenic drift, which will be dis- to 2.0 × 105 base pairs in length. As a result, herpes- cussed in Chapter 12), and changes in host range. viruses potentially evolve very slowly because few A good example of a change in host range mutations will be made, if any, during the infection adaptation occurred in 1978. Canine parvovirus cycle© of Jonesthe virus. & Bartlett Learning, LLC suddenly began© killing Jones large & numbers Bartlett of dogsLearning, glob- LLC RNANOT viruses FOR possess SALE mutation OR DISTRIBUTION rates as high as ally in 1978. TheNOT parvovirus FOR originallySALE OR had DISTRIBUTION infected one error in 103 to 104 bases. The RNA-dependent only cats, foxes, raccoons, and minks. A small num- RNA polymerases and RNA-dependent DNA poly- ber of changes in the capsid genes adapted the virus merases (for example, reverse transcriptases) used for efficient spread among dogs Fi( gure 4-19). by the RNA viruses for genome replication do not © Jones &possess Bartlett proofreading Learning, ability. LLC Hence, the presence © Jones & Bartlett Learning, LLC NOT FORof SALE mutants OR in aDISTRIBUTION virus population during each rep- NOT4.4 FOR TargetsSALE OR for DISTRIBUTION lication cycle of the virus occurs much more rapidly than in DNA viruses or cellular organisms. HIV Antiviral Therapies and coronaviruses (such as severe acute respiratory syndrome-associated coronavirus, or SARS- Modern technology allows scientists to deliberately CoV) are excellent examples© Jones of RNA & Bartlett viruses with Learning, design LLC drugs. To do this, they need© Jones to understand & Bartlett Learning, LLC high mutation rates. TheseNOT viruses FOR SALEmisincorporate OR DISTRIBUTION “the enemy.” Knowledge of the lifeNOT cycle FOR of viruses SALE OR DISTRIBUTION a nucleotide into their genome once in 103 to 104 and the mapping of them using computer-aided bases. For coronaviruses, this means that there are design (CAD) is applied toward antiviral develop- three mutations that occur during the replication ment by scientists at pharmaceutical companies. of one viral genome. For HIV, this means one to Any of the seven stages of the viral life cycle can be two mutations© Jones in& every Bartlett genome Learning, copied. The LLC high targeted for antiviral© Jones intervention. & Bartlett The stages Learning, are: LLC mutationNOT rate FOR probably SALE limits OR the DISTRIBUTION size of most RNA 1. AttachmentNOT specificity FOR SALE OR DISTRIBUTION virus genomes to approximately 104 nucleotides. 2. Penetration Many mutations are lethal because the mutated 3. Uncoating virus is unable to replicate. Nonlethal mutations 4. Replication may give the mutated virus a selective advantage. 5. Assembly © Jones &Mutations Bartlett have Learning, been associated LLC with the develop- © Jones6. Maturation & Bartlett Learning, LLC NOT FORment SALE of antiviral OR DISTRIBUTION drug resistance (such as the drug- NOT7. ReleaseFOR SALE OR DISTRIBUTION

4.4 Targets for Antiviral Therapies 87 2ND REVISE 2ND REVISE © Jones & Bartlett Learning, LLC. NOT FOR SALE OR DISTRIBUTION. Table 4-4 lists the current antiviral therapies organism. It has been difficult to develop antivirals © Jones & Bartlettavailable Learning, along LLCwith a list of which viruses© they Jones that& Bartlett have no toxicLearning, side effects LLC for the host because NOT FOR SALE ORtarget DISTRIBUTION and their mechanism of action. Key toNOT anti FORviruses SALE use OR some DISTRIBUTION of the host cellular processes for viral drug development is that the drug must target replication. Hence, drugs cannot target those cel- a process essential for viral replication and it must be lular processes because of toxicity problems for active against the virus without being “toxic” to the host the host. © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC Table 4-4 NOT PreventionFOR SALE and T reatmentOR DISTRIBUTION of Human Viral Diseases: Antiviral Drugs NOT FOR SALE OR DISTRIBUTION

Drug Virus/Disease Target Idoxuridine Herpes simplex keratoconjunctivitis Viral and cellular DNA synthesis © JonesTrifluridine & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC NOT FORVidarabine SALE OR DISTRIBUTIONHSV-1, HSV-2 NOT FORViral DNASALE polymerase OR DISTRIBUTION Acyclovir HSV-1, HSV-2, VZV, EBV, CMV Virus DNA polymerase Tromantadine HSV Viral DNA polymerase Famciclovir HSV-1, HSV-2, VZV, some activity against EBV, CMV, and HBV Viral thymidine kinase and DNA polymerase Penciclovir HSV-1, HSV-2 Viral DNA polymerase © Jones & BartlettValacyclovir Learning, LLC HSV-1, HSV-2, VZV, modestly active© Jones against EBV & andBartlett CMV Learning,Viral DNA polymerase LLC NOT FOR SALE ORGancyclovir DISTRIBUTION CMV retinitis in HIV patients NOT FOR SALE OR VirusDISTRIBUTION polymerase Foscarnet Acyclovir-resistant HSV/VZV strains Ganciclovir-resistant CMV HSV-1, HSV-2, HHV-6, EBV, VZV, parainfluenza virus CMV Viral DNA polymerase and reverse transcriptase retinitis in HIV disease © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC Docosanol HSV-labialis episodes Fusion inhibitor (10% topical NOTcream) FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION Brivudine (approved HSV-1 and VZV Viral thymidine kinase and DNA polymerase for use in Germany and other European countries) © JonesEntecavir, & Bartlett Learning,Hepatitis B LLC © JonesHepatitis & Bartlett B reverse transcriptase Learning, LLC Adefovir NOT FORAbacavir SALE OR DISTRIBUTIONHIV-1 and HIV-2 NOT FORHIV-1 SALE reverse transcriptase OR DISTRIBUTION Didanosine (ddI) Emtricitabine (FTC) Lamivudine (3TC) © Jones & BartlettLamivudine Learning, + zidovudine LLC © Jones & Bartlett Learning, LLC NOT FOR SALE OR(Combivir) DISTRIBUTION NOT FOR SALE OR DISTRIBUTION Stavudine (d4T) Tenofovir + emtricitabine (Truvada) Tenofovir DF Zalcitabine (ddC)© Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC Zidovudine (AZT)NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION Nevirapine HIV-1 HIV-1 reverse transcriptase Delavirdine HIV-1 HIV-1 reverse transcriptase Efavirenz HIV-1 HIV-1 reverse transcriptase © JonesAmprenavir & Bartlett Learning,HIV-1 LLC © JonesHIV-1 & proteaseBartlett Learning, LLC NOT FORAtazanavir SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION Fosamprenavir Saquinavir Lopinavir + ritonavir © Jones & BartlettIndinavir Learning, LLC © Jones & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION (continued)

88 Chapter 4 Virus Replication Cycles 2ND REVISE 2ND REVISE © Jones & Bartlett Learning, LLC. NOT FOR SALE OR DISTRIBUTION. Table 4-4 Prevention and Treatment of Human Viral Diseases: Antiviral Drugs (continued)

© Jones &Drug Bartlett Learning,Virus/Disease LLC © Jones & BartlettTarget Learning, LLC NOT FOR DarunavirSALE OR DISTRIBUTIONHIV-1 NOT FOR SALEHIV-1 protease OR DISTRIBUTION Nelfinavir Ritonavir Tipranavir Enfuvirtide ©HIV-1 Jones & Bartlett Learning, LLC Binds to HIV-1 gp41 surface© Jones protein, inhibiting & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTION viral entry NOT FOR SALE OR DISTRIBUTION Selzentry HIV-1 Binds to CCR5 co-receptor Raltegravir HIV-1 Integrase strand transfer inhibitor Atripola: cocktail of HIV-1 A fusion inhibitor and two reverse transcriptase efavirenz, tenofovir, inhibitors and emtricitabine© Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC RibavirinNOT FOR SALEBroad OR spectrum DISTRIBUTION (inhibits DNA and RNA viruses): RSV, mRNANOT mutagen FOR SALE OR DISTRIBUTION Influenza A andB, HCV, HSV-1 and HSV-2, measles, mumps, Lassa fever Amantadine Influenza A Inhibits penetration and uncoating of virus Rimantidine © Jones &Relenza Bartlett Learning,Influenza LLC A © Jones & BartlettNeuraminidase Learning, inhibitor LLC NOT FOR SALEand Tamiflu OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION Peramivir Influenza A (H1N1) Neuraminidase inhibitor (not FDA approved) Arbidol Influenza A andB, hepatitis C Virus-mediated membrane fusion, immune (not FDA approved) modulator? © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC Cidofovir Broad spectrum (inhibits DNA viruses): HSV-1, HSV-2, VZV, DNA polymerase NOTCMV, EBV FOR, adenovirus, SALE HPV, ORCMV retinitis DISTRIBUTION in HIV patients, NOT FOR SALE OR DISTRIBUTION experimentally used to treat poxvirus infections Interferons Hepatitis B & C Cell defense proteins activated Hairy cell leukemia, HPV, respiratory viruses Fomivirsen CMV Inhibits viral replication and translation © Jones & Bartlett Learning, LLC (antisense© Jones molecule) & Bartlett Learning, LLC PodophyllotoxinNOT FOR SALEHPV OR genital DISTRIBUTION warts BindsNOT E2 andFOR inhibits SALE E2/E3 interaction OR DISTRIBUTION Abbreviations of viruses: Herpes simplex type 1 (HSV-1), herpes simplex type 2 (HSV-2), varicella zoster virus (VZV), Epstein-Barr virus (EBV), cytomegalovirus (CMV), hepatitis B virus (HBV), hepatitis C virus (HCV), human herpes Type 6 virus (HHV-6), human immunodeficiency virus Type 1 (HIV-1), respiratory syncytial virus (RSV), hepatitis C virus (HCV), human papillomavirus (HPV). Abbreviations of antivirals: zidovudine (AZT), didanosine (ddI), zalcitabine (ddC), stavudine (d4T), lamivudine (3TC). Unless otherwise specified, drugs are approved by the FDA.

On a global scale, there are about 3.5 million severe cases of influenza illness and 300,000–500,000 deaths annually. Most at risk are the elderly, young children, and immuno- © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC compromised patients. Influenza virus can be controlled in two ways: vaccination and use of antivirals.NOT ItFOR takes atSALE least 6 monthsOR DISTRIBUTION to produce sufficient vaccine to vaccinate aNOT large proportion FOR SALE OR DISTRIBUTION of the population upon the emergence of a new strain. For this reason, antivirals are necessary to mitigate an influenza pandemic. Currently, two types of anti-influenza drugs are available: M2 ion channel blockers (amantidine © Jones & andBartlett rimantadine) Learning, and neu raminidaseLLC inhibitors: tamiflu (os©eltamivir Jones pho sphate)& Bartlett and rel Learning,enza LLC NOT FOR SALE(zanamivir). OR M2 DISTRIBUTION ion channel blockers prevent the uncoatingNOT step of FOR the lif eSALE cycle of ORinfluenza DISTRIBUTION viruses. After influenza A viruses bind to sialic acid receptors, the virus is internalized by receptor- mediated endocytosis. The low pH of the endosome causes the viral and endosomal membranes to fuse and an influx of H+ ions enter the M2 ion channels present on the influenza virus particle, allowing the viral RNA segments to be released into the cytoplasm of the host cell (uncoating). © Jones & Bartlett Learning,Antivirals LLC that block the M2 ion channel pre©vent Jones this unc &oating Bartlett step (Fi Learning,gure VF 4-3). LLC NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION(continued)

4.4 Targets for Antiviral Therapies 89 2ND REVISE 2ND REVISE © Jones & Bartlett Learning, LLC. NOT FOR SALE OR DISTRIBUTION. VIRUS FILE 4-3 Stamping Down Flu Viruses (continued) © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION

NOT FOR SALE OR DISTRIBUTION NAIs inhibit NOT FOR SALE OR DISTRIBUTION release of virions and promote clumping

Attachment Budding © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning,virion LLC NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION H+

Internalization and uncoating H+ © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning,Endoplasmic LLC H+ reticulum NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION

H+ Synthesis of Amantadine disables viral proteins M2 protein, preventing viral uncoating—virus ©rendered Jones inert & Bartlett Learning,Replication LLCof © Jones & Bartlett Learning, LLC viral RNA NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION

Figure VF 4-3 Influenza antivirals block the M2 ion channel or neuraminidase activity of influenza virus. © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION Antiviral drugs that block neuraminidase cause influenza viruses to clump at the surface of host cells, preventing its spread to other host cells at the end of the viral life cycle. Neuraminidase is an influenza protein present on the outside of the virus particle. As newly assembled viruses bud from host cells and are released, neuraminidase cleaves or removes the host cell sialic acid © Jones & Bartlett Learning, LLCreceptors present on the outside© Jones of host cells. & Bartlett When neuraminidase Learning, is blocked, LLC influenza viruses NOT FOR SALE OR DISTRIBUTIONstick to the sialic acid receptorsNOT present FOR on theSALE plasma OR membrane DISTRIBUTION of the infected host cells, causing viruses to clump and not spread to other cells of the respiratory tract (Figure VF4-3). Unfortunately, resistant strains of M2 inhibitors rapidly emerge. Resistant strains of oseltamivir (Tamiflu) are also emerging. It is believed that indiscriminate use of these drugs has led to the appearance of drug-resistant viruses. In response to the need for new anti-influenza drugs, © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC scientists are developing and testing promising new antiviral candidates. Structure-based design, NOT FORtargeting SALE the conserved OR DISTRIBUTION active site amino acids of neuraminidase, isNOT being usedFOR to SALEsynthesize OR DISTRIBUTION new high-affinity neuraminidase inhibitors. Two such candidates are CS-8958 and peramivir. Both of these are neuraminidase inhibitors. During the 2009 H1N1 pandemic, emergency authorization was given to use the FDA-unapproved © Jones & Bartlett perLearning,amivir to tre atLLC severe cases of H1N1 influenza.© PatJonesients rec &eiving Bartlett peramivir Learning, were hospitalized LLC NOT FOR SALE ORwit DISTRIBUTIONh life-threatening influenza including such comNOTplications FOR as kidSALEney fai lure.OR Per DISTRIBUTIONamivir could only be used in an injectable form.

References Bai, G. R., et al. 2009. “Amantadine- and oseltamivir-resistant variants of influenza A viruses in Thailand.” Biochem Biophys Res Comm 390:897–901. © Jones & Bartlett Learning, LLCKiso, M., et al. 2010. “Efficacy of the© new Jones neuraminidase & Bartlettinhibitor CS-8958 Learning, against H5N1 inflLLCuenza viruses.” PLoS Pathog NOT FOR SALE OR DISTRIBUTION6:2e1000786. NOT FOR SALE OR DISTRIBUTION

90 Chapter 4 Virus Replication Cycles 2ND REVISE 2ND REVISE © Jones & Bartlett Learning, LLC. NOT FOR SALE OR DISTRIBUTION. RNA Virus Mutagens: A New Class of 0 4. Replication © Jones & Bartlett Learning, LLC © Jones & BartlettAntiviral Drugs? Learning, LLC 5. Assembly NOT FOR SALE OR DISTRIBUTION NOT6. MaturationFOR SALE OR DISTRIBUTION The mechanism of action of the antiviral drug 7. Release ribavirin was a mystery for over 30 years. Drs. Joseph T. Witkowski and Roland K. Robins syn- Virus attachment occurs when a virus particle thesized ribavirin in the laboratory in 1970. In attaches to a target cell surface receptor and is the 1972, it was reported© thatJones ribavirin & Bartlett inhibited Learning, a LLC © Jones & Bartlett Learning, LLC first event in the viral life cycle. Viral receptors wide spectrum of virusesNOT in FOR vitro SALEbut its mecha-OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION are discovered via experiments that involve: nism was unknown. Today it is used to treat hepatitis C virus infections in combination with inter- • the removal of surface receptors, feron-a, SARS-CoV infections, human rabies (as • the use of monoclonal antibodies to block cell sur- in the case of the Wisconsin patient in 2004), Lassa face receptors, and fever,© and Jones some &herpesvirus Bartlett infections.Learning, It LLCis used • gene-transfer© experiments.Jones & Bartlett Learning, LLC experimentallyNOT FOR to SALE treat HBV, OR Hantaan,DISTRIBUTION Dengue, NOT FOR SALE OR DISTRIBUTION and parainfluenza virus infections. Viruses enter their target cells via fusion or In 2001 the mechanism of action was pro- receptor-mediated endocytosis. ADE has been posed and demonstrated by Shane Crotty, Craig E. shown to be an alternative mechanism of viral Cameron, and Raul Andino. Their poliovirus ex- entry into host cells. This phenomenon has been © Jones &periments Bartlett suggested Learning, that ribavirin’s LLC antiviral activ- ©observed Jones with& Bartlett Dengue, Learning, West Nile, HIV-1, LLC HIV-2, NOT FORity SALE is exerted OR through DISTRIBUTION lethal mutagenesis. In other NOTand Influenza FOR SALE A viruses. OR DISTRIBUTION words, the drug overwhelms the virus with a high Replication strategies are carried out based on mutation rate that in turn drives the virus into a the nature of the viral genome (DNA or RNA, ss genetic meltdown. Interestingly, this high muta- or ds, segmented or nonsegmented). It is often dif- tion rate does not allow the virus to escape the ficult to distinguish between the steps of assembly, inhibitory effects of the© Jonesdrug. This & discoveryBartlett pro-Learning,maturation, LLC and release of virus© particles. Jones & Bartlett Learning, LLC vides pharmaceuticalNOT companies FOR with SALE an entirelyOR DISTRIBUTION Some viruses destroy their hostNOT cells FOR (lytic SALE vi- OR DISTRIBUTION new drug strategy: RNA virus mutagens. ruses), whereas others do not. Some viruses have developed ways to overcome the conundrum of Summary entry versus exiting cell surfaces that are coated with viral receptors. © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC With the development of animal cell culture tech- Viruses are masters of mutation. Mutations can niques,NOT scientists FOR have SALE been OR able DISTRIBUTION to study virus rep- be lethal or nonlethal.NOT FOR Nonlethal SALE mutations OR DISTRIBUTION may lication via one-step growth experiments. The give rise to mutants that may increase their survi- knowledge today is very detailed and continues to vorship; for example, mutations may increase their progress so rapidly that it is impossible to cover the infectivity, result in viral drug resistance, antago- viral replication strategies used by every family of nize the host immune system, or broaden the host © Jones &viruses Bartlett in a single Learning, chapter. ThisLLC chapter presents an ©range Jones of the & virus.Bartlett Learning, LLC NOT FORoverview SALE OR of viral DISTRIBUTION replication that includes the NOTScientists FOR SALE are applying OR DISTRIBUTION the knowledge obtained seven key steps in the life cycle of a virus. They are: through molecular biology toward the design of 1. Attachment specificity antiviral drugs. These antivirals must target the 2. Penetration virus specifically to be effective in eliminating the 3. Uncoating © Jones & Bartlett Learning,virus LLC without causing harm to its© host.Jones & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION

© Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC NOT FOR SALE ORThese DISTRIBUTION questions relate to the Case Study presentedNOT FOR SALENote: ORYou DISTRIBUTIONmay need to do further research at the beginning of the chapter. to find the answers to these questions. The fol- 1. hMPV and RSV belong in the Paramyxoviridae lowing references will provide some helpful family. What type of nucleic acid genome do information. they have?© Jones Do they & Bartlett encode their Learning, own viral LLC Alto, W. A. 2004. “Human© Jones metapneumovirus: & Bartlett Learning, LLC polymerase?NOT FOR (see Figure SALE 4 ‑OR14a). DISTRIBUTION A newly describedNOT respiratory FOR SALE tract ORpatho- DISTRIBUTION 2. Draw a flowchart of the viral life cycle of gen.” JABFP 17(6):466–469. hMPV. Schildgen, O., et al. 2005. “Human metapneumo- 3. How many genes does hMPV have in its virus RNA in encephalitis patient.” EID 11(3): genome? 467–470. © Jones4. The& Bartlett first report Learning, of a fatal LLCencephalitis case Van© Den Jones Hoogen, & Bartlett B. G., et Learning,al. 2001. “A LLCnewly NOT FORassociated SALE ORwith DISTRIBUTION hMPV was published in the NOTdiscovered FOR humanSALE ORpneumovirus DISTRIBUTION isolated CDC’s Emerging and Infectious Diseases publi- from young children with respiratory tract cation in 2005. The authors recommended disease.” Nat Med 7(6):719–724. screening for patients, especially children with encephalitis symptoms of unknown © Jones & Bartlett Learning,origin. What LLC is encephalitis? © Jones & Bartlett Learning, LLC NOT FOR SALE OR5. DISTRIBUTION Have neurological symptoms been associatedNOT FOR SALE OR DISTRIBUTION with other viruses in the Paramyxoviridae family? If so, which virus(es)?

© Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION Case Study: 1 Rabies © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION In February 2009, a 17-year-old Texas teenager ar- Four days later, she continued to weaken. She rived at a hospital emergency room presenting with became agitated and “combative.” At this time, cli- symptoms of a severe headache, neck pain, dizzi- nicians did an extensive workup, collecting the pa- © Jones & Bartlettness, Learning, photophobia LLC, nausea and vomiting, and© tin-Jones tient’s& Bartlett history, Learning, and created LLCa list of possible etiolo- NOT FOR SALE ORgling DISTRIBUTION of her face and forearms. A lumbar punctureNOT FORgies SALE to explain OR DISTRIBUTIONthe encephalitis/aseptic meningitis. was performed, which revealed a very high white A breakthrough came when the teenager men- blood cell count suggesting the teen might be tioned she had been hiking and spelunking during fighting a bacterial infection. She was treated in- December (2008) and had brushed up against some travenously with the broad-spectrum antibiotic bats but didn’t recall being bitten. Serum, saliva, ceftriaxone.© No Jones bacteria & Bartlettwere cultured Learning, from her LLC and cerebral spinal fluid© Jones were drawn & Bartlett or collected Learning, LLC cerebral spinalNOT fluid FOR so SALEthe antibiotic OR DISTRIBUTION was discon- from the patient, a nuchalNOT skinFOR biopsy SALE was OR per- DISTRIBUTION tinued. After a 3-day hospital stay, she was re- formed, and the clinical specimens were sent to the leased because her symptoms had resolved. CDC to test for rabies antibodies, rabies antigens, or About a week later, she returned to the hospital the RNA genome of rabies virus. with the same symptoms. She also had a rash on The next day, no rabies RNA or virus antigens © Jonesher arms & Bartlett and back. Learning, Magnetic resonanceLLC imag- were© detected. Jones Four & Bartlett serum samples Learning, tested positiveLLC NOTing FOR (MRI) SALE was ORperformed DISTRIBUTION on her head. The MRI for anti-rabiesNOT FOR antibodies SALE byOR direct DISTRIBUTION fluorescent an- showed enlarged lateral ventricles inside her tibody tests, however. Anti-rabies antibodies were brain. The size was abnormal for her age and she also found in the cerebral spinal fluid. Four days was given the diagnosis of encephalitis. She was later, the teenager was given 1 dose of rabies vac- hospitalized and treated with ceftriaxone and anti- cine and 1,500 international units (IU) of human © Jones & Bartlettbiotics Learning, used to treat LLC tuberculosis. © Jones rabies& Bartlett immunoglobulin. Learning, LLC NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION

92 Chapter 4 Virus Replication Cycles 2ND REVISE 2ND REVISE © Jones & Bartlett Learning, LLC. NOT FOR SALE OR DISTRIBUTION. Case Study: 1 Rabies (continued)

© Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION Eight days later, her symptoms resolved and 3. Assuming the teenager was not bitten, what she was released from the hospital. Another week could be another plausible explanation for passed and she returned to the emergency room of how the teenager contracted this infection? a hospital with recurring headache but left before a 4. Rabies virus is a member of the Rhabdoviridae lumbar puncture was© performed. Jones & A Bartlettfew more daysLearning, LLCfamily. What are the molecular© Jones characteristics & Bartlett Learning, LLC passed. Again, she hadNOT a recurring FOR SALE headache. OR DISTRIBUTION A of this family of viruses (e.g.,NOT particle FOR charac- SALE OR DISTRIBUTION lumbar puncture was performed. Her headache re- teristics, life cycle)? solved and she has not been rehospitalized since. 5. How many genes do rabies viruses carry and Only her boyfriend met the criteria of requiring a what are their function(s)? series of rabies vaccinations. 6. What types of cells do rabies viruses replicate 1. ©Create Jones a hypothesis & Bartlett as toLearning, why this teenager LLC in (hint: Table© Jones 4-1)? & Bartlett Learning, LLC NOTsurvived FOR a rabies SALE virus OR infection. DISTRIBUTION 7. Rabies bindNOT to what FOR host SALE receptor(s)? OR DISTRIBUTION 2. What do the CDC researchers speculate as 8. Who should receive a rabies vaccination? to why this patient survived rabies? Refer (hint: http://www.cdc.gov/rabies/) to the CDC report related to this case published February 26, 2010 (MMWR 59[7]; For more information see Chapter 13 (Rabies). © Jones & BartlettMMWR Learning, can be found LLC at: http://www.cdc © Jones & Bartlett Learning, LLC NOT FOR SALE.gov/mmwr/). OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION

Dimiter, S. D. 2000. “Cell biology of virus entry.” Cell Resources 101:697–702. Drake, J. W., and Holland, J. J. 1999. “Mutation rates of © Jones & Bartlett Learning, LLC RNA viruses.”© PNAS Jones USA 96(24):13910–13913. & Bartlett Learning, LLC PrimaryNOT Literature FOR SALE and Reviews OR DISTRIBUTION Enders, J. F., Robbins,NOT F. C., FOR and Weller, SALE T. H. OR Nobel DISTRIBUTION Lecture, Ackermann, H-W., Berthiaume, L., and Tremblay, M. 1998. December 11, 1954. “The cultivation of the poliomyeli- “The replication cycle.” In Virus Life in Diagrams. Boca tis viruses in tissue culture.” Raton, FL: CRC Press. Flint, S. J., Enquist, L. W., Krug, R. M., Racaniella, V. R., Audelo-del-Valle, J., et al. 2003. “Infection of cultured and Skalka, A. M., eds. 2000. “Genome replication and human and monkey cell lines with extract of penaeid mRNA production by RNA viruses.” In Principles of © Jones & Bartlettshrimp infected Learning, with Taura LLC syndrome virus.” EID © JonesVirology: & Molecular Bartlett Biology, Learning, Pathogenesis, LLC and Control. NOT FOR SALE9(2):265–266. OR DISTRIBUTION NOTWashington, FOR SALE DC: ASM OR Press. DISTRIBUTION Avril, R. M. C-C., Dixon, D. W., Vzorov, A. N., Marzilli, L. G., Fu, Y-X. 2001. “Estimating mutation rate and generation and Compans, R. W. 2003. “Prevention of poxvirus infec- time from longitudinal samples of DNA sequences.” Mol tion by tetrapyrroles.” BMC Inf Dis 3:9. http://www Biol Evol 18(4):620–626. .biomedcentral.com/1471-2334/3/9. Junge, R. E., Duncan, M. C., et al. 1999. “Clinical presenta- Baranowski, E., Ruiz-Jarabo,© Jones C.M., and & Domingo, Bartlett E. 2001. Learning, tionLLC and antiviral therapy for poxvirus© Jones infection & Bartlett in Learning, LLC “Evolution of cell recognition by viruses.” Science Pudu (Pudu Pudu).” J Zoo Wildlife Med 31(3):412–418. 292:1102–1105. NOT FOR SALE OR DISTRIBUTIONKnipe, D. M., and Howley, P. M., eds. 2001.NOT “Virus FOR assembly.” SALE OR DISTRIBUTION Boriskin, Y. S., et al. 2006. “Arbidol: A broad-spectrum In Fields Virology, 4th ed. Philadelphia: Lippincott antiviral that inhibits acute and chronic HCV infection.” Williams & Wilkins. Virol J 3:56. Knipe, D. M., Samuel, C. E., and Palese, P., eds. 2001. Coffin, J. M. 1995. “HIV population dynamics in vivo: “Principles in virology and virus entry and uncoating.” Implications© Jones for & genetic Bartlett variation, Learning, pathogenesis, LLC and In Fundamental© Virology Jones. Philadelphia: & Bartlett Lippincott Learning, Williams LLC therapy.” Science 267:483–489. & Wilkins. Cohen,NOT J., and FOR Powderly, SALE W. G., OR eds. 2004.DISTRIBUTION Infectious Diseases, Martina, B. E. E., NOTHaagmans, FOR B. L.,SALE Kuiken, OR T., et DISTRIBUTION al. 2003. 2nd ed. Philadelphia: Mosby. “SARS virus infection in cats and ferrets.” Nature 425:915. Crotty, S., Cameron, C. E., and Andino, R. 2001. “RNA Mendelsohn, C. L., et al. 1986. “Transformation of a human virus error catastrophe: Direct molecular test by using poliovirus receptor gene into mouse cells.” PNAS USA ribavirin.” PNAS USA 98(12):6895–6900. 83(20):7845–7849. © Jones &De BartlettClercq, E. 2002. Learning, “Cidofovir inLLC the treatment of poxvirus ©Mendelsohn, Jones & C. Bartlett L., Wimmer, Learning, E., and Racaniello, LLC V. R. 1989. NOT FOR SALEinfections.” OR Antiviral DISTRIBUTION Res 55(1):1–13. NOT“Cellular FOR receptorSALE forOR poliovirus: DISTRIBUTION Molecular cloning,

Resources 93 2ND REVISE 2ND REVISE © Jones & Bartlett Learning, LLC. NOT FOR SALE OR DISTRIBUTION. nucleotide sequence, and expression of a new member Popular Press © Jones & Bartlett Learning,of the immunoglogulin LLC superfamily.” Cell 56:855–865.© Jones & Bartlett Learning, LLC Haseltine, W. A. 2001. “Beyond chicken soup.” Scientific NOT FOR SALE ORMothes, DISTRIBUTION W., et al. 2000. “Retroviral entry mediatedNOT by FOR SALE OR DISTRIBUTION receptor priming and low pH triggering of an envelope American, November 2001, Vol. 285, Issue 5, 56–63. glycoprotein.” Cell 103:679–689. Tiollais, P., and Buendia, M-A. 1991. “Hepatitis B virus.” Paulson, J. C., and Rogers, G. N. 1987. “Resialylated eryth- Scientific American,April 1991, Vol. 264, Issue 4, 116–123. rocytes for assessment of the specificity of sialyloligo- Villarreal, L. P. 2004. “Are viruses alive? Although viruses saccharide binding proteins.” Methods Enzymol 138: challenge our concept of what ‘living’ means, they are 162–168.© Jones & Bartlett Learning, LLC vital members of the© web Jones of life.” & Scientific Bartlett American, Learning, LLC Pluong, C. X.NOT T., et al. FOR 2004. SALE“Clinical diagnosisOR DISTRIBUTION and assess- December 2004, Vol. 291,NOT Issue FOR 6, 100–105. SALE OR DISTRIBUTION ment of severity of confirmed dengue infections in Wong, K. 2002. “Oral drug halts smallpoxlike virus in Vietnamese children: Is the World Health Organization mice.”Scientific American, News Online, March 2002. classification system helpful?”Am J Trop Med Hyg 70(2): Wright, L. “To vanquish a virus.” 2003. Scientific American in 172–179. Focus Online, July 21, 2003. Schaechter, M., ed. “Antiviral agents.” In Encyclopedia of © JonesMicrobiology & Bartlett, 3rd Edition.Learning, Amsterdam: LLC Elsevier Ltd. © Jones & Bartlett Learning, LLC NOT FOR2009. SALE OR DISTRIBUTION VideoNOT Productions FOR SALE OR DISTRIBUTION Sieczkarski, S. B., and Whittaker, G. R. 2005. “Viral Entry.” Understanding Viruses. 1999. The Discovery Channel. Curr Top Microbiol Immunol 285:1–23. The Next Plague: The Nipah Virus. 1998. The Discovery Channel. Smith, A. E., and Helenius, A. 2004. “How viruses enter Viruses: The Deadly Enemy. 1996. Available at www.insight- cells.” Science 304:237–242. media.com. Human Relations Media. Staunton, D. E., Merluzzi, V. J., Rothlein, R., et al. 1989. “A © Jones & Bartlett Learning,cell adhesion molecule,LLC ICAM-1, is the major surface© Jones The& BartlettEmerging Viruses Learning,. 1991. British LLC Broadcasting Corporation. NOT FOR SALE ORreceptor DISTRIBUTION for rhinoviruses.” Cell 56:849–853. NOT FOR SALE OR DISTRIBUTION Stine, G. J. 2002. “Anti-HIV therapy.” In AIDS Update 2002. Upper Saddle River, NJ: Prentice-Hall. eLearning Tyler, K. L. 2004. “Isolation and molecular characterization go.jblearning.com/shors2 of a novel type 3 reovirus from a child with meningitis.” The site features eLearning, an online review area J Infect Dis 189(9):1664–1675. that provides quizzes and other tools to help you Wilkin, T. J., ©et al.Jones “HIV Type & Bartlett1 chemokine Learning, coreceptor use LLC © Jones & Bartlett Learning, LLC study for your class. You can also follow useful among antiretroviral-NOT FORexp SALEerienced patientsOR DISTRIBUTION screened for NOT FOR SALE OR DISTRIBUTION a clinical trial of a CCR5 inhibitor: AIDS clinical trial links for in-depth information, or just find out the group A5211.” Clin Infect Dis 44:591–595. latest virology and microbiology news.