1 7 WS_ viral replication and antiviral targets.pdf

1/26/24, 1:13 AM Week 2: Fusion Session | Workshop: Viral Replication and Antiviral Targets: Hemtlgy Onclgy Infectn Imm - January 2024 6. Differentiate the concepts of susceptibility and permissiveness (permissibility), and discuss their influence on tissue tropism, pathogenesis, and host specific...

1/26/24, 1:13 AM Week 2: Fusion Session | Workshop: Viral Replication and Antiviral Targets: Hemtlgy Onclgy Infectn Imm - January 2024 6. Differentiate the concepts of susceptibility and permissiveness (permissibility), and discuss their influence on tissue tropism, pathogenesis, and host specificity. 7. Explain what is happening inside the host cell during the eclipse and latent phases of the viral replication process. 8. Differentiate immediate early, early, and late proteins as they apply to viral replicative cycles. 9. Explain the differences between different types of nucleic acid polymerases and why some viruses encode their own polymerases and cell-cycle regulators. 10. Compare and contrast the replication of 5 prototype viruses, distinguishing variations in mechanisms of entry, nucleic acid replication, protein production, assembly, and release. By the end of this session, you will be able to meet the following learning objectives: 1. Identify the goals of antiviral therapy. 2. Explain the mechanism of action of the included antiviral medications. 3. Describe some known mechanisms of viral resistance 4. Classify antiviral drugs based on their site of action.  Viral replication is complex and varied, and having learned basic models can be very helpful when confronted with this variability. A few questions can help keep the focus on key areas. Look for answers to these as you progress through the material. 1. How can viruses attach, enter, and uncoat? 2. How can viruses replicate their genomes, and where? 3. Where are structural and genomic components made, and how does the assembly process occur? 4. How does the virus exit? https://rossmed.instructure.com/courses/3318/pages/week-2-fusion-session-%7C-workshop-viral-replication-and-antiviral-targets 3/49 1/26/24, 1:13 AM Week 2: Fusion Session | Workshop: Viral Replication and Antiviral Targets: Hemtlgy Onclgy Infectn Imm - January 2024 Viruses differ immensely from cellular infectious agents. Viruses cannot metabolize and, therefore, cannot make energy, encoded proteins, or anything else. Instead, viruses are infectious, obligately intracellular parasites that may commandeer the machinery of living cells to replicate themselves. They must minimally contain a nucleic acid genome and a protein coating called a capsid, and all of their components derive from host cells. Unlike cells, they do not divide to replicate; rather, they assemble from individual components. Since viruses are so very different from other microbes, it is perhaps not surprising that antibacterial, antiparasitic, and antifungal drugs do not effectively prevent viral replication; viruses simply do not possess the targets of these various types of antiinfective agents. Since viruses are generally not considered to be alive, they cannot be killed; instead, they are “inactivated” or “neutralized” and, in such a state, are unable to infect a host cell. How do viruses differ from other microbes? Cannot make energy or encoded proteins Metabolically inert (not living) Require living cells to replicate (obligate intracellular parasites) Components derived from host, often virally-encoded Assemble rather than divide Unaffected by antibacterials, antifungals, and antiparasitics https://rossmed.instructure.com/courses/3318/pages/week-2-fusion-session-%7C-workshop-viral-replication-and-antiviral-targets 4/49 1/26/24, 1:13 AM Week 2: Fusion Session | Workshop: Viral Replication and Antiviral Targets: Hemtlgy Onclgy Infectn Imm - January 2024 Size comparison of viruses with other microbes. An E. Coli cell is 1-2 microns in length. Human viral pathogens are contained within 16 families of RNA viruses and 8 families of DNA viruses. Viruses from the same family can cause different diseases (e.g., different picornaviruses https://rossmed.instructure.com/courses/3318/pages/week-2-fusion-session-%7C-workshop-viral-replication-and-antiviral-targets 5/49 1/26/24, 1:13 AM Week 2: Fusion Session | Workshop: Viral Replication and Antiviral Targets: Hemtlgy Onclgy Infectn Imm - January 2024 cause poliomyelitis, common colds, hepatitis), while those from different families can cause the same disease [e.g., diarrheas (Reoviridae, Caliciviridae, Adenoviridae, Astroviridae)]. Example Viruses/Diseases by Family Family Example virus/disease Poxviridae Smallpox (variola), monkeypox, molluscum contagiosum Herpesviridae Herpes, chickenpox, EBV, CMV, roseola, Kaposi’s sarcoma Hepadnaviridae Hepatitis B virus Picornaviridae Rhinoviruses, enteroviruses (e.g., poliovirus), hepatitis A Filoviridae Ebola virus, Marburg virus Parvoviridae Human parvovirus B19 Flaviviridae Hepatitis C virus, yellow fever, West Nile virus, dengue Paramyxoviridae Measles virus, Nipah virus, Mumps virus Togaviridae Rubella (German measles) virus, Chikungunya virus Retroviridae HIV, HTLV Some basic terminology will be helpful when thinking about viruses. 1. Virus: a general term for the entity, for example, “the measles virus.” This includes both virions and defective virus-like particles, which are the outcome of assembly errors or immaturity. 2. Virion: a single, infective, complete, and mature viral particle. Virions are able to deliver the viral genome and lead to a productive infection in an appropriate host cell (more on that later). 3. Bacteriophage: a type of virus that infects bacteria. These may be important in the acquisition of virulence factors and antibiotic resistance phenotypes among bacteria and may also be used to treat infections. 4. Genome: the nucleic acid encoding the instructions to create new viral particles 5. Capsid: a protein layer that encompasses and protects the genome https://rossmed.instructure.com/courses/3318/pages/week-2-fusion-session-%7C-workshop-viral-replication-and-antiviral-targets 6/49 1/26/24, 1:13 AM Week 2: Fusion Session | Workshop: Viral Replication and Antiviral Targets: Hemtlgy Onclgy Infectn Imm - January 2024 6. Symmetry: a way to classify viral capsid shapes; common ones are helical (filamentous), icosahedral, or complex (when one of the other terms doesn’t apply) 7. Shape: a way to classify the overall shape of a virus, which may be very different from its symmetry; shapes may be quite varied and include spherical, icosahedral, filamentous, and complex 8. Nucleoprotein: a specific type of protein that complexes to nucleic acid; in the case of viruses, these may also be capsid proteins 9. Nucleocapsid: nucleoproteins complexed directly to nucleic acid form this structure 10. Icosahedron: a shape made of 20 triangles arranged into pentagons and 12 vertices, for example, a 20-sided die; several small viruses have this symmetry, but many larger ones contain arrangements of both 5 and 6 triangles to form both pentagons and hexagons, like a soccer ball. https://rossmed.instructure.com/courses/3318/pages/week-2-fusion-session-%7C-workshop-viral-replication-and-antiviral-targets 7/49 1/26/24, 1:13 AM Week 2: Fusion Session | Workshop: Viral Replication and Antiviral Targets: Hemtlgy Onclgy Infectn Imm - January 2024 Diagram of an icosahedron showing 12 vertices, 20 faces, and 30 sides. The colored balls indicate the position of protomers forming a pentamer on the icosahedron. 11. Envelope: a lipid bilayer derived from the host cell that encompasses some viral capsids 12. Naked: a way to describe a non-enveloped virus (that is, one without a lipid bilayer) 13. Viral attachment protein: an adhesin on the surface of a virus that is involved in binding to a cell receptor 14. Spike glycoprotein: another name for a viral attachment protein 15. Matrix: protein layer between the capsid and envelope of some viruses that connect these structures  https://rossmed.instructure.com/courses/3318/pages/week-2-fusion-session-%7C-workshop-viral-replication-and-antiviral-targets 8/49 1/26/24, 1:13 AM Week 2: Fusion Session | Workshop: Viral Replication and Antiviral Targets: Hemtlgy Onclgy Infectn Imm - January 2024 While a number of viruses have many different components, there are three main structures to understand well. Click below to explore the three main components of viruses. Viral genomes are quite varied and may be DNA or RNA, single- or double-stranded, linear, circular, or even segmented or polyploid. But no matter the structure, the genome contains all of the information needed to direct a host cell to build the specific viral components. The capsid can be viewed as the main protective shell of a virus, with its key role being to package and protect the nucleic acid genome and any essential proteins that must be packaged with it for a productive infection to occur (more on that later). Capsids are built from many copies of one or several proteins, which self-assemble either directly onto the nucleic acid genome (forming a nucleoprotein complex called the nucleocapsid) or into a distinct structure that surrounds it, which is frequently icosahedral in symmetry. Schematic drawing of two basic types of virions, naked capsid virus, and enveloped virus. In a naked capsid virus, the genome is condensed with a defined external capsid (coat protein), whereas enveloped virus has a nucleocapsid or capsid wrapped in a lipid bilayer envelope. https://rossmed.instructure.com/courses/3318/pages/week-2-fusion-session-%7C-workshop-viral-replication-and-antiviral-targets 9/49 1/26/24, 1:13 AM Week 2: Fusion Session | Workshop: Viral Replication and Antiviral Targets: Hemtlgy Onclgy Infectn Imm - January 2024 The capsid also plays an essential role in the delivery of nucleic acid into the host cell upon infection, as it must release the nucleic acid at the right time (called uncoating). In naked viruses, capsid proteins include the attachment protein, and therefore this structure is also responsible for adhesion. The simplest viruses consist of a genome encompassed by many copies of a single capsid protein, forming a nucleocapsid (same as nucleoprotein in this case). The variety among viruses is almost endless, with some complex viruses even containing a filamentous nucleoprotein surrounded by a second icosahedral capsid layer. Some viruses may also comprise a matrix, viral proteins and enzymes, or non-genomic nucleic acids such as specialized tRNAs or mRNA) among other things. This is another layer of protection for some viruses, and it is important to know which viruses have one, as it has important implications for how the virus replicates, how it might transmit and infect, and how it may be inactivated. Envelopes are derived from host cell membranes, either from an organelle or the plasma membrane, into which key viral proteins and glycoproteins have been placed. The viral attachment proteins, or spike glycoproteins, of an enveloped virus, will be found in the envelope as this is the structure in contact with the environment and thus in contact with host cell surfaces. The envelope also carries fusion proteins, which are important in helping enveloped viruses enter their target host cells. Usually, the envelope is acquired as the virus buds through a particular section of a membrane known as a lipid raft. These are areas of the membrane that proteins can be targeted to. The key role of the envelope is to deliver the nucleocapsid into the host cell by carrying the viral attachment proteins and then fusing with a host cell membrane (either the plasma membrane or the endosomal membrane after receptor-mediated endocytosis) to release the nucleocapsid. https://rossmed.instructure.com/courses/3318/pages/week-2-fusion-session-%7C-workshop-viral-replication-and-antiviral-targets 10/49 1/26/24, 1:13 AM Week 2: Fusion Session | Workshop: Viral Replication and Antiviral Targets: Hemtlgy Onclgy Infectn Imm - January 2024  Viruses come in different sizes (from about 18-300 nm) and shapes. Many are icosahedral, some are helical (filamentous), and some are complex (or pleiomorphic), spherical, or even ovoid. Some bacteriophages (the group of viruses infecting bacteria) also possess peculiar structures (one looks like a lunar lander). The simpler and smaller a virus is, in general, the more resistant it will be to environmental stresses such as heat and chemicals. Enveloped viruses, for example, are often susceptible to: https://rossmed.instructure.com/courses/3318/pages/week-2-fusion-session-%7C-workshop-viral-replication-and-antiviral-targets 11/49 1/26/24, 1:13 AM Week 2: Fusion Session | Workshop: Viral Replication and Antiviral Targets: Hemtlgy Onclgy Infectn Imm - January 2024 1. heat, as their lipid bilayer will melt much more easily than a protein capsid will become denatured; 2. drying, which is not an issue for naked viruses; 3. mild solvents, such as detergents or bile, which dissolve lipids but don’t affect proteins; and 4. acid, which affects the envelope much more than it does capsids. So, considering the various conditions in the external environment, in different parts of the body, and the stresses induced by different types of disinfectants, it becomes apparent that the structure of the virus can introduce significant limitations and explain many observations around pathogenic viruses. For example: 1. Enveloped viruses will survive poorly in the external environment, so will likely need to move from host to host quickly (often via direct contact or inhalation of wet droplets). Naked viruses could survive for quite a long time on fomites (inanimate objects) and be transmitted much more insidiously. 2. Enveloped viruses will not generally reach the intestine due to their acid sensitivity or survive in the intestine due to the presence of bile. Diarrhea-causing viruses are almost always naked. 3. Detergents and mild disinfectants are more likely to inactivate enveloped viruses than naked viruses, which becomes evident when considering that the naked norovirus is not susceptible to hand sanitizer and instead needs much higher levels of alcohol to disrupt. It is important to remember, however, that the envelope allows a virus to carry many more proteins on its surface than a naked virus can, conferring benefits that must more than makeup for these downsides.  Since there are SO MANY viruses, it would be very difficult to learn their characteristics all at once. However, they are grouped into families based on shared genetics and characteristics, so this greatly reduces the complexity. Viral families end in the suffix -viridae; for example, the family Retroviridae contains the retroviruses, of which human immunodeficiency virus (HIV) is an example. The family Paramyxoviridae contains paramyxoviruses, an example of which is the measles virus. Click through this interactive to learn more about the classification of viruses by family. https://rossmed.instructure.com/courses/3318/pages/week-2-fusion-session-%7C-workshop-viral-replication-and-antiviral-targets 12/49 1/26/24, 1:13 AM Week 2: Fusion Session | Workshop: Viral Replication and Antiviral Targets: Hemtlgy Onclgy Infectn Imm - January 2024 Basics of Viral Classification Start Genome Composition: DNA or RNA Strandedness: Single, double, partially double Arrangement: Linear, circular, segmented, diploid Capsid (nucleocapsid) symmetry Helical (helicoidal, filamentous) Icosahedral (spherical) Envelope Presence (enveloped) Absence (non-enveloped, naked) Shape Regular, spherical, icosahedral Complex https://rossmed.instructure.com/courses/3318/pages/week-2-fusion-session-%7C-workshop-viral-replication-and-antiviral-targets 13/49 1/26/24, 1:13 AM Week 2: Fusion Session | Workshop: Viral Replication and Antiviral Targets: Hemtlgy Onclgy Infectn Imm - January 2024 Genomes Animal viral genomes can exist as either DNA or RNA. DNA genomes can either be linear or circular, whereas genomic RNA is always linear. However, RNA genomic nucleic acids can be segmented (i.e., in pieces) in some virus groups, whereas DNA genomes are never segmented. Structures Animal viruses also come in different sizes and structures. Some viruses can be helical (tubular or helicoidal), icosahedral, or non-helical/non-icosahedral [e.g., complex (or pleiomorphic), spherical, or ovoid]. Finally, some viruses are characterized by the presence of a host-derived envelope, whereas others are referred to as naked (absence of an envelope). The following flowcharts focus on the genome nucleic acid first, then the genome structure, and then whether the family has an envelope or not. This is good to start with, and then as you progress through the remaining topics, you might find you should add information, for example, the capsid symmetry or virus shape. But for now, let’s start with the two most important types of characteristics. You will see that 10 of these families are in red text – these are the families that you can be tested on in this module, and they contain viruses that cause diseases. You will learn more about this term. Some of the terms will become more familiar as you read the following sections, and connecting family names to the replication cycles as you go through them will help in learning both the families and the replication cycles. https://rossmed.instructure.com/courses/3318/pages/week-2-fusion-session-%7C-workshop-viral-replication-and-antiviral-targets 14/49 1/26/24, 1:13 AM Week 2: Fusion Session | Workshop: Viral Replication and Antiviral Targets: Hemtlgy Onclgy Infectn Imm - January 2024 DNA Double-stranded Enveloped PoxHerpesNaked PapillomaPolyomaAdenoPartial double-stranded (gapped) Enveloped Hepadna-(iRNA) Single-stranded Naked ParvoNote: Characteristics of families in red must be learned for this module. https://rossmed.instructure.com/courses/3318/pages/week-2-fusion-session-%7C-workshop-viral-replication-and-antiviral-targets 15/49 1/26/24, 1:13 AM Week 2: Fusion Session | Workshop: Viral Replication and Antiviral Targets: Hemtlgy Onclgy Infectn Imm - January 2024 RNA Single-stranded (+) Naked PicornaCaliciHepeAstroEnveloped TogaFlaviRetro- (diploid, iDNA) Corona(-) https://rossmed.instructure.com/courses/3318/pages/week-2-fusion-session-%7C-workshop-viral-replication-and-antiviral-targets 16/49 1/26/24, 1:13 AM Week 2: Fusion Session | Workshop: Viral Replication and Antiviral Targets: Hemtlgy Onclgy Infectn Imm - January 2024 Enveloped FiloParamyxoPneumoRhabdoDeltaSegmented Enveloped ArenaBunyaOrthomyxoDouble-stranded Segmented Naked Reoviridae Note: Characteristics of families in red must be learned for this module.  https://rossmed.instructure.com/courses/3318/pages/week-2-fusion-session-%7C-workshop-viral-replication-and-antiviral-targets 17/49 1/26/24, 1:13 AM Week 2: Fusion Session | Workshop: Viral Replication and Antiviral Targets: Hemtlgy Onclgy Infectn Imm - January 2024 1. What type of molecule is found in an enveloped virus but not a naked virus? Carbohydrate Lipid (answer) Capsid protein RNA DNA 2. What is the primary role of the viral capsid? Anchor the envelope to the genome Protect the genome (answer) Attach to a host cell receptor Dampen the immune response Fusing with a host cell membrane https://rossmed.instructure.com/courses/3318/pages/week-2-fusion-session-%7C-workshop-viral-replication-and-antiviral-targets 18/49 1/26/24, 1:13 AM Week 2: Fusion Session | Workshop: Viral Replication and Antiviral Targets: Hemtlgy Onclgy Infectn Imm - January 2024 3. Drag the viral families into the correct category: dsDNA: Pox, Herpes Gapped DNA: Hepadna ssDNA: Parvo +ssRNA: Picorna, Toga, Flavi, Retro -ssRNA: Filo, Paramyxo 4. Drag the viruses into the correct category: Enveloped: Rubella virus Dengue virus Human immunodeficiency virus Ebola virus Measles virus Mpox virus Chickenpox virus Naked: Parvovirus B19 Poliovirus 5. What type of infection would a virus exhibiting tropism for pneumocytes cause? Liver infection CNS infection Gastrointestinal tract infection Respiratory tract infection (answer) Kidney infection 6. Which part of an enveloped virion would be the most likely target of an effective neutralizing antibody? Nucleic acid genome Nucleoprotein Matrix protein Host-derived membrane lipid Membrane glycoprotein (answer)  https://rossmed.instructure.com/courses/3318/pages/week-2-fusion-session-%7C-workshop-viral-replication-and-antiviral-targets 19/49 1/26/24, 1:13 AM Week 2: Fusion Session | Workshop: Viral Replication and Antiviral Targets: Hemtlgy Onclgy Infectn Imm - January 2024 As obligate parasites, viruses must multiply and be transmitted to new hosts in order to continue to exist. As obligate intracellular parasites, they must do this within a host cell, using host cell machinery and building blocks to make (1) copies of the viral genome and (2) viral proteins. This means that they must be able to enter a cell that can recognize, follow, and complete the instructions coded within the viral genome. Some viruses even modify the function of that cell to make it better suited to do so. Because they are using host cell machinery, being able to explain how host cells produce nucleic acids and proteins will be very helpful. The viral replicative cycle generally consists of 7 steps 1. Attachment (adsorption or binding) 2. Entry (penetration) 3. Uncoating 4. Replication (nucleic acid and protein production) 5. Assembly and packaging 6. Release 7. Maturation Note that, in some cases, some replication steps can take place simultaneously (e.g., assembly and release) or even be inverted (e.g., release and maturation). Each of these steps will be described in more detail below, and each is the target of at least one antiviral drug or treatment currently in use (and hopefully more to come!) https://rossmed.instructure.com/courses/3318/pages/week-2-fusion-session-%7C-workshop-viral-replication-and-antiviral-targets 20/49 1/26/24, 1:13 AM Week 2: Fusion Session | Workshop: Viral Replication and Antiviral Targets: Hemtlgy Onclgy Infectn Imm - January 2024 Virus replication cycle. A general scheme of the six discrete steps of the virus replication cycle, including attachment, penetration, uncoating, synthetic phase (transcription, translation, and replication), assembly, and release. Maturation may occur at various points during the cycle, depending on the virus. The cycle generally begins when a virion binds its cognate receptor(s) on the surface of a susceptible cell via a viral attachment protein. A susceptible cell is one that the virus is able to attach to; that is, it has the needed receptor on its surface. The virion might then be internalized, and if the host cell is permissive, that is, is able to follow and complete the viral instructions, infective viral particles will be produced. If the host cell entered is not permissive, for example, does not recognize the viral promoter, then the cycle will be non-productive, and that particular viral particle will never replicate. Cell susceptibility and permissiveness determine host range, also referred to as host specificity or host tropism. These also determine organ, tissue, and cell tropism and help define important aspects of both the epidemiology and the pathophysiology of a particular viral infectious disease. These are important concepts that possess clinical relevance, for example, in explaining why some viruses are limited to infecting a certain tissue or organ or why some infect birds but not humans. Some viruses can bind more than one type of cell surface receptor or bind a receptor present on many cell types and thus can enter a wider range of cell types (SARS-CoV-2 is a perfect example of the latter). Host and cell specificity are key to understanding infection by many microbes, not only viruses. https://rossmed.instructure.com/courses/3318/pages/week-2-fusion-session-%7C-workshop-viral-replication-and-antiviral-targets 21/49 1/26/24, 1:13 AM Week 2: Fusion Session | Workshop: Viral Replication and Antiviral Targets: Hemtlgy Onclgy Infectn Imm - January 2024 The cells/tissues/organisms that will be affected by a virus are determined by: Host cell susceptibility Possesses surface receptors/co-receptors allowing viral attachment “Cell can be recognized” Host cell permissiveness Contains all components required for virion production (i.e., leads to a productive infection) “Cell allows replication” Virus needs both host cell susceptibility and host cell permissiveness in order to propagate. It is also important to realize that viruses are not like bacteria or eukaryotes in that they do not divide to propagate. Instead, once they enter the cell, they disassemble and release their genome, which directs the production of new components. Newly-produced components assemble, producing new viral particles. So, once a virus has entered a host cell, it no longer “exists” as a virus, just as a genome. The period between when an infecting virus has fallen apart and when the first progeny are assembled inside the cell is called the eclipse period. Replication may occur https://rossmed.instructure.com/courses/3318/pages/week-2-fusion-session-%7C-workshop-viral-replication-and-antiviral-targets 22/49 1/26/24, 1:13 AM Week 2: Fusion Session | Workshop: Viral Replication and Antiviral Targets: Hemtlgy Onclgy Infectn Imm - January 2024 immediately after viral entry or perhaps after a period of inactivity, such as when a viral genome enters the host DNA and does not replicate. The time between viral entry and new progeny release is called the latent period and may range from hours to years. Note that latency may also mean the time between someone being infected and becoming infectious, which might lead to confusion! In the graph, the red line measures virions in the culture medium (outside the cell), and the blue line measures virions inside the cells. The term plaque-forming unit, abbreviated as "pfu", is a way to visualize the presence of an infectious lytic viral particle and count them, as these can infect a cell within a layer of cultured cells and spread to adjacent cells, forming a "plaque" of missing cells in the layer. In this example, infectious virions appear within the cell before they begin to be released to the extracellular medium, which tells us that assembly and release occur sequentially, not simultaneously. New viral particles may be released as they are made, leading to constant production, or may build up and burst out of the cell, as shown in the example here, leading to waves of viral production.  https://rossmed.instructure.com/courses/3318/pages/week-2-fusion-session-%7C-workshop-viral-replication-and-antiviral-targets 23/49 1/26/24, 1:13 AM Week 2: Fusion Session | Workshop: Viral Replication and Antiviral Targets: Hemtlgy Onclgy Infectn Imm - January 2024 The first step in the typical viral replicative cycle is the binding of the virion to the host cell. Viral attachment to cells is a random event. Viral adhesion is mediated through receptor-ligand interactions. Some viral attachment proteins (VAPs) may be very specific, with only one known receptor, while others may bind a variety of receptors. Some may also require binding to more than one receptor (co-receptors) to trigger cell entry. VAPs may often project from the viral surface and are often called “spikes.” Receptors may be any cell surface molecule. The initial VAP-cell receptor contact is(are) likely due to electrostatic forces, but once contact is established, other short-range forces, such as hydrophobic forces, rapidly follow. These short-range forces often lead to conformational changes that initiate the molecular cascade that causes virion entry. It is important to bear in mind that oftentimes, the VAPs are not the “natural” ligand of the cell receptor, which means that often times, the molecular interactions between VAPs and cell receptors are of low affinity. Therefore, virion binding to a single receptor molecule results in very weak adhesion. This is compensated by the presence of many closely spaced VAPs at the surface of the virion. In other words, weak molecular specificity is counterbalanced by molecular avidity. Usually, VAP-specific antibodies will block binding, neutralizing the virus, which is one reason these are so often used as vaccine targets. The VAP of a naked virus will be a part of the viral capsid, whereas that of an enveloped virus will be a membrane protein, usually a glycoprotein. The next step in the viral replicative cycle is the entry of the virion into the host cell. Entry generally occurs using endocytosis, membrane fusion, or a combination of the two. It may also be blocked by neutralizing antibodies. Most naked viruses enter cells using an endocytic pathway (phagocytosis, clathrin-dependent, clathrin-independent, or caveolae-dependent; the process may be termed viropexis). As a general rule, viral penetration (entry into the cytosol) originates from the endosomes, which progressively have a lower pH as they mature. This is important for uncoating, as described in section 3. Following endocytosis, penetration by naked viruses is the result of one of three mechanisms: (1) membrane puncture, where the viral nucleic acid is inserted into the cytosol through a pore created by the virus, and entry and uncoating are one and the same process; (2) perforation, which is the transfer of the complete virion through the membrane without any major lysis incurring on the part of the membrane; and (3) lysis, a mechanism through which the viral particle escapes from an endosomal compartment by rupturing it. Some naked viruses enter directly through the plasma membrane, a form of membrane puncture. Entry of enveloped viruses can occur by (1) fusion of the viral envelope and the plasma membrane of the cell, which results in penetration, or (2) endocytosis followed by fusion of the viral envelope and the endosomal membrane, again resulting in penetration. Click each tab below to learn about the methods of entry by enveloped viruses. https://rossmed.instructure.com/courses/3318/pages/week-2-fusion-session-%7C-workshop-viral-replication-and-antiviral-targets 24/49 1/26/24, 1:13 AM Direct Fusion Week 2: Fusion Session | Workshop: Viral Replication and Antiviral Targets: Hemtlgy Onclgy Infectn Imm - January 2024 Viropexis Some enveloped viruses enter cells by direct fusion mechanism. Viral envelope proteins (spikes) bind to the receptors on the host cell followed by fusion of the viral envelope with the plasma membrane of the host cells, which is promoted by one of the viral envelope spikes (e.g., F protein of RSV and Gp41 of HIV). After fusion, the nucleocapsid complex is released in the cytoplasm. This mode of virus entry is seen in enveloped viruses such as paramyxoviruses, herpesviruses, and some retroviruses (HIV). Several enveloped viruses and all naked capsid viruses enter cells by viropexis. In viropexis, viral spikes bind to the receptors on host cells, followed by the surrounding of the adsorbed virions by a plasma membrane and the formation of an endosomal vesicle. For enveloped viruses, low pH of the endosomes leads to a conformational change in a viral spike protein followed by fusion of the two membranes and release of the nucleocapsid into the cytoplasm. For naked capsid viruses, low pH of the endosomes exposes hydrophobic domains resulting in the binding of virions to the membrane or virions promoting lysis of the vesicle followed by the release of viral genomes into the cytoplasm. https://rossmed.instructure.com/courses/3318/pages/week-2-fusion-session-%7C-workshop-viral-replication-and-antiviral-targets 25/49