Viral Replication and Antiviral Targets Workshop.pdf

Purpose of Fusion Sessions  (https://rossmed.instructure.com/courses/3571/modules/items/61297) Fusion sessions are online lea...

Purpose of Fusion Sessions  (https://rossmed.instructure.com/courses/3571/modules/items/61297) Fusion sessions are online learning activities followed by a live session where you will  (https://rossmed.instructure.com/courses/3571/modules/items/61298) translate the content into practice. These require completing learning content in Canvas  (https://rossmed.instructure.com/courses/3571/modules/items/61299) before attending live sessions. To encourage preparation, attendance, and participation, recordings of fusion sessions will not be posted; prepare accordingly so that you can fully  (https://rossmed.instructure.com/courses/3571/modules/items/61301) participate. Remember: Learning from written materials is a critical professional and personal skill that RUSM is helping you develop through these sessions.  (https://rossmed.instructure.com/courses/3571/modules/items/61302)  (https://rossmed.instructure.com/courses/3571/modules/items/61303) Suggested Process 1. Work through the content on this page.  (https://rossmed.instructure.com/courses/3571/modules/items/61304) 2. Contact faculty via email or office hours if you have questions about content to ensure  (https://rossmed.instructure.com/courses/3571/modules/items/61305) you are prepared for the session. 3. Take the quiz. (You have three attempts.)  (https://rossmed.instructure.com/courses/3571/modules/items/61307) 4. Attend and participate in the live session.   (https://rossmed.instructure.com/courses/3571/modules/items/61308) 5. Take the quiz again. 6. Study missed content.  (https://rossmed.instructure.com/courses/3571/modules/items/61309) 7. Take the quiz for the final attempt.  (https://rossmed.instructure.com/courses/3571/modules/items/61311)  (https://rossmed.instructure.com/courses/3571/modules/items/62851)  (https://rossmed.instructure.com/courses/3571/modules/items/61313)  Overview  (https://rossmed.instructure.com/courses/3571/modules/items/61314) Viruses are responsible for many of the most common and many of the most deadly infections.  (https://rossmed.instructure.com/courses/3571/modules/items/61315) Fortunately, common and deadly infections have not frequently overlapped in human history (with  (https://rossmed.instructure.com/courses/3571/modules/items/61317) a few notable exceptions). The variability encompassed within the viral world is massive, and even related viruses can differ quite a lot in terms of how they function and the diseases they  (https://rossmed.instructure.com/courses/3571/modules/items/61318)   cause. Our understanding of viruses, while growing, is limited, and we do not have effective targeted therapies for many viral infections. Fortunately, however, by understanding how viruses Fusion Session | Workshop: Viral Replication infect and replicate, more and more antivirals are being developed and put into effective clinical use. This section provides a general overview of what viruses are, how they infect and replicate, and Antiviral Targets and introduces the mechanisms of some key antiviral medications. Learning Objectives for Viral Replication By the end of this session, you will be able to meet the following learning objectives:  Top Top How to Approach Fusion Sessions 1. Explain the key differences between viruses and other microbes. 2. Describe the composition and roles of viral components. 3. Associate viral structural characteristics with transmission, environmental stability, and infection control.  4. Compare and contrast selected virus families based on their descriptors. Viruses differ immensely from cellular infectious agents. Viruses cannot metabolize and, 5. Explain each general step of the viral replication process. therefore, cannot make energy, encoded proteins, or anything else. Instead, viruses are 6. Differentiate the concepts of susceptibility and permissiveness (permissibility), and discuss infectious, obligately intracellular parasites that may commandeer the machinery of living cells their influence on tissue tropism, pathogenesis, and host specificity. to replicate themselves. They must minimally contain a nucleic acid genome and a protein 7. Explain what is happening inside the host cell during the eclipse and latent phases of the coating called a capsid, and all of their components derive from host cells. Unlike cells, they do viral replication process. not divide to replicate; rather, they assemble from individual components. Since viruses are so 8. Differentiate immediate early, early, and late proteins as they apply to viral replicative very different from other microbes, it is perhaps not surprising that antibacterial, antiparasitic, and cycles. antifungal drugs do not effectively prevent viral replication; viruses simply do not possess the 9. Explain the differences between different types of nucleic acid polymerases and why some targets of these various types of anti-infective agents. Since viruses are generally not considered viruses encode their own polymerases and cell-cycle regulators. to be alive, they cannot be killed; instead, they are “inactivated” or “neutralized” and, in such a 10. Compare and contrast the replication of 5 prototype viruses, distinguishing variations in state, are unable to infect a host cell. mechanisms of entry, nucleic acid replication, protein production, assembly, and release. Learning Objectives for Viral Drug Targets 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.  Introduction 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 Transcript  keep the focus on key areas. Look for answers to these as you progress through the material. How do viruses differ from other microbes? 1. How can viruses attach, enter, and uncoat? Cannot make energy or encoded proteins 2. How can viruses replicate their genomes, and where? Metabolically inert (not living) 3. Where are structural and genomic components made, and how does the Require living cells to replicate (obligate intracellular parasites) assembly process occur? Components derived from host, often virally-encoded 4. How does the virus exit? Top Assemble rather than divide Top Unaffected by antibacterials, antifungals, and antiparasitics What is a Virus?  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 Smallpox (variola), monkeypox, molluscum Poxviridae contagiosum​ Herpes, chickenpox, EBV, CMV, roseola, Kaposi’s Herpesviridae sarcoma Hepadnaviridae Hepatitis B virus Rhinoviruses, enteroviruses (e.g., poliovirus), hepatitis Picornaviridae 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 Transcript  errors or immaturity. 2. Virion: a single, infective, complete, and mature viral particle. Virions are able to Size comparison of viruses with other microbes. An E. Coli cell is 1-2 microns in length. 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 Top Top and may also be used to treat infections. How Do Viruses Differ From Other Microbes? 4. Genome: the nucleic acid encoding the instructions to create new viral particles Human viral pathogens are contained within 16 families of RNA viruses and 8 families of DNA 5. Capsid: a protein layer that encompasses and protects the genome viruses. Viruses from the same family can cause different diseases (e.g., different picornaviruses  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 Transcript  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 8. Nucleoprotein: a specific type of protein that complexes to nucleic acid; in the case 14. Spike glycoprotein: another name for a viral attachment protein of viruses, these may also be capsid proteins 15. Matrix: protein layer between the capsid and envelope of some viruses that connect 9. Nucleocapsid: nucleoproteins complexed directly to nucleic acid form this structure these structures 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 Toparrangements of both 5 and 6 triangles to form both pentagons Top and hexagons, like a soccer ball.  Composition and Roles of Key Viral Components While a number of viruses have many different components, there are three main structures to protein), whereas enveloped virus has a nucleocapsid or capsid wrapped in a lipid bilayer understand well. Click below to explore the three main components of viruses. envelope.   Genome  The capsid also plays an essential role in the delivery of nucleic acid into the host cell upon Viral genomes are quite varied and may be DNA or RNA, single- or double-stranded, linear, infection, as it must release the nucleic acid at the right time (called uncoating). In naked circular, or even segmented or polyploid. But no matter the structure, the genome contains all viruses, capsid proteins include the attachment protein, and therefore this structure is also of the information needed to direct a host cell to build the specific viral components. 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 Capsid  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 The capsid can be viewed as the main protective shell of a virus, with its key role being to nucleic acids such as specialized tRNAs or mRNA) among other things. 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 Envelope  acid genome (forming a nucleoprotein complex called the nucleocapsid) or into a distinct structure that surrounds it, which is frequently icosahedral in symmetry. 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. Transcript  Top Top 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  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.  Virus Characteristics by Family 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 Viral Stability Affects Transmission, Tropism, and this greatly reduces the complexity. Viral families end in the suffix -viridae; for example, the family Inactivation Retroviridae contains the retroviruses, of which human immunodeficiency virus (HIV) is an example. The family Paramyxoviridae contains paramyxoviruses, an example of which is the Viruses come in different sizes (from about 18-300 nm) and shapes. Many are icosahedral, some measles virus. are helical (filamentous), and some are complex (or pleiomorphic), spherical, or even ovoid. Top of viruses infecting bacteria) also possess peculiar structures Some bacteriophages (the group Click through this interactive Top to learn more about the classification of viruses by family. (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:  Complex 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 Basics of Viral segmented (i.e., in pieces) in some virus groups, whereas DNA genomes are never segmented. Classification 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). Viral Replication Flowcharts 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, Transcript 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 Genome 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 Composition: DNA or RNA will become more familiar as you read the following sections, and connecting family names to the Strandedness: Single, double, partially double replication cycles as you go through them will help in learning both the families and the replication Arrangement: Linear, circular, segmented, diploid cycles. Capsid (nucleocapsid) symmetry Helical (helicoidal, filamentous) Icosahedral (spherical) Envelope Presence (enveloped) Absence (non-enveloped, naked) Top Top Shape Regular, spherical, icosahedral DNA Viruses Transcript  DNA Double-stranded Enveloped Pox- Herpes- RNA Viruses Transcript  Naked Papilloma- RNA Polyoma- Single-stranded Adeno- (+) Partial double-stranded (gapped) Naked Enveloped Picorna- Hepadna-(iRNA) Calici- Hepe- Single-stranded Astro- Naked Parvo- Enveloped Toga- Top Note: Characteristics of families in red must be learned for this module. Top Flavi- Retro- (diploid, iDNA) Corona- (-)  Enveloped Filo- Paramyxo- Pneumo- Rhabdo- Delta- Segmented Enveloped Arena- Bunya- Orthomyxo- Double-stranded Segmented Naked Reoviridae Note: Characteristics of families in red must be learned for this module.  Check Your Knowledge of Viral Characteristics Transcript  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? Top Top 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  3. Drag the viral families into the correct category: The Viral Replicative Cycle dsDNA: Pox, Herpes As obligate parasites, viruses must multiply and be transmitted to new hosts in order to continue Gapped DNA: Hepadna to exist. As obligate intracellular parasites, they must do this within a host cell, using host cell ssDNA: Parvo machinery and building blocks to make (1) copies of the viral genome and (2) viral proteins. This +ssRNA: Picorna, Toga, Flavi, Retro means that they must be able to enter a cell that can recognize, follow, and complete the -ssRNA: Filo, Paramyxo instructions coded within the viral genome. Some viruses even modify the function of that cell to 4. Drag the viruses into the correct category: 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. Enveloped: Rubella virus The viral replicative cycle generally consists of 7 steps Dengue virus 1. Attachment (adsorption or binding) Human immunodeficiency virus 2. Entry (penetration) Ebola virus 3. Uncoating Measles virus 4. Replication (nucleic acid and protein production) Mpox virus 5. Assembly and packaging Chickenpox virus 6. Release Naked: 7. Maturation Parvovirus B19 Poliovirus 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 5. What type of infection would a virus exhibiting tropism for pneumocytes cause? 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!) 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 Top Top Membrane glycoprotein (answer)  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. Transcript  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. Transcript  The cells/tissues/organisms that will be affected by a virus are determined by: 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 Host cell susceptibility attach to; that is, it has the needed receptor on its surface. The virion might then be internalized, Possesses surface receptors/co-receptors allowing viral attachment and if the host cell is permissive, that is, is able to follow and complete the viral instructions, “Cell can be recognized” infective viral particles will be produced. If the host cell entered is not permissive, for example, Host cell permissiveness does not recognize the viral promoter, then the cycle will be non-productive, and that particular Contains all components required for virion production (i.e., leads to a productive viral particle will never replicate. Cell susceptibility and permissiveness determine host range, infection) Top Top “Cell allows replication” 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 Virus needs both host cell susceptibility and host cell permissiveness in order to propagate. 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   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 General Stages of Viral Replication genome, which directs the production of new components. Newly-produced components 1. Attachment (adhesion, adsorption, or binding) 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 The first step in the typical viral replicative cycle is the binding of the virion to the host cell. Viral and when the first progeny are assembled inside the cell is called the eclipse period. Replication attachment to cells is a random event. Viral adhesion is mediated through receptor-ligand may occur immediately after viral entry or perhaps after a period of inactivity, such as when a viral interactions. Some viral attachment proteins (VAPs) may be very specific, with only one known genome enters the host DNA and does not replicate. The time between viral entry and new receptor, while others may bind a variety of receptors. Some may also require binding to more progeny release is called the latent period and may range from hours to years. Note that latency than one receptor (co-receptors) to trigger cell entry. VAPs may often project from the viral surface may also mean the time between someone being infected and becoming infectious, which might and are often called “spikes.” Receptors may be any cell surface molecule. The initial VAP-cell lead to confusion! 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. 2. Entry 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 In the graph, the red line measures virions in the culture medium (outside the cell), and the blue rule, viral penetration (entry into the cytosol) originates from the endosomes, which progressively line measures virions inside the cells. The term plaque-forming unit, abbreviated as "pfu", is a way have a lower pH as they mature. This is important for uncoating, as described in section 3. to visualize the presence of an infectious lytic viral particle and count them, as these can infect a Following endocytosis, penetration by naked viruses is the result of one of three mechanisms: (1) cell within a layer of cultured cells and spread to adjacent cells, forming a "plaque" of missing cells membrane puncture, where the viral nucleic acid is inserted into the cytosol through a pore in the layer. In this example, infectious virions appear within the cell before they begin to be created by the virus, and entry and uncoating are one and the same process; (2) perforation, released to the extracellular medium, which tells us that assembly and release occur sequentially, which is the transfer of the complete virion through the membrane without any major lysis not simultaneously.​New viralTop particles may be released as they are made, leading to constant incurring on the part of the membrane; Top and (3) lysis, a mechanism through which the viral particle production, or may build up and burst out of the cell, as shown in the example here, leading to escapes from an endosomal compartment by rupturing it. Some naked viruses enter directly waves of viral production. 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. 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). Once inside a cell, the viral particle must reach the site needed for the replicative cycle to continue. Some viruses must make it all the way to the nucleus, whereas others replicate in the cytoplasm; replication in the cytoplasm usually occurs within the cytosol, sometimes in close association with specific organelles (e.g., endosomes, endoplasmic reticulum, or Golgi), or within the organelles themselves. In order to achieve this, viruses rely on the cytoplasmic transport system of the cell, either directly or within endosomal vesicles. Peripheral movement is mediated by actin filament transport (short-distance; myosin motor-dependent), whereas movement toward deeper compartments of the cell, such as the nucleus, is accomplished by microtubules (long- distance; dynein/dynactin/kinesin-dependent). 3. Uncoating For many viruses, uncoating begins during viral entry, but it will end only when the genome has Several enveloped viruses and all naked capsid viruses enter cells by viropexis. In viropexis, been delivered to its intended destination. For enveloped viruses, uncoating starts with the viral spikes bind to the receptors on host cells, followed by the surrounding of the adsorbed shedding of the envelope during the fusion process, followed by the progressive, stepwise virions by a plasma membrane and the formation of an endosomal vesicle. For enveloped destabilization of the capsid until the replication-competent form of the genome (nucleic acid viruses, low pH of the endosomes leads to a conformational change in a viral spike protein with or without accessory proteins, depending on individual viruses) is released in its appropriate Topmembranes and release of the nucleocapsid into the cytoplasm. followed by fusion of the two Top involves conformational modifications, enzymatic proteolytic location. Capsid uncoating usually For naked capsid viruses, low pH of the endosomes exposes hydrophobic domains resulting cleavages, and weakening of intermolecular interactions, which are often pH driven, leading to in the binding of virions to the membrane or virions promoting lysis of the vesicle followed by progressive loss of structural proteins. Endosome acidification is a common event during viral the release of viral genomes into the cytoplasm. entry that may trigger uncoating. For some non-enveloped viruses, uncoating can even be initiated upon viral adhesion, as some cellular receptors induce conformational changes that trigger capsid destabilization (e.g., many picornaviruses). For most RNA viruses, replication occurs in the cytoplasm, but the genome of most DNA viruses must reach the nucleus intact. Two separate mechanisms allow viruses access to the nucleus. The first one involves entry via the nuclear pore complex, a process requiring binding to nuclear import receptors, the nuclear pore complex, and nuclear localization signals recognized by importins or karyopherins. This mechanism allows infection of non-dividing, terminally differentiated cells. The second way through which viruses can gain access to the nuclear region is to linger in the cytosol or in the nucleus until the nucleus disintegrates in preparation for cell division. Such viruses are restricted to dividing cells or must induce cell cycling. 4. Replication The replication step refers to genome duplication, gene transcription, protein translation, and post- transcriptional events. Most viruses make extensive use of the host’s cell machinery, although poxviruses encode many of the needed components themselves. There are almost as many variations on viral replication as there are viruses. In general, viral proteins are categorized into early proteins, which are used for genome replication, and late proteins, which are structural and are used for purposes such as building capsids or envelope. Some viruses also encode immediate-early proteins, which are transcription factors that direct the host cell to bind viral promoters better than its own - the host cell will “prefer” the virus to itself! Genome replication happens early in the infection process, sometimes even before mRNA and protein production. Nucleic acid replication schemes can fall into five broad categories: dsDNA viruses, gapped dsDNA viruses, ssDNA viruses, RNA viruses, and retroviruses. This can be complicated, so before proceeding, ensure that you understand the general processes of DNA Viral Genomes and Replication and RNA polymerization and the nomenclature of the enzymes used in these processes. For any Watch this video carefully to learn more about viral genomes and replication. Make note of viral protein to be produced, viral mRNA must be present, and this figure summarizes how each their major mistake – we will review it in detail during class. group of viruses produces this necessary molecule. Viral Genomes and Replication for the USMLE Step 1 (https://www.youtube.com/watch?v=j2k8cavi44s&t=1s)) Click below to learn more about virus types.   Top Top Double-stranded DNA viruses  Replication of double-stranded DNA (dsDNA) viruses is what most resembles that of normal cells. Those viruses may use the host’s cell machinery to replicate their DNA. Those viruses that require the host’s DNA polymerase need to infect cells that divide rapidly for this enzyme Most DNA viruses generate mRNA through splicing because they replicate inside the to be active. Viruses that do not infect rapidly dividing cells solve the problem of DNA nucleus using host cell machinery. polymerase accessibility by inducing the cell cycle (as they need cells to be in the S phase) or by encoding their own DNA polymerase. Most dsDNA viruses encode their own DNA- dependent DNA polymerases. Viral dsDNA is transported to the nucleus where it can either These proteins can be involved in many aspects of the viral replicative cycle, such as be (1) amplified by cellular DNA-dependent DNA polymerase (requires dividing cells) or (2) gene regulation, virulence, and the actual assembly of new viral particles, to name but a transcribed into mRNA by cellular DNA-dependent RNA polymerase (RNA pol II), which is few. Glycoproteins destined to be embedded into viral envelopes go through the secretory used to synthesize viral DNA-dependent DNA polymerase, which is then used to amplify pathway of the Golgi apparatus. Note that the dsDNA viruses that replicate in the viral dsDNA. cytoplasm, as opposed to those that replicate in the nucleus, must encode their own DNA- Some of the dsDNA will eventually be packaged into newly formed capsids, but some will dependent DNA polymerase and DNA-dependent RNA polymerase (e.g., poxviruses). serve as templates for further mRNA transcription. That mRNA will be transported to the ER, Important examples of dsDNA viruses are herpesviruses and papillomaviruses. where it will, in turn, serve as a template for protein synthesis (translation). Single-stranded DNA viruses  Replication of single-stranded DNA (ssDNA) viruses is very similar to the replication of dsDNA viruses, but, because DNA-dependent RNA polymerase cannot bind ssDNA, the incoming viral ssDNA must first have its complementary strand synthesized by the DNA-dependent DNA polymerase (which can bind either ssDNA or dsDNA) before any transcription can occur. These require hairpin structures (palindromic sequences) functioning as primers for the DNA- dependent DNA polymerase. There is only one virus of medical importance that does this, and this is the Erythrovirus (Parvovirus B19; Parvoviridae) responsible for erythema infectiosum, also called fifth disease or slapped-cheek disease, a common childhood infection. Gapped, or partially double-stranded DNA viruses  Replication of gapped (partial) dsDNA viruses is a particularly odd replication strategy but important as these include the Hepatitis B virus (Hepadnaviridae). First, the partially dsDNA must be repaired in order to create an entire dsDNA that can be used by the cellular DNA- dependent RNA polymerase (RNA pol II). This is most likely achieved by the cellular DNA repair system and some viral proteins (evidence shows that the viral reverse transcriptase is Transcript  involved). Once the gaps in the DNA have been filled, the cellular DNA-dependent RNA polymerase can transcribe the DNA into mRNA so that protein synthesis can occur. Among the proteins being synthesized is a virus-specific reverse transcriptase; this reverse Top Top Protein production strategy in DNA viruses. (Image A). Human viruses follow the transcriptase is responsible for polymerizing gapped dsDNA from pre-genomic mRNA. Both eukaryotic rule of mRNA synthesis, which means one mRNA encodes one protein. pre-genomic mRNA and the reverse transcriptase enzyme itself are encapsidated, and reverse transcription of pre-genomic mRNA yields partly dsDNA within the capsid.  (a) HBV infects liver cell. (b) Polymerase completes DNA strand. (c) Complete viral genome is transported to cell nucleus, messenger RNA and RNA copy of genome transcribed. (d) Reverse transcriptase and other viral proteins are translated; assembly of viral capsid begun. (e) Reverse transcription of RNA genome copy is packaged in newly forming capsid; RNA strand degraded. (f) Synthesis of complementary DNA strand begun. RNA viruses  Whether they are ssRNA viruses of positive polarity, negative polarity, or double-stranded, all RNA viruses must encode their own RNA-dependent RNA polymerase (except for retroviruses which encode a reverse transcriptase; see the next section), because cells do not possess such enzymes. Negative-polarity (-) ssRNA viruses absolutely must carry their RNA- dependent RNA polymerase when they penetrate the host cell; (-) ssRNA is unusable by ribosomes and will be degraded rapidly (remember that (-)ssRNA is the “antisense” strand; translating it would make no sense). If the virus penetrates the cell carrying its polymerase (which is usually complexed with the RNA, the best way to ensure that the polymerase will follow the genome!), then (-) ssRNA soon gets amplified via a (+) ssRNA intermediate. Once capsid proteins have been synthesized by translation from (+) ssRNA, the (-) ssRNA will be packaged into newly synthesized capsids to form progeny viral particles. Top Top Transcript   viruses’ genome is translated into a polyprotein that is cleaved to mature proteins by protease enzyme. Positive-polarity (+) ssRNA viruses, on the other hand, immediately initiate protein synthesis by connecting to ribosomes after penetration (the + strand is the sense strand, and will have the correct initiating sequences and result in a functional protein). In this case, the viral particle does not need to carry its own polymerase upon penetration because the (+) ssRNA acts as an mRNA (it has a structure that mimics a 5’ cap and is polyadenylated) that can be translated directly. Once the RNA-dependent RNA polymerase has been synthesized, then RNA amplification can begin. Amplified (+) ssRNA will serve as both mRNA and genomic RNA to be packaged into progeny particles. Double-stranded RNA viruses follow the (-) ssRNA replication strategy; ribosomes only recognize ssRNA, and dsRNA requires the polymerase for denaturation. Knowing which viruses have RNA genomes is important when considering disease. RNA polymerases do not have proofreading capability, so RNA viruses are much more likely to replication errors, and, consequently, mutate more quickly than DNA viruses do. This can be important to virulence, such as with a virus attachment protein becoming either more efficient in binding its receptor or perhaps gaining the ability to bind different receptors, to the ability to change a surface protein and be less recognized by pre-existing antibodies, or to treatment, in terms of becoming resistant to an antiviral drug. Transcript  Protein production strategies in RNA viruses. RNA viruses use three mechanisms to generate mRNA. B. For viruses Top with a segmented genome, one segment encodes one Top protein. C. The viral RNA polymerase of negative-sense RNA viruses initiates transcription at the start of each gene, pauses at the end of the gene, and continues to the end of the genome. This results in synthesis of a nested set of mRNAs. D. Positive sense RNA  synthesized, then RNA amplification can start. Amplified (+) ssRNA will serve both as mRNA and genomic RNA to be packaged into progeny particles. Double-stranded RNA viruses follow the (-) ssRNA replication strategy because ribosomes only recognize ssRNA and dsRNA requires polymerase activity (under natural conditions) to separate the strands. Retroviruses  The last important replication scheme is that of retroviruses. In this case, the (+) ssRNA genome is first reverse transcribed by the virally-encoded reverse transcriptase carried by the virus upon penetration. The product of reverse transcription is dsDNA, and that DNA becomes integrated into the host’s genome via another virally-encoded enzyme, an integrase. The integrated viral DNA is referred to as a provirus (analogous to the prophages of bacteriophages). When the regulatory sequences controlling the provirus are activated, cellular DNA-dependent RNA polymerase (RNA pol II) transcribes the DNA into mRNA. Again, some of that RNA will go into protein synthesis, whereas the rest will be used as genomic RNA to be packaged into new virions. Nucleic Acid Replication in RNA viruses Whether they are ssRNA viruses of positive polarity (red bars), negative polarity (orange bars), or double-stranded, all RNA viruses must encode their own RNA-dependent RNA polymerase (protein shown as green ovals, gene as green bars) with the exception of retroviruses and hepadnaviruses which encode reverse transcriptase, because cells do not possess such enzymes. Negative-polarity ssRNA viruses absolutely must carry their RNA- dependent RNA polymerase enzyme when they penetrate the host cell; otherwise, the RNA gets degraded without any product being made. This is because (-) ssRNA is unusable by ribosomes (in blue) and cells have no RNA-dependent RNA polymerase to produce a mRNA. If the virus penetrates the cell carrying its polymerase (which is usually complexed with the RNA-that’s the best way to ensure that the polymerase will follow the genome!), then (-) ssRNA soon gets amplified via a (+) ssRNA intermediate. At some point, the (-) ssRNA will be packaged into newly synthesized capsids to form progeny viral particles, whereas the (+) ssRNA intermediates serve as mRNA. Positive-polarity ssRNA viruses, on the other hand, immediately initiate protein synthesis by connecting to ribosomes after penetration. In this Top Top case, the viral particle does not need to carry its own polymerase upon penetration because the (+) ssRNA acts as mRNA, and the RNA-dependent RNA polymerase can be translated directly from that RNA. This is possible because the RNA of these viruses is capped & polyadenylated or carries an internal ribosome entry site. Once the polymerase has been  genomic nucleic acid into the protective capsid of the virus. Packaging requires packaging signals Transcript  (nucleic acid sequences recognized by viral proteins) and, for icosahedral viruses, is limited by capsid size. Envelopment is the process whereby enveloped viruses acquire an external Retroviral (HIV-1) life cycle. A. Viral entry and postentry (reverse transcription, DNA membrane layer. This envelope, into which viral proteins (usually transmembrane glycoproteins synthesis, and integration) events; B. Viral gene expression (transcription and protein involved in virus attachment and/or entry; “spikes”) have been inserted and accumulated, is synthesis); C. Virus assembly and release derived from a host organelle membrane during the assembly and exocytic pathway or the plasma membrane during release (see below). Assembly can occur either sequentially or simultaneously. During sequential assembly, genomic nucleic acid is incorporated into a preformed capsid, which then might acquire an envelope; these 5. Assembly and packaging steps occur one after the other and often in different locations. Sequential assembly of icosahedral viruses starts with individual structural proteins assembling into more This stage illustrates some of the paradoxical properties of viruses: viruses must assemble into complex protomers. Protomers, in turn, assemble into capsomers and, finally, the capsomers metastable entities capable of protecting their nucleic acid cargo from one cell to another, assemble into the capsid, into which the genome is encapsidated. During simultaneous or sometimes through very harsh environmental conditions. At the same time, they must be able to coordinated assembly, the structural proteins attach to genomic nucleic acid as it is being readily disassemble upon entry into a new host cell. For replication to be successful, this process synthesized, and for enveloped viruses, this often occurs as the virus is budding through a must only occur in one direction and at the right time. This is ultimately determined by the membrane. biochemical structure of the virus, which controls the stability of the viral particle and causes steps to be triggered successively and is aided by the compartmentalization of several steps, such as uncoating, assembly, and maturation. Factors contributing to the stability of the structure include pH (e.g., the structure might be stable at nearly neutral pH but unstable at lower pH), hydrophobicity, etc. The amino acid sequence of the viral structural proteins determines the assembly process; the viral assembly blueprint is contained within the amino acid sequence of its structural proteins. This means that these elements usually dock or interlock by themselves when their concentration is high enough. In order to assemble properly, individual subunits must be present in the appropriate cellular compartment in appropriate concentrations. This has two effects: (1) it increases the rates of assembly reactions (remember these are random ), and (2) it increases the specificity of the assembly reactions (fewer errors). Self-Assembling Virus Video Watch this video to learn more about self-assembly driven by random motion. Self Assembling Virus (https://www.youtube.com/watch?v=X-8MP7g8XOE) Human enveloped viruses often acquire lipid bilayer membranes by budding, generally from the plasma membrane. Viral spikes are expressed on the cell surface, followed by the synthesis of matrix protein that associates near the plasma membrane where viral spikes are present. The matrix protein attracts the assembled nucleocapsid (genome + nucleoprotein) near the plasma Top viral assembly can occur in different cellular compartments Depending on individual viruses, Top followed by envelope membrane wrapping and virus particle membrane, expressing viral spikes (microenvironments) and, again, viruses rely on the cytoplasmic transport system of the cell to release. traffic the individual viral subunits from their site of synthesis (cytosolic ribosomes, membrane- 6. Release (Egress, exit) bound ribosomes, the nucleus), to sites of partial and/or final assembly (Golgi, cytosol, cell membrane, virus factories, etc.). Packaging, or encapsidation, relates to the incorporation of In order to spread within a host or transmit to another one, viral particles must move from one cell The maturation step is required by many viruses in order to pass from a non-infectious form to to another. Viruses exit cells by one of three mechanisms: cell lysis, exocytosis, or budding infectious virions, and often occurs late in the process or even after exit and normally involves the (budding can only be achieved by enveloped viruses). proteolytic processing of surface viral proteins. This step is often needed to ensure a subsequent round of infection, by preparing the virus for uncoating upon entry into the next infected cell. In the As a general rule, naked viruses are released following cell destruction and death by a process of case of many enveloped viruses, such as the influenza virus (Orthomyxoviridae), maturation cell lysis, or cytolysis (in other words, the host cells burst...); this tends to induce important involves modification of both virus and host cell. Sialic acid (neuraminic acid) is on the surface of cytopathic effects. The process is poorly understood, but some evidence points towards the most mammalian cells and consequently ends up in the viral envelope. Influenza virus, along with capacity of some of these viruses to increase membrane permeability and/or weaken the numerous other viruses, uses sialic acid as a receptor, so its attachment protein would reattach to cytoskeleton. Of course, exceptions exist, and some important non-enveloped viruses, such as the producing cell, or bind to other viral particles if the sialic acid on these was not removed. hepatitis A virus and papillomaviruses, are capable of exiting their host cells by exocytosis, Some enveloped viruses actually finish their assembly after exiting the cell (e.g., HIV maturation, without lysing them. Many diseases caused by naked viruses also tend to be self-limited, with whereby large polyprotein precursors are packaged and processed into functional enzymes and important exceptions such as, again, human papillomavirus, which is chronic and can cause structural components only after viral release. Protease inhibitors are, therefore, key in the cervical carcinoma. On the other hand, enveloped viruses may kill their host cells quickly or treatment of HIV patients. You will see details around retrovirus replication and antiretroviral slowly. Many are released by exocytosis or budding, both of which leave the host cell intact. treatments in a later activity. During budding, viral components induce membrane curvature, followed by bud growth, membrane fusion, and pinching off of the particle. Some enveloped viruses never “exit” their cells, instead causing cells to fuse together, via the viral fusion protein, and allowing them to spread. This last pathway often causes cells to either bridge or even completely fuse, leading to visible  microscopic effects (for example, multinucleated giant cells in the case of some herpesviruses). Check Your Knowledge of Viral Replication Cells in which enveloped viruses are replicating will usually die due to the immune response against them and the build-up of damage caused by resources being diverted to virus production, and the timeline for this varies due to several factors, including potential latency and immune privilege. Immune-privileged sites are partially protected from the inflammatory response so as to preserve them; the benefit derived from killing infected cells to try to eradicate infection would almost always be less than the damage caused. The central nervous system and eyes are prime examples. Viruses that induce latency will tend to have no cytopathic effect during their latent phase. It would be the equivalent of viral suicide to heavily dysregulate or kill the cells harboring them while latent, and their quiescence does not alert the immune response or produce metabolic stress. However, even a latent virus can replicate at some point, usually in response to signals from the host cell that it is stressed. For example, in herpes simplex infections, which are chronic, the virus is usually latent in the neurons that harbor the virus (viral reservoir). When replication does occur, they are not killed; replication is not overwhelming, and neurons are protected from the inflammatory response as the CNS is a privileged immune site. On the other hand, when the virus infects epithelial cells, replication is rapid, causing them to be destroyed and contributing to the characteristic lesions of herpes. Many enveloped viruses are unable to induce latency at all and, therefore, rapidly destroy their host after replicating; these are responsible for acute Top by influenza viruses. infections such as the flu caused Top 7. Maturation  Nucleoprotein: Protein 3. An antiviral drug is developed that inhibits the herpesvirus fusion protein. Which stage of the viral replication cycle is most likely affected? Binding Entry (answer) Uncoating Genomic replication Assembly Exit 4. What event frequently triggers uncoating of the viral genome? Binding to cell surface receptor Release into cytoplasm Endosome maturation (answer) Docking at nuclear pore Binding of polymerase enzyme 5. Match the polymerase to the role. DNA virus genome replication: Viral DNA-dependent DNA polymerase Production of mRNA from DNA: Host DNA-dependent RNA polymerase (+)ssRNA virus genome replication: Viral RNA-dependent RNA polymerase (-)ssRNA virus genome replication: Viral RNA-dependent RNA polymerase Retrovirus DNA production: Viral RNA-dependent DNA polymerase Translation: Host RNA-dependent amino acid polymerase Transcript  Retrovirus genome production: Host DNA-dependent RNA polymerase 6. Which virus would most likely cause lysis of the host cell? 1. Completion of which phase of the viral replication cycle marks the end of the eclipse phase? Epstein-Barr virus Ebola virus Attachment Measles virus Release Hepatitis A virus (answer) Assembly (answer) Human immunodeficiency virus Uncoating 2. Matching : Top Top Naked virus adhesion: Protein Genome: Nucleic Acid  Enveloped virus adhesion: Glycoprotein Self Review of Viral Replication Nucleocapsid: Protein and nucleic acid Review your answers to the questions posed at the beginning of this session. proliferation of other immune cells to help transition into an adaptive immune response. Once this response is fully underway, the host is usually able to clear the infection through the humoral 1. How can viruses attach, enter, and uncoat? Consider general differences between naked response to free the virus and the cytotoxic response to infected cells. and enveloped viruses. 2. How can viruses replicate their genomes, and where? Consider the nature of the genomic However, in immunocompromised patients, these mechanisms are either weakened or not in material. place. Without any checks in place to clear the viral infection, viruses may continue to proliferate 3. Where are structural and genomic components made, and how does the assembly process in the host. When the viral burden is high, it can lead to dysfunction or failure of the affected occur? Consider these points: nucleus vs. cytoplasm, through organelles, sequential vs. organs and even death. simultaneous. 4. How does the virus exit? Consider these points: naked vs enveloped, cytopathic effects, and maturation.  What Are the Different Types of Antiviral Medication? Antiviral medications are geared toward preventing the virus from completing its metabolic  process for self-replication. Antiviral medications work in a variety of ways and are classified by What is Antiviral Therapy? their mechanism of action. Viruses can cause a wide range of illnesses that vary drastically in severity. The common cold, liver failure, AIDS, and a slew of other medical problems can all arise as a consequence of viral   infection, depending on the infecting virus. So, what do all these viruses have in common? One Attachment and Entry  unique trait of all viruses is the inability to live independently without having a host. Viruses do not contain any organelles, so they cannot synthesize their own deoxyribonucleic acid (DNA), Palivizumab ribonucleic acid (RNA), or proteins. To maintain themselves, viruses must infect host cells, and these host cells then process the viral nucleic acids, ultimately permitting the virus to proliferate Palivizumab is a humanized monoclonal antibody that is directed against respiratory syncytial and infect new host cells. virus (RSV). RSV causes infections of the lungs and respiratory tract and is most common in children under the age of 2 years. Adults and healthy children usually have a relatively benign With this system in place, viruses can reproduce and infect other cells in the host. Antiviral