Viral Replication & 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 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, wher...

1/26/24, 1:13 AM Week 2: Fusion Session | Workshop: Viral Replication and Antiviral Targets: Hemtlgy Onclgy Infectn Imm - January 2024 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). For many viruses, uncoating begins during viral entry, but it will end only when the genome has been delivered to its intended destination. For enveloped viruses, uncoating starts with the shedding of the envelope during the fusion process, followed by the progressive, stepwise destabilization of the capsid until the replication-competent form of the genome (nucleic acid with or without accessory proteins, depending on individual viruses) is released in its appropriate location. Capsid uncoating usually involves conformational modifications, enzymatic proteolytic cleavages, and weakening of intermolecular interactions, which are often pH driven, leading to progressive loss of structural proteins. Endosome acidification is a common event during viral https://rossmed.instructure.com/courses/3318/pages/week-2-fusion-session-%7C-workshop-viral-replication-and-antiviral-targets 26/49 1/26/24, 1:13 AM Week 2: Fusion Session | Workshop: Viral Replication and Antiviral Targets: Hemtlgy Onclgy Infectn Imm - January 2024 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. The replication step refers to genome duplication, gene transcription, protein translation, and posttranscriptional 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 immediateearly 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 and RNA polymerization and the nomenclature of the enzymes used in these processes. For any viral protein to be produced, viral mRNA must be present, and this figure summarizes how each group of viruses produces this necessary molecule. https://rossmed.instructure.com/courses/3318/pages/week-2-fusion-session-%7C-workshop-viral-replication-and-antiviral-targets 27/49 1/26/24, 1:13 AM Week 2: Fusion Session | Workshop: Viral Replication and Antiviral Targets: Hemtlgy Onclgy Infectn Imm - January 2024 Watch this video carefully to learn more about viral genomes and replication. Make note of their major mistake – we will review it in detail during class. 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. 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 to https://rossmed.instructure.com/courses/3318/pages/week-2-fusion-session-%7C-workshop-viral-replication-and-antiviral-targets 28/49 1/26/24, 1:13 AM Week 2: Fusion Session | Workshop: Viral Replication and Antiviral Targets: Hemtlgy Onclgy Infectn Imm - January 2024 be active. Viruses that do not infect rapidly dividing cells solve the problem of DNA 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 be (1) amplified by cellular DNA-dependent DNA polymerase (requires dividing cells) or (2) transcribed into mRNA by cellular DNA-dependent RNA polymerase (RNA pol II), which is used to synthesize viral DNA-dependent DNA polymerase, which is then used to amplify viral dsDNA. Some of the dsDNA will eventually be packaged into newly formed capsids, but some will serve as templates for further mRNA transcription. That mRNA will be transported to the ER, where it will, in turn, serve as a template for protein synthesis (translation). Protein production strategy in DNA viruses. (Image A). Human viruses follow the eukaryotic rule of mRNA synthesis, which means one mRNA encodes one protein. Most DNA viruses generate mRNA through splicing because they replicate inside the nucleus using host cell machinery. These proteins can be involved in many aspects of the viral replicative cycle, such as gene regulation, virulence, and the actual assembly of new viral particles, to name but a few. https://rossmed.instructure.com/courses/3318/pages/week-2-fusion-session-%7C-workshop-viral-replication-and-antiviral-targets 29/49 1/26/24, 1:13 AM Week 2: Fusion Session | Workshop: Viral Replication and Antiviral Targets: Hemtlgy Onclgy Infectn Imm - January 2024 Glycoproteins destined to be embedded into viral envelopes go through the secretory pathway of the Golgi apparatus. Note that the dsDNA viruses that replicate in the cytoplasm, as opposed to those that replicate in the nucleus, must encode their own DNA-dependent DNA polymerase and DNA-dependent RNA polymerase (e.g., poxviruses). Important examples of dsDNA viruses are herpesviruses and papillomaviruses. 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 DNAdependent 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. 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 DNAdependent 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 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 transcriptase is responsible for polymerizing gapped dsDNA from pre-genomic mRNA. Both pre-genomic mRNA and the reverse transcriptase enzyme itself are encapsidated, and reverse transcription of pre-genomic mRNA yields partly dsDNA within the capsid. https://rossmed.instructure.com/courses/3318/pages/week-2-fusion-session-%7C-workshop-viral-replication-and-antiviral-targets 30/49 1/26/24, 1:13 AM Week 2: Fusion Session | Workshop: Viral Replication and Antiviral Targets: Hemtlgy Onclgy Infectn Imm - January 2024 (a) HBV infects liver cell. (b) Polymerase completes DNA strand. ( )C l t i l i t t dt ll l RNA https://rossmed.instructure.com/courses/3318/pages/week-2-fusion-session-%7C-workshop-viral-replication-and-antiviral-targets d RNA f 31/49 1/26/24, 1:13 AM Week 2: Fusion Session | Workshop: Viral Replication and Antiviral Targets: Hemtlgy Onclgy Infectn Imm - January 2024 (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. 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. https://rossmed.instructure.com/courses/3318/pages/week-2-fusion-session-%7C-workshop-viral-replication-and-antiviral-targets 32/49 1/26/24, 1:13 AM Week 2: Fusion Session | Workshop: Viral Replication and Antiviral Targets: Hemtlgy Onclgy Infectn Imm - January 2024 Protein production strategies in RNA viruses. RNA viruses use three mechanisms to generate mRNA. B. For viruses with a segmented genome, one segment encodes one 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 viruses’ i t l t di t l t i th t i l dt t t i https://rossmed.instructure.com/courses/3318/pages/week-2-fusion-session-%7C-workshop-viral-replication-and-antiviral-targets b t 33/49 1/26/24, 1:13 AM Week 2: Fusion Session | Workshop: Viral Replication and Antiviral Targets: Hemtlgy Onclgy Infectn Imm - January 2024 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. https://rossmed.instructure.com/courses/3318/pages/week-2-fusion-session-%7C-workshop-viral-replication-and-antiviral-targets 34/49 1/26/24, 1:13 AM Week 2: Fusion Session | Workshop: Viral Replication and Antiviral Targets: Hemtlgy Onclgy Infectn Imm - January 2024 Photo Credit: Jolyne Drummelsmith & Marc Bergeron 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 case, the viral particle does not need to carry its own polymerase upon penetration because the (+) ssRNA acts as mRNA, and the RNAdependent 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 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. 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. https://rossmed.instructure.com/courses/3318/pages/week-2-fusion-session-%7C-workshop-viral-replication-and-antiviral-targets 35/49 1/26/24, 1:13 AM Week 2: Fusion Session | Workshop: Viral Replication and Antiviral Targets: Hemtlgy Onclgy Infectn Imm - January 2024 Retroviral (HIV-1) life cycle. A. Viral entry and postentry (reverse transcription, DNA synthesis, and integration) events; B. Viral gene expression (transcription and protein synthesis); C. Virus assembly and release This stage illustrates some of the paradoxical properties of viruses: viruses must assemble into metastable entities capable of protecting their nucleic acid cargo from one cell to another, sometimes through very harsh environmental conditions. At the same time, they must be able to readily disassemble upon entry into a new host cell. For replication to be successful, this process must only occur in one direction and at the right time. This is ultimately determined by the biochemical structure of the virus, which controls the stability of the viral particle and causes steps https://rossmed.instructure.com/courses/3318/pages/week-2-fusion-session-%7C-workshop-viral-replication-and-antiviral-targets 36/49 1/26/24, 1:13 AM Week 2: Fusion Session | Workshop: Viral Replication and Antiviral Targets: Hemtlgy Onclgy Infectn Imm - January 2024 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). Watch this video to learn more about self-assembly driven by random motion. Self Assembling Virus (https://www.youtube.com/watch?v=X- 8MP7g8XOE) Depending on individual viruses, viral assembly can occur in different cellular compartments (microenvironments) and, again, viruses rely on the cytoplasmic transport system of the cell to traffic the individual viral subunits from their site of synthesis (cytosolic ribosomes, membranebound 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 genomic nucleic acid into the protective capsid of the virus. Packaging requires packaging signals (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 membrane layer. This envelope, into which viral proteins (usually transmembrane glycoproteins involved in virus attachment and/or entry; “spikes”) have been inserted and accumulated, is 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 steps occur one after the other and often in different locations. Sequential assembly of icosahedral viruses starts with individual structural proteins assembling into more complex protomers. Protomers, in turn, assemble into capsomers and, finally, the capsomers assemble into the capsid, into which the genome is encapsidated. During simultaneous or coordinated assembly, the https://rossmed.instructure.com/courses/3318/pages/week-2-fusion-session-%7C-workshop-viral-replication-and-antiviral-targets 37/49 1/26/24, 1:13 AM Week 2: Fusion Session | Workshop: Viral Replication and Antiviral Targets: Hemtlgy Onclgy Infectn Imm - January 2024 structural proteins attach to genomic nucleic acid as it is being synthesized, and for enveloped viruses, this often occurs as the virus is budding through a membrane. 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 membrane, expressing viral spikes followed by envelope membrane wrapping and virus particle release. In order to spread within a host or transmit to another one, viral particles must move from one cell to another. Viruses exit cells by one of three mechanisms: cell lysis, exocytosis, or budding (budding can only be achieved by enveloped viruses). As a general rule, naked viruses are released following cell destruction and death by a process of cell lysis, or cytolysis (in other words, the host cells burst...); this tends to induce important cytopathic effects. The process is poorly understood, but some evidence points towards the capacity of some of these viruses to increase membrane permeability and/or weaken the cytoskeleton. Of course, exceptions exist, and some important non-enveloped viruses, such as hepatitis A virus and papillomaviruses, are capable of exiting their host cells by exocytosis, without lysing them. Many diseases caused by naked viruses also tend to be self-limited, with important exceptions such as, again, human papillomavirus, which is chronic and can cause cervical carcinoma. On the other hand, enveloped viruses may kill their host cells quickly or slowly. Many are released by exocytosis or budding, both of which leave the host cell intact. During budding, viral components induce membrane curvature, followed by bud growth, membrane fusion, and https://rossmed.instructure.com/courses/3318/pages/week-2-fusion-session-%7C-workshop-viral-replication-and-antiviral-targets 38/49 1/26/24, 1:13 AM Week 2: Fusion Session | Workshop: Viral Replication and Antiviral Targets: Hemtlgy Onclgy Infectn Imm - January 2024 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). 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 infections such as the flu caused by influenza viruses. The maturation step is required by many viruses in order to pass from a non-infectious form to infectious virions, and often occurs late in the process or even after exit and normally involves the 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 case of many enveloped viruses, such as the influenza virus (Orthomyxoviridae), maturation involves modification of both virus and host cell. Sialic acid (neuraminic acid) is on the surface of most mammalian cells and consequently ends up in the viral envelope. Influenza virus, along with numerous other viruses, uses sialic acid as a receptor, so its attachment protein would reattach to the producing cell, or bind to other viral particles if the sialic acid on these was not removed. Some enveloped viruses actually finish their assembly after exiting the cell (e.g., HIV maturation, whereby large polyprotein precursors are packaged and processed into functional enzymes and structural components only after viral release. Protease inhibitors are, therefore, key in the treatment of HIV patients. You will see details around retrovirus replication and antiretroviral treatments in a later activity. https://rossmed.instructure.com/courses/3318/pages/week-2-fusion-session-%7C-workshop-viral-replication-and-antiviral-targets 39/49 1/26/24, 1:13 AM Week 2: Fusion Session | Workshop: Viral Replication and Antiviral Targets: Hemtlgy Onclgy Infectn Imm - January 2024  1. Completion of which phase of the viral replication cycle marks the end of the eclipse phase? Attachment Release Assembly (answer) Uncoating 2. Matching : https://rossmed.instructure.com/courses/3318/pages/week-2-fusion-session-%7C-workshop-viral-replication-and-antiviral-targets 40/49 1/26/24, 1:13 AM Week 2: Fusion Session | Workshop: Viral Replication and Antiviral Targets: Hemtlgy Onclgy Infectn Imm - January 2024 Naked virus adhesion: Protein Genome: Nucleic Acid Enveloped virus adhesion: Glycoprotein Nucleocapsid: Protein and nucleic acid 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 Retrovirus genome production: Host DNA-dependent RNA polymerase 6. Which virus would most likely cause lysis of the host cell? Epstein-Barr virus Ebola virus Measles virus Hepatitis A virus (answer) Human immunodeficiency virus  https://rossmed.instructure.com/courses/3318/pages/week-2-fusion-session-%7C-workshop-viral-replication-and-antiviral-targets 41/49 1/26/24, 1:13 AM Week 2: Fusion Session | Workshop: Viral Replication and Antiviral Targets: Hemtlgy Onclgy Infectn Imm - January 2024 Review your answers to the questions posed at the beginning of this session. 1. How can viruses attach, enter, and uncoat? Consider general differences between naked and enveloped viruses. 2. How can viruses replicate their genomes, and where? Consider the nature of the genomic material. 3. Where are structural and genomic components made, and how does the assembly process occur? Consider these points: nucleus vs. cytoplasm, through organelles, sequential vs. simultaneous. 4. How does the virus exit? Consider these points: naked vs enveloped, cytopathic effects, and maturation.  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 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), 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 and infect new host cells. With this system in place, viruses can reproduce and infect other cells in the host. Antiviral medications take advantage of these mechanisms of cell entry and virus proliferation to control infection.  Viral infection typically is self-resolving. In a healthy host, innate immunity and adaptive immunity response will be mounted against viral infection. One of the first steps is the presentation of viral antigen or the lack of self-antigen on the surface of infected cells by the major histocompatibility complex (MHC) class I molecule. Presentation of viral antigen by MHC class I molecules to https://rossmed.instructure.com/courses/3318/pages/week-2-fusion-session-%7C-workshop-viral-replication-and-antiviral-targets 42/49 1/26/24, 1:13 AM Week 2: Fusion Session | Workshop: Viral Replication and Antiviral Targets: Hemtlgy Onclgy Infectn Imm - January 2024 cytotoxic (CD8+) T lymphocytes will induce a cell-mediated immune response. In contrast, an absence of MHC class I molecules will trigger an innate response from natural killer (NK) cells. In addition, both infected and immune cells secrete interferon α, which helps combat viral infection by halting protein synthesis in uninfected cells (thereby protecting them) and upregulating the 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 response to free the virus and the cytotoxic response to infected cells. However, in immunocompromised patients, these mechanisms are either weakened or not in place. Without any checks in place to clear the viral infection, viruses may continue to proliferate in the host. When the viral burden is high, it can lead to dysfunction or failure of the affected organs and even death.  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 their mechanism of action. Click each tab below to learn more about antiviral medications and their effect on the viral life cycle. Palivizumab Palivizumab is a humanized monoclonal antibody that is directed against respiratory syncytial 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 and self-limited course of infection, but at-risk persons can have serious symptoms. Those at greatest risk for severe infection include premature infants, children younger than 2 years with lung disease, congenital heart disease, compromised immune systems, or certain neuromuscular disorders. Palivizumab targets an epitope in the A antigen site on the fusion protein surface protein(F) of RSV, which prevents fusion to the host cell membrane. Palivizumab is used clinically as a prophylactic therapy against RSV in high-risk children. Like other monoclonal antibodies, palivizumab is administered by injection. https://rossmed.instructure.com/courses/3318/pages/week-2-fusion-session-%7C-workshop-viral-replication-and-antiviral-targets 43/49 1/26/24, 1:13 AM Week 2: Fusion Session | Workshop: Viral Replication and Antiviral Targets: Hemtlgy Onclgy Infectn Imm - January 2024 Amantadine Amantadine is a medication used in the treatment of influenza type A by blocking the uncoating of the virus in the host cell. Specifically, amantadine inhibits the viral membrane protein M2 which is required at the onset of infection to acidify the virus core leading to its uncoating. Amantadine is not effective against influenza B. Clinically, amantadine is used in patients that have been diagnosed with influenza A and have had symptoms present for less than 48 hours. Treatment may reduce the duration of infection by approximately 1-2 days, and reduce the risk of hospitalization and complications associated with influenza. Amantadine is administered orally. Adverse effects associated with amantadine include nausea, anorexia, nervousness, insomnia, and seizures. Amantadine can also cause livedo reticularis, an atropine-like peripheral reddish-blue mottling of the skin with edema around the ankle. In addition to its antiviral effect, it has also been used to treat Parkinson’s Disease. https://rossmed.instructure.com/courses/3318/pages/week-2-fusion-session-%7C-workshop-viral-replication-and-antiviral-targets 44/49 1/26/24, 1:13 AM Week 2: Fusion Session | Workshop: Viral Replication and Antiviral Targets: Hemtlgy Onclgy Infectn Imm - January 2024 Nucleic acid synthesis inhibitors consist of multiple drug classes that all inhibit DNA and RNA synthesis of viruses. Each class has a unique mechanism for blocking the nucleic acid synthesis pathway. These drugs have the suffix -cyclovir or -ciclovir. Included in this class are acyclovir and ganciclovir, and these drugs are specific for herpes viruses because they are activated by viral kinases. Their chemical structure mimics nucleosides and can become incorporated into growing DNA strands. When added to DNA, their chemical structure prevents the lengthening of the DNA strand and thereby reduces viral proliferation. Because they are not specific to viral DNA, they also affect mitochondrial DNA and have side effects. Acyclovir is a purine analog that needs to be converted to nucleoside triphosphate for its activity. The initial monophosphorylation takes place by a viral thymidine kinase, and then host cell kinases are used to convert to a nucleotide triphosphorylated form. The acyclovirtriphosphate is a substrate and inhibitor of viral DNA-polymerase. When the acyclovirtriphosphate is incorporated into the DNA molecule, it acts as a chain terminator. Acyclovir is used to treat infections caused by herpes simplex virus (HSV), varicella-zoster virus (VZV), and Epstein-Barr virus (EBV). It can be administered topically, orally, or by injection. Up to 50% of HSV strains may be resistant to acyclovir due to a lack of thymidine kinase. Other resistance mechanisms to acyclovir include modified DNA polymerase or a tyrosine kinase with reduced activity. Adverse effects associated with acyclovir include gastrointestinal, nephrotoxicity, and neurotoxicity. https://rossmed.instructure.com/courses/3318/pages/week-2-fusion-session-%7C-workshop-viral-replication-and-antiviral-targets 45/49 1/26/24, 1:13 AM Week 2: Fusion Session | Workshop: Viral Replication and Antiviral Targets: Hemtlgy Onclgy Infectn Imm - January 2024 Ganciclovir is a purine analog that has a similar mechanism of action to acyclovir. The initial monophosphorylation of ganciclovir is virus-specific and involves thymidine kinase in HSV and the phosphotransferase UL97 in cytomegalovirus (CMV). Acyclovir is not able to utilize UL97 and is not effective against CMV. After the first phosphorylization, host cell kinases convert to a triphosphate which inhibits DNA polymerase and causes chain termination. Ganciclovir can be used for HSV infection, but its primary clinical use is for prophylaxis and treatment of CMV. Ganciclovir is administered intravenously. The primary and dose-limiting toxicity of ganciclovir is bone marrow suppression. This class of antivirals acts on the viral DNA polymerase by imitating nucleotides. Foscarnet is a pyrophosphate analog used primarily to treat herpesvirus infections. It acts as a noncompetitive inhibitor of many viral RNA and DNA polymerases. It also works against HIV reverse transcriptase. Because it is not an antimetabolite or nucleoside analog, it does not require phosphorylation for its antiviral activity. Foscarnet has activity against a number of different viruses, including HSV, VZV, and CMV. Foscarnet has poor oral bioavailability and poor https://rossmed.instructure.com/courses/3318/pages/week-2-fusion-session-%7C-workshop-viral-replication-and-antiviral-targets 46/49 1/26/24, 1:13 AM Week 2: Fusion Session | Workshop: Viral Replication and Antiviral Targets: Hemtlgy Onclgy Infectn Imm - January 2024 tolerability with oral administration, so is given intravenously. The dose-limiting toxicity of this drug is nephrotoxicity and electrolyte imbalances. Neurotoxicity, including headache, tremors, hallucinations, and seizures, is also possible. These drugs act on both DNA and RNA viruses by mimicking purine structure. Ribavirin. Chemically, ribavirin mimics native adenosine or guanosine. Its monophosphorylated form inhibits Inosine monophosphate dehydrogenase, while its triphosphate form inhibits viral RNA polymerase and end-capping of viral RNA. Its mechanism of action against DNA viruses is less clear. Ribavirin is used to treat chronic hepatitis C infection, severe respiratory syncytial virus infection, and a variety of other DNA and RNA viruses. Adverse effects of ribavirin include anemia, bone marrow suppression, jaundice, and upper airway irritation. Ribavirin is a known teratogen and should not be used during pregnancy. Interferon- α Interferon α is an immune cell–signaling protein (i.e., cytokine) produced in the human body, primarily in response to viral infection or cancer. Synthetic forms of the protein are used as a medication in the treatment of hepatitis B, and C. Interferon α has many cellular signaling effects. Its main function is to halt protein synthesis in uninfected cells, allowing for the virus to be cleared before taking over the host cell. Secondarily, it helps the body mount an immune response by altering cell surface antigen expression, increasing macrophage phagocytosis activity, and increasing the cytotoxicity of lymphocytes. Interferon α has a wide range of antiviral and chemotherapeutic uses but most commonly for hepatitis B and C infections. The most common adverse effect of this therapy is an acute flu-like syndrome. Bone marrow suppression, neurotoxicity, and cardiotoxicity are also potential risks of treatment. Oseltamivir inhibits influenza virus neuraminidase, a viral enzyme that releases budding viruses from an infected cell’s outer membrane to form a viral envelope. This prevents newly formed viruses from exiting infected cells and traveling to healthy cells. These medications are given to a patient with known or suspected influenza A or influenza B who presents within 48 hours of symptom onset. After this window, the drug does not provide any additional benefit. The potential benefit of treatment with oseltamivir is reduced duration of symptoms and reduced https://rossmed.instructure.com/courses/3318/pages/week-2-fusion-session-%7C-workshop-viral-replication-and-antiviral-targets 47/49 1/26/24, 1:13 AM Week 2: Fusion Session | Workshop: Viral Replication and Antiviral Targets: Hemtlgy Onclgy Infectn Imm - January 2024 risk of hospitalization or complications from influenza. Oseltamivir is generally well tolerated and is safe to use in pregnancy.  Antiviral drugs have been developed to target various phases of viral replication. This introduction to the pharmacology of viral drug targets has highlighted some of the key anti-viral therapies. Still, it is not a comprehensive review of the therapies available to treat viral infections. Knowledge of the steps in viral replication and the mechanisms of action for the pharmacologic agents is the key to beginning to understand how viral infections are treated. Stage of Replication Anti-Viral Agent Cell entry Palivizumab Uncoating Amantadine Acyclovir Ganciclovir Foscarnet Ribavirin Transcription Translation Interferon α Release Oseltamivir  Quiz | Viral Replication and Antiviral Targets (https://rossmed.instructure.com/courses/3318/quizzes/19393)  https://rossmed.instructure.com/courses/3318/pages/week-2-fusion-session-%7C-workshop-viral-replication-and-antiviral-targets 48/49