Microbiology II - Replication of Viruses (Al-Turath University PDF)

Al-Turath University /College of Pharmacy nd nd Microbiology II-2 semester/2 year students (2023/2024) Assist. Prof. Dr. Shaymaa Abdalwahed Replication of Viruses Lec 2 Virus Life Cycle – Viruses multiply only in living cells. – The host cell must provide the energy and synthetic machinery and the l...

Al-Turath University /College of Pharmacy nd nd Microbiology II-2 semester/2 year students (2023/2024) Assist. Prof. Dr. Shaymaa Abdalwahed Replication of Viruses Lec 2 Virus Life Cycle – Viruses multiply only in living cells. – The host cell must provide the energy and synthetic machinery and the low-molecular-weight precursors for the synthesis of viral proteins and nucleic acids. – This “growth cycle” involves specific attachment of virus, penetration and uncoating, nucleic acid transcription, protein synthesis, maturation and assembly of the virions and their subsequent release from the cell by budding or lysis. Specific events during the growth cycle The infecting parental virus particle attaches to the cell membrane and then penetrates the host cell. The viral genome is “uncoated” by removing the capsid proteins, and the genome is free to function. Early mRNA and proteins are synthesized; the early proteins are enzymes used to replicate the viral genome. Late mRNA and proteins are then synthesized. These late proteins are the structural, capsid proteins. The progeny virions are assembled from the replicated genetic material, and newly made capsid proteins and are then released from the cell. Another, more general way to describe the growth cycle is as follows: (1) early events or Initiation Phase includes the following steps: (attachment, penetration, and uncoating); (2) middle events (i.e., gene expression and genome replication); and (3) late events (i.e., assembly and release). General Steps in Viral Replication Cycles Attachment/Adsorption The first step in viral infection is attachment, interaction of a virion with a specific receptor site on the surface of a cell (i.e Viral attachment protein recognizes specific receptors on the cell surface.) 1 Receptor molecules differ for different viruses but are generally glycoproteins. In some cases, the virus binds protein sequences (eg, picornaviruses) and in others oligosaccharides/carbohydrate (eg, orthomyxoviruses and paramyxoviruses) or lipid components of the cell surface. – Cells without the appropriate receptors are not susceptible to the virus. The presence or absence of receptors plays an important determining role in cell tropism and viral pathogenesis. The attachment step may initiate irreversible structural changes in the virion. Penetration (Virus enters the cell) After binding, the virus particle is taken up inside the cell. This step is referred to as penetration or engulfment. – Virions are either engulfed into vacuoles by “endocytosis” or the virus envelope fuses with the plasma membrane to facilitate entry. So the viral entry by: (A). Entry via membrane fusion (Fusing): Entry by fusing with the plasma membrane. Some enveloped viruses fuse directly with the plasma membrane. Thus, the internal components of the virion are immediately delivered to the cytoplasm of the cell. – Those systems involve the interaction of a viral fusion protein with a second cellular receptor or “coreceptor” (eg, chemokine receptors for human immunodeficiency virus (HIV)). Fusing Endocytosis 2 (B). Entry via Endocytosis Viruses with no viral envelope enter the cell generally through endocytosis; they are ingested by the host cell through the cell membrane. –Entry via endosomes at the cell surface. –In some systems, the step of penetration accomplished by receptor-mediated endocytosis, with uptake of the ingested virus particles within endosomes. – Many enveloped viruses, such as COVID-19, also enter the cell through endocytosis. – Entry via the endosome guarantees low pH and exposure to proteases which are needed to open the viral capsid and release the genetic material inside. Further, endosomes transport the virus through the cell and ensure that no trace of the virus is left on the surface, which could be a substrate for immune recognition. There are also examples of direct penetration of virus particles across the plasma membrane. (C) Entry via Genetic Injection ❑ A third and more specific example, is by simply attaching to the surface of the cell via receptors on the cell, and injecting only its genome into the cell, leaving the rest of the virus on the surface. ❑ This is restricted to viruses in which only the gene is required for infection of a cell (most positive-sense, single-stranded RNA viruses because they can be immediately translated) and further restricted to viruses that actually exhibit this behavior. ❑ The best studied example includes the bacteriophages; for example, when the tail fibers of the T2 phage (Enterobacteria phage T2 is a virus that infects and kills E. coli) land on a cell, its central sheath pierces the cell membrane and the phage injects DNA from the head capsid directly into the cell. Uncoating occurs concomitantly with or shortly after penetration. – Uncoating is the physical separation of the viral nucleic acid from the outer structural components of the virion so that it can function. The genome may be released as free nucleic acid (picornaviruses) or as a nucleocapsid (reoviruses). – When the nucleic acid is uncoated, infectious virus particles cannot be recovered from the cell this is the start of the Eclipse phase – which lasts until new infectious virions are made. 3 – Uncoating is usually achieved by cellular proteases “opening up” the capsid, which required low PH in the endosome. Note: The nucleocapsids usually contain polymerases. Uncoating may require acidic pH in the endosome. The infectivity of the parental virus is lost at the uncoating stage. Viruses are the only infectious agents for which dissolution of the infecting agent is an obligatory step in the replicative pathway. Biosynthesis Expression of Viral Genomes and Synthesis of Viral Components The first step in viral gene expression is mRNA synthesis. It is at this point that viruses follow different pathways depending on the nature of their nucleic acid and the part of the cell in which they replicate. – mRNAs must be transcribed from the viral nucleic acid for successful expression and duplication of genetic information. – Then, viruses use cell components to translate the mRNA. – All of the virus specified macromolecules are synthesized in a highly organized sequence. In some viral infections, notably those involving double-stranded DNA-containing viruses, early viral proteins are synthesized soon after infection and late proteins are made only late in infection after viral DNA synthesis begins. – Viral protein is synthesized in the cytoplasm on polyribosomes composed of virus-specific mRNA and host cell ribosomes. – Many viral proteins undergo modifications (glycosylation, acylation, cleavages, etc). – Viral DNA is usually replicated in the nucleus and use the host cell DNA-dependent RNA polymerase to synthesize their mRNA. The poxviruses are the exception because they replicate in the cytoplasm, where they do not have access to the host cell RNA polymerase. They therefore carry their own polymerase within the virus particle. The genome of all DNA viruses consists of double stranded DNA, except for the parvoviruses, which have a single-stranded DNA genome. – Viral genomic RNA is generally duplicated in the cell cytoplasm, although there are exceptions 4 (retroviruses and influenza viruses, both of which have an important replicative step in the nucleus). In addition, the mRNA of hepatitis delta virus is also synthesized in the nucleus of hepatocytes. Gene expression is the process by which gene sequences are transcribed into functional gene products such as proteins or functional RNAs. Maturation – The stage of viral replication at which a virus particle becomes infectious; nucleic acids and capsids are assembled together. Assembly – The stage of replication during which all the structural components come together at one site in the cell and the basic structure of the virus particle is formed. Release – Disintegration: naked (nonenveloped) viruses accumulate in infected cells, and the cells eventually cause host cell lysis and release the virus particles. – Budding: enveloped viruses mature by a budding process. – Budding viruses do not necessarily kill the cell. Thus, some budding viruses may be able to set up persistence. Virus specific envelope glycoproteins are inserted into cellular membranes; viral nucleocapsids then bud through the membrane at these modified sites and in so doing acquire an envelope. – Enveloped viruses are not infectious until they have acquired their envelopes. Virus-induced mechanisms may regulate apoptosis (cell death), a genetically programmed event that makes cells undergo self-destruction. Some virus infections delay early apoptosis, which allows time for the production of high yields of progeny virus. Additionally, some viruses actively induce apoptosis at late stages, which facilitates spread of progeny virus to new cells. 5 Products of viral replication: 1– Virion. 2– Defective virus. 3– Abortive infection. 4– Interference. – Defective Virus: deficiency in some aspects of replication. eg, hepatitis D virus. Defective virus like Viroids (are molecule of RNA and lack a protein coat). Hepatitis D virus is a defective virus cannot replicate without the help of hepatitis B virus as a helper virus for virion assembly and infectivity. – Abortive Infection: ❑ When a virus infects a cell (or host), but cannot complete the full replication cycle (lacking some functional viral gene), i.e. a non-productive infection (did not produce any progeny virus as a result of the infection). ❑ Defective virus can cause abortive infection (fail to produce infectious progeny). – Interference means the Infection of a cell with two viruses often leads to an inhibition of multiplication of one of the viruses. Interference in animals is distinct from specific immunity. Furthermore, interference does not occur with all viral combinations; two viruses may infect and multiply within the same cell as efficiently as in single infections. 6 Several mechanisms have been elucidated/explain as causes of interference: (1) One virus may inhibit the ability of the second to adsorb to the cell, either by blocking its receptors (retroviruses, enteroviruses) or by destroying its receptors (orthomyxoviruses). (2) One virus may compete with the second for components of the replication apparatus (eg, polymerase, translation initiation factor). (3) The first virus may cause the infected cell to produce an inhibitor (interferon) that prevents replication of the second virus. Example, Interferons (IFNs) are a group of signaling proteins made and released by host cells in response to the presence of several pathogens, such as viruses, bacteria, parasites, and also tumor cells. Interferons proteins cause prevents replication of the second virus. Culture of Viruses Viral growth curve when one virion (one virus particle) infects a cell, it can replicate in approximately 10 hours to produce hundreds of virions within that cell. This remarkable amplification explains how viruses spread rapidly from cell to cell. Note that the time required for the growth cycle varies; it is minutes for some bacterial viruses and hours for some human viruses. The first event of growth cycle is: the virus disappears, as represented by the solid line dropping to the x axis. Although the virus particle, as such, is no longer present, the viral nucleic acid continues to function and begins to accumulate within the cell, as indicated by the dotted line. The time during which no virus is found inside the cell is known as the Eclipse period. 7 The eclipse period ends with the appearance of virus (solid line). The latent period, in contrast, is defined as the time from the onset of infection to the appearance of virus extracellularly. Note that infection begins with one virus particle and ends with several hundred virus particles having been produced; this type of reproduction is unique to viruses. Alterations of cell morphology accompanied by marked derangement of cell function begin toward the end of the latent period. This cytopathic effect (CPE) culminates/end in the lysis and death of cells. CPE can be seen in the light microscope and, when observed, is an important initial step in the laboratory diagnosis of viral infection. Not all viruses cause CPE; some can replicate while causing little morphologic or functional change in the cell. One step growth curve to study viral replication: 1. Attachment/Adsorption of virus (initial phase). 2. Eclipse phase: This lasts for 10-12 hours, and it corresponds to the period during which the input virus becomes uncoated. As a result, no infectious virus can detected during this time (any infectious virus detected is simply virus that is still stuck on the cell membrane). 3. Synthetic phase: This starts around 12 hours post-infection and corresponds to the time during which new virus particles are assembled. 4. Latent period: during this period, no extracellular virus can be detected. After ~18 hours, extracellular virus is detected. Ultimately, production will reach a maximum plateau level. Virus latency (or viral latency): is the ability of a pathogenic virus to lie dormant (latent) within a cell, denoted as the lysogenic part of the viral life cycle. 􀀀A latent viral infection is a type of persistent viral infection which is distinguished from a chronic viral infection. 􀀀Latency is the phase in certain viruses' life cycles in which, after initial infection, proliferation of virus particles ceases. However, the viral genome is not fully eradicated. 􀀀The result of this is that the virus can reactivate and begin producing large amounts of viral progeny (the lytic part of the viral life cycle) without the host being infected by new outside virus, , and stays within the host indefinitely. 8 􀀀Virus latency is not to be confused with clinical latency during the incubation period when a virus is not dormant. Virus latency example is Bacteriophages (viruses that infect bacteria), may undergo a lytic or lysogenic cycle. Latency: The ability of a pathogenic virus to lie dormant within a cell. Lytic cycle: The normal process of viral reproduction involving penetration of the cell membrane, nucleic acid synthesis, and lysis of the host cell. Lytic bacteriophage is T4, which infects E. coli found in the human intestinal tract. Lytic phages are more suitable for phage therapy. Lysogenic cycle: A form of viral reproduction involving the fusion of the nucleic acid of a bacteriophage with DNA of a host, followed by proliferation of the resulting prophage (endogenous phages) that become active when host conditions deteriorate or stressful condition. ` Anti-Viral Therapy When to use antiviral therapy: 1. When vaccines are not available or not highly effective. 2. Multiplicity of serotypes (eg, rhinoviruses classify to 3 species (A, B, C), including 160 serotypes that differ in their surface proteins). 3. Constantly changing virus (eg, influenza, HIV). Who need to treat with antiviral agents: 1. When vaccines would not be effective. 2. Immunosuppressed patients. 9 Antiviral Chemotherapy can be classified to: 1. Nucleoside and Nucleotide Analogs: The majority of available antiviral agents are nucleoside analogs. They inhibit nucleic acid replication by inhibition of polymerases essential for nucleic acid replication. In addition, some analogs can be incorporated into the nucleic acid and block further synthesis or alter its function. Examples of Antiviral nucleoside analogs include Acyclovir (acycloguanosine), Lamivudine (3TC), Ribavirin, Vidarabine (adenine arabinoside), and Zidovudine (azidothymidine; AZT). – Acylovir is antiviral use to treat infectious cause by Herpes simplex virus (HSV) and Varicella zoster virus (VZV). – Gancyclovir ia antiviral use to treat infectious cause by Cytomegalovirus (CMV). Nucleotide analogs differ from nucleoside analogs in: - having an attached phosphate group, - persist in cells for long periods of time, - Resistance arise, - The use of combinations of antiviral drugs can delay the emergence of resistant (e.g. Highly active antiretroviral therapy (HAART)) These agents are generally safe and well tolerated as they are used by the viral, but not human polymerases in DNA replication. 2. Reverse Transcriptase Inhibitors (RTI) Antiviral Nevirapine was the first member of the class of nonnucleoside reverse transcriptase inhibitors. RTI Characteristics: - It does not require phosphorylation for activity and does not compete with nucleoside triphosphates. RTI Action: - It acts by binding directly to reverse transcriptase (RT) and disrupting the enzyme’s catalytic site. -Resistant mutants emerge rapidly. Note: Reverse transcriptase (RT), also known as RNA-dependent DNA polymerase, is a DNA polymerase enzyme that transcribes single-stranded RNA into DNA. 10 3. Protease Inhibitors (PI) is a peptidomimetic agent (synthetic) designed by computer modeling as a molecule that fits into the active site of the HIV protease enzyme. PI Action: - inhibit the viral proteases. - Prevent mature virion core formation. - Prevent activation of reverse transcriptase (RT). PI Yields: - It Inhibition of the protease yields noninfectious virus particles. Ex: Saquinavir drug was the first protease inhibitor to be approved for treatment of HIV infection - Protease inhibitors include indinavir and ritonavir D. Other Types of Antiviral Agents - Fuzeon: is a large peptide that blocks the virus and cellular membrane fusion step involved in entry of HIV-1 into cells. - Amantadine and Rimantadine: synthetic amine, they specifically inhibit influenza A viruses by blocking viral uncoating. They must be administered prophylactically to have a significant protective effect. - Foscarnet: (phosphonoformic acid) is an organic analog of inorganic pyrophosphate. It selectively inhibits viral DNA polymerases and reverse transcriptases at the pyrophosphate binding site. - Methisazone: is of historical interest as an inhibitor of poxviruses. It was the first antiviral agent to be described and contributed to the campaign to eradicate smallpox. It blocked a late stage in viral replication, resulting in the formation of immature, noninfectious virus particles (e.g. poxvirus). – Interferons (IFNs) represent proteins that are secreted from host cells in response to various stimuli (bacteria, virus) The IFNs are host-coded proteins that are members of the large cytokine family and that inhibit viral replication. They are produced very quickly (within hours) in response to viral infection or other inducers and are one of the body’s first responders in the defense against viral infection. 11 IFN was the first cytokine to be recognized. IFNs are central to the innate antiviral immune response. They also modulate humoral and cellular immunity and have broad cell growth regulatory activities, but the focus here is on their antiviral effects. There are multiple species of IFNs that fall into three general groups, designated IFN-α, IFN-β, and IFN-γ (Alpha and Beta & Gamma) is produced mainly by lymphocytes, especially T cells and natural killer (NK) cells. – IFNs induced during viral infection. – RNA viruses are stronger inducers of IFNs than DNA. – IFNs Degrade viral mRNA. IFNs Inhibit protein synthesis of the virus. – IFNs prompting the synthesis of other proteins in the host cell which inhibit viral replication. – IFN itself is not the antiviral agent. – IFN Enhance major histocompatibility complex (MHC) class I, II expression to present viral Antigens. – IFN Induce nitric oxide synthetase (plays a key role in the control of intracellular pathogens). – IFN Used for hepatitis B virus (HBV) and hepatitis C virus (HCV). Viruses display different mechanisms that block the inhibitory activities of IFNs on virus replication, processes necessary to surmount/defeat this line of host defense. Examples include: specific viral proteins may block induction of expression of IFN (herpesvirus, papillomavirus). Thank You 12