Viral Replication PDF

Viruses: Replication Dr. Lorina Badger-Emeka KFU Learning objectives Students should be able to know: 1. how a virus enter its host cell 2. what viropexis is 3. define “uncoating of the viral genome 4. the difference between a positive-sense and negative-sense RNA virus...

Viruses: Replication Dr. Lorina Badger-Emeka KFU Learning objectives Students should be able to know: 1. how a virus enter its host cell 2. what viropexis is 3. define “uncoating of the viral genome 4. the difference between a positive-sense and negative-sense RNA virus 5. consequences that differences between a plus-sense and a minus- sense RNA virus have for the composition of the virus particle  Learning objectives cont’d 6. what a retrovirus is 7. be able to enumerate how viral components are synthesized 8. Know what the difference between “early” and “late” viral gene products are. 9. how new viral particles leave the infected cell 10. be able to explain the term “budding” of viral particles 11. what defective interfering particles are 12. define the concept of mutation and selection 13. explain the role of viruses as vectors for therapy. Introduction Viral replication process or circle is generally separated into phases 1. the early phase infection, recognition of an appropriate target cell by the virus; attaching to the cell; penetrating the plasma membrane and gets taken up by the cell; the virus releases (uncoat) its genome into the cytoplasm; And if there is a need, delivers the genome to the nucleus Introduction 2 2. Late phase infection The late phase commences with genome replication and viral macromolecular synthesis Then proceeds through viral assembly and release.  Steps in Viral Replication Courtesy Murray all editions. How a virus enter its host cell 1 Internalisation of a virus into a cell is initiated by Interaction between multiple VAPs and cellular receptors This mechanism of internalisation depends on 1. the structure of the virion and 2. the type of the cell How a virus enter its host cell [envelope viruses] They [envelope viruses] fuse their membranes to Those of the cellular membranes and then, Delivers the genome or nucleocapsid, directly into the cytoplasm  How a virus enter its host cell [non-envelope viruses] Most enter the cell by either Receptor-mediated endocytosis which is the normal process used by cells to the uptake of receptor molecules. Or by Viropexis  Courtesy google image Receptor-mediated endocytosis  Penetration: endocytosis (engulfment). Courtesy: Marija Milovanovic et al., 2017 https://doi.org/10.1016/B978-0-323-52733-0.00014-8 what is viropexis Defined as “the process by which different classes of viruses, [e.g., picornaviruses and Papovaviruses] enters a cell and is incorporated into it by phagocytosis. Viropexis, is made possible by exposed hydrophobic structures of capsid proteins Which help the virus to penetrate the cell membrane Viral replication refresh steps of the process  Virus Replication Cycle 1. Adsorption or Attachment 2. Penetration, Entry, and Uncoating 3. Production of Virion Components 4. Transcription 5. Genome Replication 6. Assembly of Naked Capsid Viruses and Nucleocapsids 7. Release of Viral Particles 1. Attachment or adsorption Virus must recognize and interact with a specific receptor site on the surface of the host cell. Receptor molecules differ for different viruses but are generally glycoproteins. The receptors may also be proteins or oligosaccharides. 1. Attachment or adsorption cont’d There is specificity of attachment (key and lock interaction) This explains specificity of the viruses to certain cells and tissues. e.g., HIV binds to the CD4 protein on T helper cells, rabies virus attach to the acetylcholine receptor and poliovirus binds to CNS cells. The presence or absence of receptors plays an important role in viral pathogenesis. 2- Penetration After binding, the virus particle is taken up inside the cell [internalization]. Mechanism of internalization of a virus depends on its structure. The nucleocapsid of the non-enveloped viruses is taken up inside the cell by a process of endocytosis (engulfment). Enveloped viruses are internalized by fusion of the virion envelope and plasma membrane of the cell. Penetration by fusion 3. Uncoating of the viral genome As soon as the virus enters the host cell, The nucleocapsid must be delivered to the replication site inside the cell Then, The capsid or the envelope are removed For DNA viruses, the genome is delivered to the nucleus While that of the RNA, remains in the cytoplasm Uncoating process can now be initiated  Uncoating [Define] It is therefore, a physical separation of the viral nucleic acid from the outer structural components of the virion by host cell enzymes contained in endosomes or lysosomes. The infectivity of the parental virus is lost at this point. Uncoating cont’d Viruses are the only infectious agents for which dissolution of the infecting agent [i.e., uncoating] is Obligatory in the replication pathway. This phase of the replication process is called eclipse period (loss of infectivity). The eclipse duration period depends on both the virus and the host cell Eclipse phase is followed by an interval of rapid accumulation of infectious progeny particles Or synthetic phase. 4. Gene expression and genome replication The phase occurs after uncoating of the viral genome. Most RNA viruses undergo their full replication cycle in the cytoplasm Except for retroviruses, influenza viruses and hepatitis D virus. Replication of viral DNA occurs in the nucleus, Except for poxviruses and herpes viruses. Positive-sense and negative-sense RNA virus  Positive-sense and negative-sense RNA virus [introduction] The ribosome only reads mRNA Therefore, either the DNA or RNA virus must first transfer Their genome into mRNA. So that the ribosome can read them And make the viral protein  A positive-sense RNA virus Positive [+ve] sense RNA that comes from a virus is in itself, An mRNA and is readable directly into the ribosome Therefore, It goes directly from the virus into the ribosome and Translates into protein  A negative-sense RNA virus 2 For negative sense [-ve] RNA refers to RNA from the virus That is not readable for the ribosomes And Most first be transferred to +ve RNA All must depend on an enzyme to transfer the –ve sense RNA to +ve sense RNA A) + ssRNA (positive sense single stranded RNA): This viral nucleic acid is infectious and serves as mRNA. B) – ssRNA (negative sense single stranded RNA): These viruses contain RNA polymerases to synthesize mRNA. They must supply their own RNA polymerase (RNA dependent RNA polymerase)  Difference between a positive-sense and negative-sense RNA virus Positive [+ve] sense RNA Negative [-ve] sense RNA Has –ve sense [3’to5’] RNA genome Has +ve sense[5’to3’] RNA genome. Referred to as – strand or an The positive strand RNA viral genome antisense Acts as mRNA Negative strand RNA genome is not Binds to ribosomes infectious And Cannot bind to the ribosome Directs protein synthesis A polymerase must be carried into The naked +ve stand viral genome is the cell with the genome to Make the sufficient to initiate infection mRNA for different viral proteins Difference between a positive-sense and negative-sense RNA virus. cont’d Positive [+ve] sense RNA Negative [-ve] sense RNA Does not require RNA polymerase Needs RNA dependent RNA Replication is through double polymerase to transcript the genome stranded intermediate RNA into +ve sense RNA E.g., Replication is with aid of RNA –ve dependant RNA polymerase Polio virus E.g., mumps virus, Coxackie virus Ebola, Rabies, influenza Etc. C) Double stranded RNA ± dsRNA These viruses use viral RNA polymerases to synthesize mRNA. What is a retrovirus Retroviruses have a positive-strand RNA genome, However, the virus provides no means for replication of the RNA in the cytoplasm. Instead, the retroviruses carries two copies of the genome, two transfer RNA (tRNA) molecules, and an RNA-dependent DNA polymerase (reverse transcriptase) in the virion.  Retrovirus Retrovirus, therefore, are a type of virus They use a special enzyme called reverse transcriptase to translate its genetic information into DNA. The DNA can now be integrated into the DNA of the host cell. On integration, the virus will use the components of the host cell to make additional viral particles. General scheme of viral replication. summary Courtesy Medical Microbiology by Murray 9th Ed. And all editions. How viral components are synthesized Once inside the cell, the genome directs the synthesis of viral mRNA and protein and generates identical copies of itself. The process of transcription into a functional mRNAs Or translation into protein Transcription and translation steps occur in two stages A- Formation of early proteins: e.g., Polymerases, regulatory proteins, viral antigens. B- Formation of late proteins: e.g., Structural capsid proteins, enzymes required for the release of the virus from the cell.  Viral macromolecular synthesis steps: 1, Double-stranded DNA (DS DNA) uses host machinery in the nucleus (except poxviruses) to make mRNA 2, Single-stranded DNA (SS DNA) is converted into DS DNA and replicates like DS DNA. 3, (+) RNA resembles an mRNA that binds to ribosomes to make a polyprotein that cleaves into individual proteins. 4, (−) RNA is transcribed into mRNAs, a full-length (+) RNA template by the virion RNA polymerase 5, DS RNA acts like (−) RNA. For details see Medical Microbiology, 7th Ed. Pg 482 Differences between early and late viral gene products Early gene products are frequently DNA-binding proteins and enzymes, that includes virus-encoded polymerases. They are catalytic with only a few are required. Replication of the genome usually initiates the transition to transcription of late gene products. Late viral genes encode structural and other proteins. Many copies of the proteins are required to package the virus, But are however, generally not required before genome replication. Replication of a complex enveloped DNA virus. Transcription and translation occur in three phases: immediate early, early, and late. For details see Medical Microbiology, 7th Ed. Pg 483 5- Assembly and maturation It is the irreversible combination of the viral nucleic acid with the protein coat to form new infectious virions. Components of capsid synthesis directed by late genes Assembly of enveloped viruses will need to interact with plasma membrane which has been modified How new viral particles leave the infected cell  6- Release: Final stage of the infection There are two methods for the release of viruses 1. Lysis [known as cytolytic viruses] at the death of the cell 2. Budding [by cytopathic viruses] they do not kill the infected cell. None killing of the cell allows continual release of virus from the factory Non-enveloped viruses are released from the infected cells after lysis by exocytosis. Enveloped viruses are released by budding where virus specific envelope glycoproteins are inserted into cellular membrane. Re-initiation of the Replication [Spread of infection] Virus released to the extracellular medium of cell Through cell-to-cell fusion (merging of infected cells into multinucleated giant cells syncytia Becoming huge virus factories Vertically to daughter cells transmitting gene copy on cell division Involves alternate routes of the transmission of viral nucleocapsid, or genome Defining the concept of viral mutation Mutations spontaneously and readily occur in viral genomes, Result are new viral strains Whose properties could differ from those of the parental, or wild-type, virus. Resultant variants can be identified by their nucleotide sequences, antigenic serotype differences, or by functional or structural property differences. Mutations in essential genes could 1. inactivate the virus [termed lethal mutations] 2. cause resistance to antiviral drugs 3. alter the antigenicity or pathogenicity of the virus Concepts of mutation and selection Viral mutants are selected by several factors including The ability to use the host cell machinery In withstanding body and environmental conditions Also, Cellular properties acting as pressure selections. Includes cell growth rate and tissue-specific of proteins required by the virus (e.g., enzymes, glycoproteins, transcription factors) viruses which cannot endure specific or evade the host defenses are eliminated. What are Defective interfering particles (DIPs)? These are also known as defective interfering viruses, They are the result of unexpectedly generated virus mutants Here, a critical portion of the genome particle is lost Either as a result of defective replication Or non-homologous recombination. Viruses as vectors for therapy Modification of viral genetic composition can be excellent for creating foreign genes. Resultant can provide gene replacement therapy, used for vaccines to promote immunity etc. Remove viral genes causing the disease and replace it by desired gene Viral vectors used (attenuated / defective viruses) Gene therapy clinical trials - Retroviruses or Adenoviruses to deliver the desired gene Summary Consequences of Viral Properties Steps in Viral Replication References Medical Microbiology by Murray 7th Edition, section 5, page 471 onwards 8th Edition, chapter 36; page 416. 9th Edition, Chapter- 36; page 362 onwards