Viral+ReplicationS24.pdf

Viral Replication Chapter 2 from the recommended textbook (Fenner's Veterinary Virology 2017) Lecture Learning Outcomes Describe how do we study Recognize the sequence of...

Viral Replication Chapter 2 from the recommended textbook (Fenner's Veterinary Virology 2017) Lecture Learning Outcomes Describe how do we study Recognize the sequence of Explain the concept of Describe how do we study viral replication in viruses events of the one step List and describe the basic susceptible and permissive Explain the eclipse time the replication of viruses that do not grow in cell growth curve, what do you steps in viral replication cells culture measure? Explain the concepts of genome replication, Explain what does uncoating transcription and translation Determine what is required List and explain different mean and how do virus Describe the replication of Describe the replication of applied to viruses (including for attachment methods of viral penetration translocate (cellular DNA viruses RNA viruses what is the source and what chaperones, signaling) is the product). Explain what are the Explain what the packing Describe how do most naked Identify the major Describe the concept of + and Appreciate the replication structures of mRNAs (cap, sequence in the viral genome viruses exit the cell vs characteristics of retrovirus – sense RNA in replication mechanism of Retroviruses poly AAAA) and their is used for enveloped viruses replication functions  Viral replication is studied In vitro using cell cultures (in suspension or Growth curve of viruses monolayer) in what is known as the growth curve of viruses. The increase in infectious virus is measured over time by sampling the virus in the supernatant. The sequence of events are: binding the receptors and entering the cells, eclipse (replication of the genome, production of structural and non structural proteins) assembly and exit of the cells to infect other cells where virus particles are observable once again. The eclipse period is the time after the virus has penetrated the cell but cannot be observed for hours until the first progeny of virions become visible again. The eclipse time is variable between families/genus and constant within (2-12 hrs depending on the virus genus/family). This is an important time where antiviral drugs can interfere with viral replication The eclipse time is unrelated to the incubation time of the virus which is the time between the infection of the host until it shows clinical signs all of which occurs in vivo There is a difference between susceptible cell lines (virus can infect but not complete the replication cycle) and permissive cell lines (virus can infect and complete the cycle) Unculturable viruses and other means of studying viruses If a virus does not grow in cell culture, we can construct an infectious clone; which means inserting the viral genome into a plasmid: this circumvents the need of the necessary receptors to enter that specific cell type and promotors to start replication and transcription Basics steps of viral replication  1) Viral attachment Receptors on the viral envelope or capsid become connected to complementary receptors and coreceptors on the cell membrane expressed in susceptible cells 2) Viral penetration the viral capsid or genome enters the host cell's cytoplasm https://www.youtube.com/watch?v=xqIxZruKpm0 Entry via membrane fusion: Viral receptors attach to the receptors on the surface of the cell and secondary receptors may also be present to initiate the fusion with the host cell. The virus's envelope blends with the cell membrane, releasing its contents into the cell. Enveloped viruses usually enter this way Entry via endocytosis: This is a receptor mediated process. Most naked viruses use this process. Entry via genetic injection (FYI https://www.youtube.com/watch?v=oqYKPf7hnHs): injecting only its genome into the cell, leaving the rest of the virus on the surface, for example phages 3) Viral uncoating The viral capsid opens and frees the genome (it can be partial or complete). Viruses are not motile thus, cellular proteins called chaperones may take the viral genome to the nucleus if it is a virus that replicates at that site 4-8) Transcription (genome to mRNA), translation (mRNA to protein) and replication (genome copying itself) these steps can differ in the order depending whether viruses have DNA or RNA genome and if it is positive or negative polarity (5-3' or 3-5') DNA viruses that replicate in the nucleus use the cellular DNA-dependent RNA polymerase to produce their mRNAs (click here for a short animation) https://www.youtube.com/embed/blQirc3Oasw?wmode=opaque The exception are DNA viruses that replicate in the cytoplasm which carry a DNA-dependent RNA polymerase of their own (which has the same function as the cellular enzyme but is localized in the cytoplasm such as Poxviruses) In most single stranded + RNA viruses the nucleic acid can directly bind to ribosomes and start translating either partially or fully. Some are capped and have polyA tails so they look and behave like cellular mRNAs. Once they translate the mRNA they usually code for their own viral polymerase to replicate their genome (a specific RNA-dependent RNA polymerase). After translation by the cellular machinery the enzyme is produced to carry out the RNA transcription and replication (click here for a short animation) https://www.youtube.com/embed/v7Av-TfnjII?wmode=opaque Single stranded negative sense RNA viruses must carry their own polymerase enzyme inside their nucleocapsid, a specific RNA- dependent RNA polymerase (the protein itself not just the gene) (click here for a short animation) https://www.youtube.com/embed/tSf6wbojFK8?wmode=opaque Polymerases  Post translational processing: A cap facilitates the formation of a stable complex with the 40S ribosomal unit necessary for initiation of translation (FYI Most viruses use cap-dependent translation machinery to ensure the expression of their messenger RNAs (mRNAs). The addition of a cap structure at the 5' end of viral mRNAs is therefore an essential step for the replication of many viruses. Indeed, the cap protects mRNAs from degradation by cellular nucleases and neutralizes the detection of viral mRNAs by the mechanisms of innate immunity. The acquisition of the viral RNA cap is done either by using the cell-forming enzymes of the cap of the infected cell or by stealing the mRNA cap of the infected cell, or by dedicated viral enzymatic machinery. Many viral enzymes involved in cap synthesis have recently been characterized structurally and functionally. These studies have revealed original synthesis mechanisms that pave the way for the development of specific inhibitors with antiviral potential. This summary is from https://pubmed.ncbi.nlm.nih.gov/22549871/#:~:text=Viral%20RNAs%20acquire%20their%20cap%20structure%20either% 20by,process%20have%20recently%20been%20structurally%20and%20functionally%20characterized. 50-100 adenylate residues added to the 3’end. This poly A tail is a recognition signal for transport from the nucleus to the cytoplasm and protects the mRNA against degradation FYI https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7158166/ Methylation: methyl group added to the 6 position to about 1% of the adenylate residues throughout the mRNA Splicing: removal of introns (only exons left)- alternative splicing Post translational modifications (phosphorylation, glycosylation, proteolytic cleavage) Cellular mRNAs are monocistronic (1 mRNA= 1 Protein) but viral mRNA are usually polycistronic (mRNA carrying information for the synthesis of more than one protein) Ribosomal frameshifting 9) Assembly The structural proteins of simple icosahedral viruses associate spontaneously to form capsomers which self-assemble to form capsids into which the viral nucleic acid is packaged (click here for an example) https://www.youtube.com/watch?v=X-8MP7g8XOE In helical structured viruses, the RNA molecule self- assembles as a cylindrical helix bound to their structural proteins (think of a pearl necklace) Completion may need proteolytic cleavage of one or more viral proteins One end of the viral genome has a “packing sequence” that binds to a protein that enables the entering to the procapsid 10) Release Naked viruses usually accumulate in the nucleus or the cytoplasm until they exit from the cell by lyses Hydrophilic and hydrophobic portions of the envelope viral proteins localize to the pertinent cellular membranes Matrix proteins also align on the inside of the membrane where the nucleocapsid is going to bud from Thousands of virions can shed per hour or days without cell damage (many can be persistent infections) Usually, viruses carrying envelopes from golgi or RER (flavi, corona, arteri, bunya) travel in vesicles to the cell membrane which they fuse, releasing the virus by exocytosis Herpes is an exception: buds from nuclear membrane exits directly through the cisternae of the endoplasmic reticulum (ER). Enveloped viruses exit from the cell by budding (A) or exocytosis (B)  Retrovirus replication chapter 2 pgs 33-36 Although retroviruses have single stranded RNA genome, they do not act as mRNAs……instead they use their own retrotranscriptase (RT) enzyme with RNA dependent DNA polymerase capability) to first produce a RNA-DNA hybrid and then a double stranded DNA that can insert into the host genome. FYI https://en.wikipedia.org/wiki/Reverse_transcriptase In summary: Retrovirus genomes have 2 copies of single stranded + polarity RNA but they replicate through a DNA intermediate These viruses are unique in that they carry a reverse transcriptase (RT) enzyme that uses a tRNA molecule as primer to make a ss DNA copy The same enzyme then (acting as a ribonuclease) removes the parental RNA from the RNA:DNA hybrid and then copies the negative sense (-) single stranded DNA to form a linear double stranded DNA which contains an additional sequence called LTR (long terminal repeats) at each end These LTRs are important for 2 reasons A) allows viral genome to integrate to host genome B) act as a strong promotor https://en.wikipedia.org/wiki/Long_terminal_repeat and just in case https://en.wikipedia.org/wiki/Promoter_(genetics) The resulting double stranded DNA then integrates into the cellular chromosomal DNA Transcription of viral RNA occurs from this integrated (proviral) DNA From there, the cellular polymerase will transcribe mRNA and full length (genomic) RNAs that will pair to form the diploid genome of the new progeny virus