Virus Replication-1.pdf

Virus Replication Rob Noad [email protected] For the exam you should be able to: 1. Describe the stages of virus replication in a host cell. 2. Outline how viruses are cultured in the laboratory. 3. Explain the effects that virus infection has on cells. - What is a virus? Example: FMDV RNA + protein s...

Virus Replication Rob Noad [email protected] For the exam you should be able to: 1. Describe the stages of virus replication in a host cell. 2. Outline how viruses are cultured in the laboratory. 3. Explain the effects that virus infection has on cells. - What is a virus? Example: FMDV RNA + protein shell (=capsid, both together=nucleocapsid) RNA =Instruction to make protein copy me package me proteins, capping enzymes) Viruses need a host. What is it that the virus specifically needs from the host cell? from DNA virus ↓ transcription. RWA RNA from ↑ URNA/DNA virus + ↓ ribosome translationa ↓ from host I cell Varus to higarks. prote production make new virus in host. capsed. well reral/virus capsed protes (attachment potem) D (bunding Viruses normally interact with specific receptors on the surface of host cells ↑ well protein (reception : Lee H, et al., Proc Natl Acad Sci U S A. 2019;116(41):20462-20471. doi: 10.1073/pnas.1904918116. The type of proteins (and sometimes glycans) on the surface of the cell can help determine whether that cell is susceptible to virus infection. Normal cell biology of receptor activation and recycling. · normal endosomal prodway Normally when a receptor is activated by destroyed by the cell. Gradual steration Image from: https://doi.org/10.1016/j.cytogfr.2016.07.002 This prevents the receptor being permanently activated and allows the cell to respond to a dynamic environment. Example: Canine parvovirus entry (nonenveloped virus) - 1. attachment. Receptor used= Transferrin receptor (normally expressed highly on rapidly dividing cells). 1 4 3 2 Lee H, et al., Proc Natl Acad Sci U S A. 2019;116(41):20462-20471. doi: 10.1073/pnas.1904918116. 3 : camme only carrovirus I infect eating wells membrane breaks down reforms & nuclear 2. Virus is taken up into endosomes via endocytosis. #x 3. Acidification of the endosome as it matures leads to a conformational shift in the virus particle which is trafficked to the nucleus. 4. The viral ssDNA is released into the nucleus (probably through a pore which opens at the 5 fold axis of symmetry) can uses other hallicked exception To in DNA the Pox V DWA cytoplasm. elication rel is nucleus DNA viruses in : , only the in to will RNA for translation cytoplasm transport usually exceptional Example: Influenza A virus entry (enveloped virus) case virus. - > - carbohydrate 1. attachment. Receptor used= sialic acid (glycan present on the surface of many cell types including those lining the epithelia in mammals). 1 2 coated under any sializ proteins. receptor protein w/ and influenza internalizes verus 2. Virus is taken up into endosomes via endocytosis. 4 3. Acidification of the endosome as it matures leads to a conformational shift in the HA protein which leads to fusion between the endosomal membrane and the viral membrane. : both liped membrane 3 ↳ real mucheocapsed released 4. The viral nucleocapsid trafficked to the nucleus. S ummary initial & : stages of attachment virus of veral attachment receptor protein ⑦ Membrane virus infection on well protein to surface penetration effect I delevers nucleic and across well membrance Example: SARS-CoV-2 (enveloped virus) ACE-2 receptor Pattach pro Spike is cleaved by proteases to allow fusion between viral and endosomal membranes cellular surface - I in e.g. TMPRSS2 and lysosomal proteases SARS-CoV-2 - brings 2mumbe an eged eng Credit: Janet Iwasa, University of Utah interaction between & virus. host Attachment is the first step in any virus infection, it is a major determinant of tropism - 6 determines it FMDV Y : infect can rabbits of absence receptor ⑳ speci ficity/ what N infect Summary of attachment and entry Attachment is an important step in the biology of viruses because it is a major determinant of tropism (which cell in the body and which type of host) the virus can infect. Attachment involves the interaction between a virus attachment protein (on the surface of the virus, sometimes referred to as a viral glycoprotein) and a host cell receptor (this is usually a protein but can be a glycan which is present on cellular proteins, such as sialic acid). The cell membrane is a major barrier to virus entry. To overcome this many (but not all) viruses trick the cell into internalising them by receptor mediated endocytosis. The acidification of the endosome triggers a conformational change in the proteins of the virus that allow them to escape the endosome by fusion or lysis depending on whether the virus is enveloped or nonenveloped. What happens next? Influenza CPV 1. Transcription (production of mRNA) SsDNA 3 3 DNA 1 1 2. Translation (production of viral structural and nonstructural proteins) 3. Genome replication 4 5 Lysis 2 4 O 2 5 Budding I used in some replication of in nucleus : very trafficked back to nucleus some proteins form 4. Assembly of new virus particles viral capsed 5. Release from infected cells. What happens next? FMDV 1. Transcription (production of mRNA) 3 2. Translation (production of viral structural and nonstructural proteins) 1 3. Genome replication i 2 5 Lysis 4 3,1 4 4. Assembly of new virus particles 2 2 4 WRWA translated 5. Release from infected cells. 5 Budding to pan a Enzyme > - f new a viral. particle Although the order of the step varies most viruses will go through the following steps in their replication inside cells Attachment Release (lysis or budding) Assembly new virus particle Penetration (fusion) Uncoating (may be partial) Translationg Transcription In general DNA viruses replicate in the nucleus (pox viruses are an exception) In general RNA viruses replicate in the cytoplasm (influenza virus and some other negative sense viruses are an exception) Most enveloped viruses exit via budding, this is usually either at the cell membrane, or into the golgi, or sometimes through the nuclear membrane. * enveloped virus membrane when : picks it up cellular leaves virusmaynot carry well a ey In contrast, non-enveloped (naked) viruses usually exit via lysis. There is also evidence that some nonenveloped viruses can spread from infected cells by non-lytic routes (for most of these, the only mode of release. Veterinary examples: Rotavirus, Bluetongue y J. Gen. Virol. 102, 001557 (2021); https://doi.org/10.1099/jgv.0.001557 do the ly egress no. Virus infection can have impacts on the function and morphology of the host cell. Kedney wells Virus infection can have severe consequences for cells grown in culture. Typical signs include: BHK21 Venhance :speedlysisof spready Rounding and detachment of cells. : some Formation of giant multi-nucleated cells (Syncytia) - programmed ↓ Apoptotic cell death. dismantley avoud recognise inflammation active autonomous vellular " to elicting BHK21+FMDV -o Inclusion bodies (protein+nucleic acid deposits inside cells. For some viruses these are implicated as sites of virus replication). Sakamoto et al., J. Vet. Med. Sci. 64(1): 91 94, 2002 · metabolic load viral protein : in cell some virus I inhibit viral presence gene expression response accumulation time to b4 trigger use up early cormote resources replicate leaving. Syncytia form when the virus infected cell fuses with adjacent cells. 88 Measles virus Paramyxoviridae Syncytia (respiratory syncytial virus) : buddia ng plasma membrane > - V rus attachment protesa > - bind to next cell fuse membrances b ~ giant cellswl multiple. nucleus. Many viruses form lipid and protein based structures that act as virus replication centres. diff virus diff names for the structures. I j A brain tenacious Inclusion Nevers Q, Albertini AA, Lagaudrière-Gesbert C, Gaudin Y. Negri bodies and other virus membrane-less replication compartments. Biochim Biophys Acta Mol Cell Res. 2020;1867(12):118831. doi:10.1016/j.bbamcr.2020.118831 Rabies virus b travel through nervous system. autopsy Cytopathic effect has implications for pathology and virus growth and quantification.. Totructural changes in host wells caused by viral Invasion Pathology- Direct effects on target cell (more on this in the next lecture I give you this year) Virus quantification- Plaque assay/TCID50) TCIDJU dont back calculate original amount Infectivity assays, like plaque assays quantify the amount of infectious virus present in a sample. Example of method; plaque assay What happens when a virus infection does not cause CPE? Virus infection does not always lead to CPE it depends on the virus species, cell line and sometimes virus strain. Example: Bovine Viral Diarrhea virus has cytopathic and non cytopathic strains. It is possible to detect if cells are infected with a noncytopathic strain of a virus but it is necessary to use an antibody that recognises virus proteins. 3 detect where anibody binds. Viruses: a possible culture problem : hard to grow For a virus to grow in culture you need a sample of virus AND a suitable cell line Primary cells 1. Take tissue sample -STERILE In use Cell culture medium Buffered salt solution containing: antibiotics glucose amino acids 2. Cut tissue into 3. Treat with enzymes to very small pieces vaccine break up tissue further and to obtain individual cells 4. Put cells into culture flasks with cell culture medium. prep Lancerous well line "guarantee X adventations foetal calf serum (growth factors) pathogens ag an on a chep : tissue manerate & feed differentiate Primary cells are good for following host response to virus infection in real time (although arguably organoids are better). > ↑ authentic of well - variety types hormone cocktail to a - back original organs mimics but of what's the. the animal in : -usually cells have deferentiation when > - I divisionn.. However they have limitations (they can typically only be passaged 10-20 times before senescence, you need to reisolate from animals when the cells stop growing). into X last reached isolated limited well long terminal Immortalised cells are spontaneous or engineered mutants which do not undergo senescence. Some immortalised cells e.g. Vero cells are particularly good for virus culture because they lack a functional type I interferon system. Immortalised cells do not always have the correct receptors or surface proteases for culture of specific virus so sometimes genetically engineered derivatives are used. Culture of viruses in chicken eggs Fertilised eggs (approx.10 days) Sterile inoculation e.g. growth of influenza viruses in allantoic cavity Influenza vaccine production Then....1940s Australia With thanks to Frederick A Murphy for image And now...... Sample from site of clinical signs: e.g. nasal swab, faeces, tissue from post mortem case..... but X the site of necessarily inoculation very Keep on ice or freeze for long-term storage Suspend sample in cell culture medium with antibiotics Inoculate cell cultures and wait for changes if culture sample b stone nudear in and fred ge analyses > store qPCR rather than culture of electron microscopy is the current default detection method for most viruses. infreezer For the exam you should be able to: 1. attachment to exit Describe the stages of virus replication in a host cell. plague assay (primple 2. Outline how viruses are cultured in the laboratory. 3. Explain the effects that virus infection has on cells. & how et works