Viruses Flashcards
Give examples of the different structures viruses can have:
- Helical capsid, filament like (TMV)
- Icosahedral capsid (Polio)
- Icosahedral capsid with lipid envelope (HSV)
- Helical capsid with lipid envelope, spherical (Influenza)
How can the type of nucleic acid determine the size of a virus?
- Number of sections (monopartite = one, segmented)
- Error rate: RNA genomes are error prone (particularly RNA dependent RNA polymerases)
- Some viruses have developed checking capabilities
- Therefore the largest viruses are dsDNA
Detail ways that viruses increase coding efficiency in their genome:
Coding increases efficiency of information to reduce genome size.
- Small intergenetic spacing (few non-coding regions)
- Overlapping reading frames: ambi-sense RNAs where +ve and -ve sense produce different proteins or ORF within larger ORFs
- Post-transcriptional mRNA editing (hijack the host splicing system to create different polyproteins).
- Ribosomal frameshift: nucleotides can be added by polymerase stutter, resulting in higher diversity of protein formation.
Name some methods to study virus structure
- Cryo-EM: Shows crystalline structure and can be used to calculate virion concentration (mix sample with known concentration of small beads and count)
- PCR: when section of sequence is known, specific primers used to identify and quantify genomes (does not measure infectivity).
Suggest some ways to detect a specific virus:
- PCR: using specific primers used to identify and quantify genomes
- Haemagglutination: for RBC binding viruses
- Immunological evidence of infection: adaptive immune response will leave antibody/T cell traces to specific virus
- Cryo-EM to determine structural markers
How might viral concentration and infectivity be determined (4 methods)?
Concentration:
- PCR (number of cycles produced)
- Cryo-EM: concentration estimated by mixing virions with beads to compare count (in virion/mL)
- Haemagglutination: for RBC binding viruses, crude concentration measured by mixing with known RBC concentration. Determine maximum dilution where haemagglutination still occurs.
Infectivity:
- Plaque Assay: A series of dilutions are applied to lawns of susceptible cells. Eventually a visible area of cells are destroyed (plaque forms due to cytopathic effect of virus). Each plaque is from one infective virus. Infectivity = pfu/mL (particle/pfu ratio important)
- Observe effect in host: ID50 number
What are the general stages of viral replication (giving specific examples)?
- Find appropriate host (shows tropism)
- Adsorption to host cell: bind to cell surface receptor e.g. HIV gp120 binds to CD4 receptor. Influenza HA binds to sialic acid
- Penetration: either endocytosis, macropinocytosis or fusion of virus envelope and cell membrane
- Eclipse phase: viral genome inside cell but no viral particles yet.
- Assembly of new virions
- Release (giving release burst size)
Detail the difference ways that viruses can enter a cell, giving examples:
- Extracellular Uptake: Endocytosis (e.g. macropinocytosis)
- Fusion (enveloped virus) with cell membrane:
Influenza: penetrates (endocytosis) allowing acidification of endosome, causing movement of HA1, exposing HA2 and allowing fusion pore formation.
HIV: gp120 binds to CD4, causing a conformational change to gp120/41 (bringing virus closer to cell) - Disruption to host membrane: picornavirus (FMDV, Polio) binds to receptor causing a conformational change to disrupt the membrane
- Injection of nucleic acid: bacteriophage T4 injects using a syringe like sheath
Describe the general genome replication cycle of -ve sense ssRNA viruses and give some examples.
- RNA transcribed to +ve sense by virus encoded RdRp
- +ve RNA used as a template strand for new -ve strands, replicates in cytoplasm
- Packaged into new virus
Purified viral RNA is not infectious
Examples: rabies, measles, ebola
Describe the general genome replication cycle of +ve sense ssRNA viruses and give examples.
- RNA impersonates mRNA so translated directly
- Translated proteins include RdRp (since they use -ve strand as template)
- Either translated into proteins or packaged into new virions
Purified viral RNA is infectious
Examples: poliovirus, FMDV, hepatitis A, rubella
Describe the general genome replication cycle of retroviruses (e.g. HIV)
- +ve ssRNA converted to intermediate DNA using viral encoded reverse transcriptase
- This DNA is inserted into host genome = a provirus
- mRNA produced using host RNA polymerase (from the provirus)
- Full length transcripts are either translated, spliced or packaged into new virus capsids
Examples: HIV, Visna virus
Describe the general genome replication cycle of dsDNA viruses and giving examples.
- Viral capsid transported to nucleus (except for Poxviruses!)
- Transcribed using host RNA polymerase II
- Some proteins (e.g. DNA polymerase and capsid) are taken back to nucleus for virion formation. DNA replicated – through rolling circle replication concatemeric DNA (later cleaved).
- Progeny genomes produced and packaged.
Viral DNA alone NOT infectious (needs to get to nucleus)
Examples: HSV, Adenovirus, HPV
What is rolling circle replication and which viruses may use it?
Method of replicating circular DNA, used by T4 bacteriophages as well as human dsDNA viruses such as HSV and HPV
1. One strand of ds circular DNA is ‘nicked’
2. ‘unnicked’ circle acts as template strand and is elongated, displacing 5’ end of nicked strand
3. Displaced DNA (‘nicked’) is effectively lagging strand and is replicated using Okazaki fragments
4. Displaced DNA is circularised
5. Replicated circles may be cleaved if DNA was concatemeric (repeating copies)
Describe poxvirus replication
Complex and large DNA viruses
- Have replication ‘factories’ = creates a microenvironment for DNA replication.
- Segregates DNA in cytoplasm (DNA in the cytosol is a powerful PAMP)
- Contain their own vDdRp for capping their RNAs
- They perform cap-snatching from host mRNA. Makes them less likely to be recognised (RLRs do not bind)
What are the ways that viruses control their gene expression (name 5)?
- Temporal control: proteins for host cell modification produced earlier than capsid proteins
- Quantitative control: early proteins (e.g. viral DdRp in a poxvirus) produced in low concentration as reusable enzymes, whereas large amounts of capsid proteins needed (noroviruses produce 180VP1 (capsid), 12VP2 and 1 viral RNA)
- Polyprotein processing: many proteins from one mRNA (post-translationally cleaved)
- RNA splicing: coding region placed close to 5’ end (increases chance of ribosome reading it). Splicing efficiency further controls protein concentration
- Ribosomal Frameshift: ribosomes pause and may slip onto another frame to restart (-1 nucleotide) producing a different protein. Retroviruses exploit this to make polyproteins.
How might transformation of a host cell lead to a tumour?
- HPV 16 or 18 induce proliferation in host cells before viral replication (increase susceptible hosts): causes host cell to not respond to contact inhibition from neighbouring cells.
- Retrovirus oncogene capture: virus can obtain a host oncogene during replication and express it at high levels (with no regulation).
- Disruption of tumour suppressor genes: due to integration of retrovirus DNA in the middle of gene (rare)
What are acute transforming retroviruses and why are they more likely to cause cancer?
Oncogene capture and expression can occur:
- Retroviruses which acquire a host gene during replication, allowing that gene to be expressed at unregulated and high levels
- When this is an oncogene, a tumour may result e.g. Rous sarcoma virus expressing src (a tyrosine kinase) at high levels – involved in proliferation and differentiation.
- Oncogene capture often associated with loss of viral sequences so may require co-infection by a helper virus to replicate.
What conditions to viruses need for assembly and how do they assemble themselves?
High concentrations of dNTPs (e.g. for large DNA viruses (poxviruses)):
- Have viral thymidine kinases
- Have ribonucleotide reductase (makes dNTP from ribonucleotides)
- Secretes moleculest o increase host production of dNTPs
Virion assembly:
- Spontaneous (non-catalytic) and may involve progressive addition of subunits (e.g. TMV)
Discuss some ways that viruses have a cytopathic effect (7 general points):
- Subversion of host cellular metabolism
- Stimulation of biochemistry to enhance viral yield
- Increase nucleotide concentrations for viral synthesis
- Cell membrane modifications/morphological changes (e.g. hypertrophy or viral protein insertion into membrane)
- Evasion of host sensing of infection (blocking innate immunity)
- Cell transformation
- Suppression of host innate immune signalling pathways.
Give examples of how a virus has a cytopathic effect on host genomes.
Altering mRNA
- Poliovirus mediated host shut off: cleaves 5’ end off host mRNA so they cannot recruit ribosomes.
- Poliovirus also inserts internal ribosome entry sites (IRES) into its RNA so it can be translated in a cap-independent manner = instead of host.
- Destruction of host mRNA/DNA
- Poxviruses de-cap host mRNA, using it as a signal to switch from early to late gene expression and subverting host proteins
What are the methods of release used by viruses?
Budding: enveloped capsid viruses ‘bud’ from host cell over prolonged periods, stealing cell membrane. E.g. influenza
Cell fusion (cell associated viruses): viruses passes directly from one cell to the other e.g. measles)
Cell lysis: spreads virus. Common for bacteriophages. Proteins causing lysis must be expressed late in infection.
What is a latent viral infection? Contrast latent HIV and HSV
Time when virus is quiescent – cell contains viral genome but no virus replication occurs. Virus has potential to switch from latency to productive replication cycle.
Retroviruses (HIV): provirus in host chromosome cannot be recognised by host immune system.
- Enables vertical transmission
HSV: viral DNA is quiescent but not integrated = an episome. HSV-1 causes cold sores, VZV causing shingles.
What are HERVs?
Human endogenous retroviruses make up 8% of genome – relics of evolutionary infection.
E.g. synctin-2 which is essential for placental formation
How do viruses beat physical barriers of a host? What are these physical barriers?
Replicate without beating barrier = superficial infection:
- Virus replication occurs in epithelium at initial entry site; short incubation, short duration. E.g. influenza, rotavirus
Systemic Infection: breaks through:
- Skin: structural barrier, microbiome (importance shown by sterile mice), sweat (contains lysozyme and dermcidin)
- Epithelial cells: cillia, mucous cells, producing mucus (high in carbs which is hard to navigate)
- Stomach acid (low pH and proteases)
How might a virus spread in a human?
Primary viraemia = spread via lymph, blood or through nerves
Secondary viraemia = infects internal organs for excessive replication (+ high viral blood count)
Describe the ways that a cell recognises viral infection.
- Nucleic acid in unusual place (e.g. DNA in cytoplasm)
- Nucleic acid with unusual structure e.g. RNA with 5’ triphosphate
- Sensed by PRRs which activates signalling cascade/TFs: E.g. TLR-4 detects dsRNA in endosome –> nuclear factor NF-kB/interferon response factors activated.
Name ways that the innate immune system works against a viral infection (8 ways)
- Complement: destroys viral particles using MAP/opsonisation
- Phagocytosis: of viral debris or infected cells
- Apoptosis: stops viral replication
- Chemokines: chemoattractant molecules to recruit leukocytes to infection site
- Cytokines: particularly IL-1/12/18, TNF and IFNs)
- Interferons: activate virus into an antiviral state and promote adaptive immunity.
- NK cells: antigen independent killing of infected cells
- Fever: restricts the replication of microorganisms
Detail how viruses evade the innate immune system (giving examples):
Against complement:
- Complement binding proteins (e.g. HSV produces glycoprotein C which binds Cb3).
- Steal host complement suppressor proteins and package into virions (CD46/55/59 produced by HIV and poxviruses)
Phagocytosis: some viruses replicate inside (want recruitment!) E.g. influenza.
Apoptosis blocking: block Bcl-2 functioning and caspase action.
Chemokines:
- Viral blocking e.g. CK protein binds glycosamide glycan (GAG) on endothelial cell
- Viral blocking of CK receptor on leukocyte
- Reduced immune cell recruitment
Against cytokines
- Transformation of host cell to secrete cytokine binding molecules
- Block signalling pathways
- EBV secretes a viral cytokine (vIl-10) to drive response towards Th2 rather than Th1.
Detail the pathway leading to IFNβ release from an infected cell:
- PRR detects viral PAMP, activating IRF3 and NF-kB.
- These move to nucleus to activate IFNβ genes
- IFNβ produced which can bind to type I IFN receptor (including as an autocrine molecule)
