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Virology (MD PhD) (Subject) / Hangartner (Lesson)
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- Solutions for the end problem 1. Circular(ize) genome- Herpesviridae- Papillomaviridae- Polyomaviridae 2. Hairpins/linkage of ends- Poxviridae- Parvoviridae 3. Protein primers - Adenoviridae
- Simian Virus 40 (SV40) - Polyomavirus - closed circular ds DNA genome - replication fork-typ - With exception of initiation via large-T, replication of the SV40 genome depends on the cellular DNA synthesis apparatus (S phase only!) - The large-T antigen/SV40 ORI system is used to increase expression of transfected plasmids. - commonly used cell lines that express Large T and will amplify and plasmid containing a SV40 Ori:- COS-7 (African green monkey)- HEK 293T (Human embryonic kidney)
- Adeno-associated virus 2 (AAV2) - Parvovirus:Dependovirus - ss DNA genome - Primer-independent (Hairpin-type of replication) - Requires a helper virus (adeno-, herpes-, or poxvirus) for replication - Because superinfection is rare, AAV-2 needs a strategy to persist in a cell until this cell becomes superinfected; it does this by integrating its genome into the genome of the host cell (latency)
- Adenovirus - linear double-stranded genome of 22-45 kb exists as 55 serotypes that group in seven species (A to G) in humans can cause - respiratory disease - conjunctivitis - gastroenteritis - replicates in the nucleus - Primer-dependent (uses a protein primer to initiate replication) - replicates by strand displacement - provides four proteins for DNA replication1. DNA Polymerase2. Pre-terminal protein3. Terminal protein 4. Single-stranded-DNA-binding Protein
- Licensed replication: Epstein-Barr virus - Goes into latency by inactivating its genome via CpG-methylation as a stable autosomal episome The EBV episome- is maintained at 10-50 copies per cell. - uses a special origin (OriP) and ori-binding protein (EBNA-1) to allows licensed replication (replication of the episome during division of the host cell) - Reactivation occurs by transcription of viral transcriptional activators Zta and Rta that initiate expression of the viral polymerase and other proteins required for replication from OriLyt
- Modulation of polyadenylation - regulated production of protein isoforms - Adenoviridae- Herpesviridae- Papillomaviridae
- Modulation of splicing - regulated production of protein isoforms - Adenoviridae- Herpesviridae- HIV (Rev)
- Modulation of RNA export - export of exonless viral RNAs - inhibition of cellular protein synthesis (incl. innate antiviral responses) - Adenovirus- VSV- Picorna
- Antigen half-life - soluble immunogens usually only have a poor half-life therefore,- they are emulsified- injected into a intra-muscular depot
- Adjuvants Aluminium salt precipitates (Aluminium hydroxide or aluminium phosphate) - improves antigen-delivery to APC- activates stroma cells- does not directly activate APC--> triggers inflammation AS03 Adjuvant- Squalen - DL-Tocopherol (Vitamin E)- Polysorbat 80 (emulsifier) MF59C.1--> induces extracellular ATP (danger signal)
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- Immunogenicity B-cells triggered best:- rigid two-dimensional arrays - more than 12-16 moieties- optimally spaced by 5-10 nm --> virions and VLP are better immunogens than soluble proteins Soluble immunogens usually only have a poor half-life --> emulsified--> injected into a intra-muscular depot
- Passive immunisation Application - Post-exposure prophylaxis (RV, hepatitis A virus, and HBV) - Prophylaxis against severe illness (RSV, Varicella-Zoster, measles virus in high-risk individuals) - preventing congenital infection with HBV - protecting organ recipients from emerging cytomegalovirus infection - Rabies post-exposure - ZMAb® against Ebola
- Attenuation can be caused by - Loss of countermeasures against the innate immune system (e.g. interferon) loss of viral fitness - replication velocity - translational efficiency (Polio)- reduced receptor affinity (Rubella) - use of different receptor/altered tropism (Measles, Mumps) - temperature sensitivity (FluMist)
- Viruses that provide viral DNA synthesis apparatus - Usually only observed in viruses with large genomes (e.g. Herpes or Poxviruses), as it comes at a high genetic expense - allows replication outside of the nucleus in the cytoplasm - provides targets for antiviral drugs (e.g. Acyclovir -> HSV thymidin kinase)
- Positive autoregulatory loop: HIV-1 Tat/TAR In the absence of Tat, only 10% of all transcripts cover the full length of proviral RNA, the rest terminates within the first 60 nt Thus, HIV-1 LTRs are unable to support efficient RNA synthesis in the absence of Tat
- RNA capping by viral enzymes can be performed by - dedicated capping enzyme (eg. Reoviridae)- viral RNA polymerase (e.g. Rhabdoviridae)
- cap-snatching - Hijacking cellular machinery - 5‘ end of viral RNA binds to PB1 - Binding activates the other two binding sites - cellular mRNA binds to PB2- site in PB1 binds to 3’ end of genome, which activates nuclease activity --> The capped mRNA stump from the cellular mRNA serves as primer for mRNA synthesis
- IRES - Tricking machinery with cap independent initiation - can be found in picorna-, pesti- and hepaciviridae - IRES form extensive secondary structures that are required for their function - there is little sequence conservation between different IRES - all IRES share a oligopyrimidine tract of 25nt close to 3’ end
- Polyadenylation by cellular enzymes - CPSF is recruited to AAUAAA consensus - CSTF binds to GU or U rich sequences downstream - Poly(A) polymerase is recruited and mRNA cleavage is triggered - Pol(A) polymerase slowly adds 12-15 A residues - Poly(A) binding protein (PABII) binds to the short poly(A) stretch and tethers Pol(A) pol to end of mRNA - rapid synthesis of 200-250 A (protects mRNA from degradation) - Viral alternative: 'stuttering' at AAUAA sequences
- No viral analogues - all viruses rely on the cellular splicing machinery for viral mRNA export and splicing - viruses may modify cellular splicing/nuclear exporting Problematic cases: - retroviruses, herpesviruses and orthomyxoviruses depend on export of viral RNA without concomitant splicing - single exon viral mRNAs
- Export w/o splicing - HIV uses Rev to protect the mRNA from being spliced in the cytoplasm
- Ribozyme cleavage Hepatitis D - viroid, requires Hepatitis B co-infection for replication - (-) ssRNA genome that is transcribed and replicated by RNA Pol II(!)Latter is tricked by the small Hepatitis Delta Antigen (SHDAg) - employs RNA editing of the antigenome to remove stop codon and to make two products from the single gene - replicates by rolling circle, encodes for a ribozyme that autocatalytically resolves concatemers
- Ribosomal frameshifting - requires "slippery sequence": usually two consequent homopolymeric tripplets XXXYYY that is followed by an RNA secondary structure - frequently found in retrovirus (generation of read-through protein eg HIV gag-pol)
- Dealing with starvation - Amino acid-levels are constantly monitored - replication of viruses such as Yellow Fever lead to a depletion of intracellular amino-acid levels - starvation response is induced that shuts down the translations machinery by phosphorylation of eIF2a - Yellow-fever interferes with GCN2 and prevents translational shut-down
- Vaccination serotypes - Measles: 1 serotype, monovalent vaccine (Edmonston-Zagreb, Schwarz)- Rubella: 1 serotype, monovalent vaccine (Wistar RA 27/3)- Mumps: 1 serotype, monovalent vaccine (RIT 4385)- Varicella: 1 serotype, monovalent vaccine (OKA)- Rabies: 1 serotype, monovalent vaccine - Polio: 3 serotypes (Type 1, 2 and 3), trivalent vaccine (Mahoney, MEF-1, Saukett) - Influenza: 3 circulating serotypes (H1, H3, B), trivalent vaccine- Papilloma: >20 serotypes, di- or tetravalent vaccine
- Flu vaccine production in eggs - ‘High-growth’ Influenza virus injected into fertalized eggs (usually superinfectant of fast replicating virus and strain with desired surface composition) ---> large lot of virus-containing allantoic liquid --> purified viral proteins --> add adjuvant/assemble to VLP --> formulated vaccine