Lecture 6 - Features of viral genomes Flashcards
What are the features of viral genomes?
- can be DNA or RNA
- can be single or double stranded
- genome smaller than host cell
- range between 5000 and 23, 000 bp
Why are virus genomes so small?
Under a size pressure
- Genomes of viruses that infect prokaryotic cells are compact (have overlapping genes) in achieve maximum genetic capacity - they need to replicate fast to keep up with their hosts
- Genomes of viruses that infect eukaryotic cells are also under pressure, they have to fit into the virion - limit is packagin size
What is the difference in size in RNA and DNA genomes?
RNA viral genomes are typically smaller than DNA viruses because:
- RNA is fragile, so long strands can easily break. The biochemical structure means that it is more sensititvve to hydrolysis and therefore unstable.
- RNA genomes have in general higher mutation rates than DNA genomes
Are viral genome mutations benefitical or detremental?
Mutation is a mixed blessing
- beneficial because mutations may allow an escape from immune system and increased fitness
- but deleterious mutations can limit viral replication and spread
Have to be combined with other features to be a positive feature
What are the typical rates of viral genome mutation?
Range from: 1nt/1000-10,000nt in retroviruses; to 1nt/10,000,000,000nt in herpes virus (similar to eukaryotic DNA replication)
Why do RNA genomes mutate more frequently than DNA genomes?
RNA-dependent RNA polymerases have higher error rates than DNA-dependent DNA polymerases (these can sometimes be from the host)
- How many mutations per genome are there if a HIV genome is 9.7kb and the mutation rate is 1nt/1000-10000nt?
- How can the possibility of deterimental mutations be overcome?
- Everytime have a replication cycle, get between 1-10 mutations.
Very quickly a lot of mutations build up (as many viruses per cell), therefore within an infected person, there are lots of different vairants of HIV (Quasispecies)
- Has to be able to replicate quickly, if is slow, a bad round of replication may wipe out a lot of viruses. Combination of quick repication and high mutation rate = success
What are the different types of viral gene products?
- Structural proteins (formation of the capsid and envelope)
- Enzymes (RNA/DNA polymerases, proteases, integrases)
- Regulators of viral gene expression
- Regulators of host gene expression
Some are more sensitive to mutation than others e.g. mutation in the catalyctic site of an enzyme = bad
When do genetic interactions between viruses occur?
During superinfection (infection of one cell with more than one mutant)
Define viral complementation
Interaction of viral gene products of two viruses that result in increased production of one or both of these viruses (e.g. one virus provides a gene that is defective in the other virus)
Both viruses remain unchanged genetically.
Define viral recombination during superinfection
Physical interaction between enomes resulting in genes that were not present in either parent virus. Joining of parts os the genome from different viruses.
Intramolecular (homologous or non-homologous) recombination or reassortment (viruses with segmented genomes)
Why do herpes viruses exists of a mixture of 4 isotypes?
Herpes virus genomes have a unique long (UL) and unique short sequence (US) region bounded by inverted repeats. These repeats allow rearrangments of the unique regions and Herpesviruses therefore exist as a mix of four isomers (HSV: 150kbp; V2V: 120-130kbp; CMV: 220kbp; EBV: 170 kbp)
What are the features of herpes virus genomes?
- Genomes are tethered (not integrated) to the host chromatin, often in circular episomes
- Genomic clustering of genes expressed in different lifecycle stages (Latent vs. Lytic)
- Major latency genes mediate viral genome tethering (only require genes required during latency)
- Lytic transactivator genes initiate the cycle leading to virion production
- Latency genes often inhibit lytic transactivation
- Frequently encode for cellular homolog genes
How is most dsDNA viral genomes found in the cell? (structure)
- Mostly found circularised. Have clustered genes, genes expressed in one section are all the genes expressed during a particular part of the virus life cycle.
- contain cellular genes stolen from the host cell (cellular homologues): vFLIP, vCYCLIN, vOX2, vGPCR, VIL6, vMIR1/2, vCCL2/3, vIAP, vBCL2
- KSHV episome contains laten cluster (polycistronic) and a lytic activator (ORF50)
What is the lytic transctivator as part of the dsDNA viral genome?
RTA or ORF50
inhibited by LANA(major multifunctional latency protein that tethers the episome)
Why do dsDNA viruses have cellular homologues? (KSHV episome)
Have low mutation rates so must find other ways to interact with the host cell
What are the features of ssDNA genomes?
- Commonly (but not always) circular
- Genome: 2 - 30kb, encoding for 4-15 ORFs (with some exceptions)
e.g. M13 filamentous bacteriophage
Describe the genome organisation of M13 filamentous bacteriophage as an example of a ssDNA virus
Host RNA and DNA polymerases convert the +ve ssDNA viral genome into a covelently closed dsDNA called the replicative form of DNA (RF) [intermediate essential for replication and transcription)
Viral g2p protein nicks RF DNA strand at the origin of replication. +ve strand replication occurs. New +ve ssDNA genomes are converted into new RF molecules and further transcription occurs.
When enough g5p protein is synthesised, conversion into RF dsDNA is inhibited as newly synthesised genomic ssDNA is covered with g5p. g5p is replaced by g8p to trigger the assembly into the viral capsid.
What is the structure of the M13 filamentous bacteriophage genome?
- 6.4kb circular genome
- requires dsDNA intermediate for replication
- has infrequently transcribed region and frequently transcribed regions, these are clusters of genes.
- highly structured viral genome
What are the efatures of dsRNA genomes?
- commonly but not always fragmented
- genome 20-30kb, encoding for approximately 10 proteins
Describe the genome of Rotavirus as an example of dsRNA viruses
Rotavirus (the most common cause of gastroenteritus in children)
- 8 viral species (A - H), A-V are human infective
- classified based on VP7 (G serotypes) and VP4 (P serotype)
- 11dsRNA fragment encoding for 12 proteins
- Viral mRNAs are 5’ capped but lack a poly-A tail - instead have 3’ end conscenus sequence (UGACC) which is conserved in all 11 viral genomes (helps in the identification of what genetic information is viral)
- fragmented genetic structure allows reassortment
what are the features of viral transcription of the rotavirus genome?
- 11 segments
- easly transcription by the viral polymerase is initatied in partially uncoated virions and within the host lysosome (double layered particles)
- dsRNA is not exposed in the cytoplasm
- each gene is (almost) part of a separate segment - genomic clustering: VP4 is broken up into VP8* and VP5* on segment 4; segment 9 contains VP7 (1), VP7(2); segment 11 contains NSP5 and NSP6 - these last two are acheived by leaky scanning
Give examples of +ve ssRNA viruses and features of their genomes
- Picornaviruses
- Togaviruses
- Flaviviruses
- Coronoviruses
Essentially mRNA (no intermediate step). All have a polyA tail, sometimes with a cap structure/protein. As soon as it enters the cytoplasm, can make viral proteins
What are the differences between the viral genome organisation of Hep C (+ve ssRNA virus) and Hep B (Class VII)?
Hep C
- protected by a 5’ UTR
- as soon as enters the cytoplasm start to transcibe the polypeptide, protease used to cleave into proteins
- get some genome clustering of NS (non structural genes)
- Make NS2 = protease then other proteins are made
Hep B
- Not genomically similar to Hep C - Hep B: circular, Hep C linear
- bidirectional transription
- unclear stucture
- retrovirus
- goes through mRNA intermediate
What are the features of -ve ssRNA viruses?
- usually larger than +ve sense, more complex
- commonly segmented
- reverse of mRNA
- sometimes ambisense (i.e. one or more segments are +ve sense)
- NS proteins are clustered
- Main regions of a -ve ssRNA virus: 3’ - I, N, NS (M2), NS (M2), G, L, 5’ UTR - 5’
What is the viral genome organisation of Ebola?
- -ve ssRNA virus (class )
- 5/6 gene products
- Virus is dangerous because of its tropism: infects different types of cells leading to organ failure, bleeding
Put Ebola in context of other worldwide killers - why was it so scary?
Current Ebola outbreak: 7000 infections, 4500 deaths
Hep: about 1 in 3 people infected, 1000000 deaths per year
AIDS: approx. 35 million living with aids, 1.6 million deaths per year
Flu: 3-5 million cases per year, 250-500 000 deaths/year
Measles: 120 000 deaths in 2012
Suicide: 800 000 deaths per year
Ebola: alarmed by the high rate of spread, normally infects and takes down a village and then stops.
What are the features of Paramyxoviruses (influenza)?
- -ve ssRNA viruses (class V)
- 8 segments, used as a form of extreme clustering, NS regions in the same segment
- several types of genetic information, very common in flu, especially as you can have hot infeted with different types of flu e.g. human virus in pigs, avian flu in humans. Have the possibility to be in the same host/cell and physically exchange genetic information
Segments:
- Segment 1: RNA polymerase subunit (PB1) and the PB1-F2 protein
- Segment 2: RNA polymerase subunit (PB2)
- Segment 3: RNA polymerase subunit (PA)
- Segment 4: Hemagglutinin (HA)
- Segment 5: Nucleoprotein (NP)
- Segment 6: Neuraminidase (NA)
- Segment 7: Matrix proteins (M1 and M2)
- Segment 8: Nonstructural proteins (NS1 and NS2)
What is the importance of genetic reassortment in flu?
Allows the infection of multiple hosts
e.g. alpha 2-6 for human infection, alpha 2-3 and alpha 2-6 for infection in pigs, alpha 2-3 for avian infection
How is retroviral genome organisation unquie to that of organisms?
- truly diploid (have 2 copies of RNA in the virion)
- genome is produced by the cellular transcriptional machinery (no viral polymerase)
- genome requires a specific cellular RNA (tRNA(lys)) for replication (the initiation of reverse transcription)
- although they are positive strand, their genome is not used as an mRNA immediately after infection
- e.g. HIV structure: 3 regions (gag, pol, env), have enzymes clustered together
What are the significant differences between Class VI and Class VII viruses?
Both classes use reverse transcription (transformation of RNA to DNA) as part of the replication cycle: this is an early event for class VI viruses (HIV), before integration. In class VII viruses, this is a late event, during virion formation.
e.g. HBVis transmitted similarly to HIV but up to 50 more infectious
How is the pathogenic outcome of an infection determined?
Determined by the interaction between the pro-inflammatory and anti-inflammatory mechanisms of the immune systems.
If the immune response is over the top, person will get ill e.g. influenza virus colonises deep areas in the lungs, this initiates inflammation response (cytokine storm) causes problems.
What are the specific parts of the retroviral genome structure?
gag: capsid and packaging
pol: protease, reverse transcriptase, integrase - essential for replication
env: envelope
What genes does HIV have and what are their functions?
Gag: p24 (capsid protein), p17 (matrix protein), p7 (capsid protein), p6 (capsid protein)
Pol: Reverse transciptase (transcribes viral RNA into dsDNA), Integrase (integrates viral dsDNA into host genome), Protease (cleaves gag and pol precursors)
Vif: Vir protein (Degrades host cell defense protein APOBEC3G)
Vpr: Vpr protein (Regulates nuclear import of pre-integration complex)
Tat: Tat protein (promotes transcription of viral DNA)
Rev: Rev protein (Allows export of unspliced viral RNA from nucleus)
Vpu: Vpu protein (Intracellular degradtion of CD4)
Env: gp120 (binds CD4, CCR5, CXCR4), gp41 (promotes fusion of virion with plasma membrane)
Nef: Nef (downregulates cluster of differentiation 4, MHC-1 and other rececptors)
What are LTRs?
Long terminal repeats
Important feature of the retoviral genome
Identical sequences that are repeated hundreds of times at the end of the genome
What is the funtion of LTRs?
- Facilitate the viral integration of the genome
- serve as the first promoter of the viral genome (5’ LTR)
- mediate polyadenylation (3’ LTR)
- Encode Nef (3’ LTR)
What is the structure of LTRs?
consist of three regions U3, R, U5
U5 region contains essential RNA motifs including the transactivation response element (TAR) which is essential for HIV transcription, and other elements controlling revenerse transcription, polyadenylation, and genome packaging
What is the Tat gene in HIV (retroviruses)?
Tat: transactivator of transcription
Enhances HIV transcription by more than 100 fold through binding the TAR element and enhancing transcriptional elongation
What is the Nef gene in HIV (retroviruses)?
Nef: negative regulatory factor
Inhibits viral transcription through binding the LTR
Directly induces a host transcriptional programme that prepares the cell for HIV transcription enhanced by TAT.
Also a major immunomodulatory factor.
Why does HIV have a positive and negative regulator? (TAT and Nef)
More stable
Timing is crucial: If transcription happens and the host is unprepared, host may go into senescence or find a way to escape.
What factors of viral infection favour tissue damage?
- An overwhelming immune response
- Age of the host - old people have a very different immune system; babies get protection from their mother, immune sustem not fully developed; some spidemics of avian flu those most at risk were in 25-40 age group to do with an overwhelming immune response
- Dose of infection
- Route of infection
- heterlogous immunitry (co-infections, infection history) - people are never exposed to just one infection, past infections can determine outcome of future infections e.g. HIV
- host genetics - everybody is slightly different, polymorphism in one gene could cause a completely different outcome of infection e.g. RSV normally contract when young, most children are fine but a small group can get it very badly, have a polymorphism on gene TRL4
What is the multiplicity of infection?
MOI
The number of infectious virions used per target cell in an experimental setting
You prepared a virus stock that you have quantified at 1000000 virions/ml. You want to infect 10000 cells at an MOI of 5. What volume of your stock will you use?
1000000 : 1ml
50 000 : 5 ml
5 * 10 000 * 1 000 000 = 0.05ml
What is the most accurate method od determining viral copy number before performing the experiment to make sure you are using the correct volume?
look at the plaque forming units
