Replication of (-)-strand viruses Flashcards
Characteristics of Rhabdovirus
- Unsegmemted -strand RNA serves as template for mRNA synthesis, is always in complex with proteins ans is replicated in cytoplasm
- viral RdRp (L-Protein) transcribes antigenome/genome, transcribes 5 monocistronic mRNAs, capping & polyadenilation (is always packed in the particle)
- virus is bullet-shaped, has lipid bilayer, glycoproteins (G), matrix protein (M) and nucleocapsid protein (N). P and L proteins are part of the RdRp
Genome organisation and mRNA-synthesis of VSV(vesicular stomatitis virus-Rhabdovirus) →Attenuation model
- start of mRNA synthesis always at 3‘ end of N gene (not at 3‘ end of genome)
- inefficient reinitiation after termination of transcription
- Attenuation: decreasing amounts of transcript from 3‘ to 5‘ (N>P>M>G>L) leading to severely reduced amounts of L (RdRp)
- PolyA via stuttering of viral polymerase at the 7U sequence in the intervening region → ~200 A‘s
VSV: Switching between mRNA and genome synthesis
- start of genome replication exactly at 3‘ end of (-)-strand genome
- switching between mRNA synthesis (transcription; early) and replication (late) via changes in composition of the replicate complex: L+P = transcription, L+N+P = replication
- Complex of N and P attaches during its synthesis to the +strand and inhibit poly adenylation and termination at IGs
- intercellular accumulation of amount of N decisive for switch to replication
Explain the antigenic switch happening in influenza virus and it’s benefit
The viral surface proteins (HA/NA) are main antigens for immunsystem
The exchange of surfaceproteins by reassortment Leads to massive change in antigenicity of the virus and therefore no acquired protective immunity via previous infections or vaccinations
Explain the genetic reassortment of influenza viruses
Genetic reassortment is the mixing of genome segments of influenza viruses of fowl, swine and humans. Pigs are serving as „mixing vessel“
Orthomyxoviridae: Structure and genome organisation of influenza A Virus
- Segmented genome: 8 segments, each coding 1-2 proteins, each associated with a polymerase and NP, splicing,RNA replication in the nucleus, reassortment
- Viral RdRp transcribes anti.genome/genome and mRNAs, cap snatching:5‘ ends of cellular pre-mRNA in nucleus is cleaved off and serves as primer for viral mRNA synthesis, three subunits PB1, PB2 and PA
Ribonucleoprotein complex (RNP) of Influenza
248 bases mini RNA-Segment in complex with 9 NP and Pol-complex
- genomic RNA never naked, always associated to nucleocapsid protein (NP)
- polymerase complex (PB1, PB2 and PA) mediates circularisation of genome segments
Procedure of cap snatching in mRNA synthesis of influenza A virus
Hydrolysis of random, capped nuclear mRNA via nuclease activity in the viral polymerase complex (PA)
Viral pol initiates +strand synthesis by integration of a GTP compl. To the second last nucleotide (C) of the (-)Strand: cap-primer dependent initiation of transcription
Longatiom by viral polymerase
What are the parts of the RNA polymerase complex of influenza A virus and what are their functions.
PB1, PB2 and PA
PB1 has binding site for 5‘ and 3‘ ends of (-) RNA and is activated upon its binding furthermore it catalysers the synthesis of cRNA
PB2has a cap binding domain, capped host mRNAs bind and are cleaved off
PA catalysed mRNA cleavage and binds to the NP attached to RNA
What are the activation steps of the influenza a polymerase complex for transcription?
The inactive polymerase complex binds the specific 5‘ end sequence and induces the cap binding activity of PB2 and the 3‘ end binding activity of PB1.
The specific binding of 3‘ end of (-)strand induces the endonuclease activity of PA the initiation of the transcription and the elongation of the mRNA.
What is the current model of influenza virus transcription?
Primary transcription. Following nuclear entry, viral ribonucleoproteins (vRNPs) that were associated with incoming viruses transcribe viral mRNAs in cis using the resident polymerase complex bound to the double-stranded genomic ends and cellular pre-mRNA caps obtained by cap- snatching from cellular RNA polymerase II (Pol II) (known as initiation). The vRNA is then threaded through (and copied by) the viral polymerase complex (known as elongation). When the 5′ end of the vRNA is reached, it is held by the polymerase to promote the generation of the poly-A tail in conjunction with the cellular SFPQ protein (known as poly-A addition).
What is the current model of influenza virus replication?
Genome replication. After new viral proteins are translated by the cellular machinery, soluble polymerases mediate genome replication in trans,
which is promoted by the activity of the cellular minichromosome maintenance
(MCM) complex and the Tat-SF1 and UAP56 cellular proteins. The FMR1 protein stimulates the assembly of polymerase complexes and
nucleoprotein (NP) in the presence of vRNA. The specific contributions of CLE and importin-α (IMPα) are currently unknown.
How does the packaging of the new synthesised RNA segments into new visions work (influenza virus).
Segment-specific packaging signals at the 5 ́and 3 ́end (NTRs) of the segments as well as ca. 80 bases of the coding sequence in the 3 ́region
- RNA-structure for specific packaging?
- Interaction of different segments needed for packaging?
- ordered structure of segments in structure:-8helical rods with different length -anchored at the top of the virion -placement of segments not conserved
Describe the replicative cycle of influenza A virus
- virus binds to sialic acid resedues via HA
- Endocytosis and pH induced fusion via HA
- Nuclear Transport of nucleocapsid via NLS in NP and PB2
- synthesis of PA,PB1 and PB2 in cytoplasm and nuclear Import
- synthesis of 8 mRNAs in the nucleus, cap snatching
- mRNA splicing for NS2/M2
- export and translation of viral mRNAs
- processing and localisation of HA and NA (through ER and golgi -> vesicle with embedded proteins to cell membrane) leads to budding of new virion
What are the two main reasons for genetic diversity of negative strand RNA viruses?
→Missing accuracy of RNA-polymerase
- RdRp without proofreading activity leads to one mistake in 10^3 –10^4 bp therefore 1 to 10 mistakes per genome
- formation of a quasispecies
- influenza: Antigenic drift
→Segmentation allows reassortment
- co-infection with two related segmented viruses allows reassortment
- influenza: Antigenic shift
Name the Main Features, viruses and caused diseases of the Bunyaviridae
Features: genome is three segments: M, L and S, proliferates in insects and mammals
Viruses: Hantaan virus, Rift Vally fever virus
Ausbrüche and disease: hantavirus-infections in Yosemite national Park 2012 Überträgen:Hirschmaus, puumala virus-infections 2007 - 2019 in south/west Germany zoonotic host: Rötelmaus, Hemorrhagic fever with renal failure syndrome (HFRS)
Cap-snatching mechanism of Hantaviruses
- viral N protein binds to cap of cellular mRNA
- after transport of mRNAs to P-bodies it is degraded
- during mRNA degradation the 5‘ end region and cap is protected by viral N protein
- P-bodies serve as stock for N-cap complexes
- protein in Hantavirus preparation shortens 5‘cap-RNA to ca 17 bases
- N-bound 5‘-RNA fragment with cap serves as primer for viral mRNA
- N mediates initiation of translation during transcription
Compare the genomes of negative and positive RNA viruses
Negative strand RNA viruses:
- genome packaged in viral proteins ribonucleoprotein (RNP)-complexes with RdRp packed in particle, nucleocapsid RNA
- stabilised against RNase
- only recognised and amplified from pol. As RNP-complexes; naked RNA non-infectious
- serves as matrix for positive strand RNA synthesis
- Nucleoproteins are cooperative ss-RNA binding proteins and are required in RNA synthesis to maintain the sis form (no stable das made of template and newlysynthesised strand)
Positive strand RNA viruses:
- genome not packaged in RNP; no RdRp in particle (exeption retrovirus)
- instabile against RNase
- naked RNA is infectious
- serves as matrix for protein synthesis and (-) strand RNA synthesis
- codes in most cases for a helicase which is essential for replication and strand separation
- RNA replication in cytoplasm
- dsRNA can be detected in infected cells
