Replication and RNA synthesis of (-)-strand RNA viruses Flashcards

Question

Activation of Influenza virus polymerase complexes for transcription

Answer 1

1) Inactive polymerase complex 2) Binding of specific 5 ́sequence induces activation of: - Cap-binding activity of PB2 - 3 ́(-)-strand binding activity of PB1 -> Temporal coordination! 3) Selective binding of specific 3 ́-(-)-strand sequence induces activation of: - Endonuclease activity of PA - Initiation of transcription - Elongation of mRNA 4) Elongation of mRNA to the 7 U sequence in the template 5) - RNA signal (7xU) blocks elongation - 7 U transcribed multiple times = polyA

Answer 2

vRNAs: (-)-strand segments in virion cRNAs: (+)-strand copies; replication intermediates - RNA promotor: „corkscrew structure“ of communicating ends RNA promotor: Bases 1-9 at 5 ́ and 3 ́ ends as well as 11-16 at 5 ́ and 10-15 at 3 ́end -> differently efficient RNA promotors in vRNAs and cRNAs Promotor on vRNA weak (for synthesis of cRNA) Promotor on cRNA strong (for synthesis of vRNA) Consequence: ratio of 10:1 for vRNA:cRNA By increasing the efficiency of the cRNA-promotors, equal amounts of RNA can be generated in the cell Base paired with the other end of the same genome segment!

Answer 3

Each influenza viral ribonucleoprotein (vRNP) consists of one single-stranded, negative-sense genomic RNA associated with multiple nucleoprotein (NP) monomers and a single trimeric polymerase complex (composed of PB1, PB2 and PA). The 5′ and 3′ vRNA ends are complementary and base pair to form a double-stranded structure, which is bound by the polymerase complex at one end of the vRNP filament. The internal vRNA region is organized into an antiparallel double helix, the formation of which is driven by contacts between NP monomers (known as the 'minor' groove), and a loop can be observed at the end of the filament opposite to that bound by the polymerase complex.

Answer 4

- pol complex already present in vRNP makes transcription only in cis - pol remains stably bound to 5 ́end of vRNA - proceeds after cap snatching with elongation of mRNA - when it comes close to the end of the vRNA a steric hindrance supports stuttering of pol at 7U sequence to produce poly A - after termination reinitiation takes place at 3 ́end of vRNA template (permanent contact of pol also with 3 ́end of vRNA?) - mRNA splicing and export into cytoplasm - translation and import of pol subunits into nucleus - cRNA synthesis at incoming vRNPs can occur by already associated pol (in cis) - vRNA synthesis is started by newly synthesised pol complex (in trans) at 3 ́end of cRNA; 5 ́end of the newly synthesised vRNA also recruites pol; this pol complex recruits NP molecules via protein/protein interaction - the new protein coat of the vRNA including pol complex at 5 ́end is essential for packaging - RNA synthesis leads to replacement of pol at 5 ́end of template

Answer 5

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).

Answer 6

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.

Answer 7

- 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 the genome segments in the virion! - 8 helical rods in the virion - differ in length Multiple interactions are found among the RNPs. The position of the eight RNPs is not consistent among virions, but a pattern suggests the existence of a specific mechanism for assembly of these RNPs.

Answer 8

- Foreign genes can be linked up with those signals and so be co-packaged reliably into virions Experiment: - Instead of hemagglutinin (HA) and neuraminidase (NA) GFP and VSV-G coded on the „HA“ and „NA“ segments That means: - Virion contains still 8 genome segments - Is infectious (HA and NA functional replaced by VSV-G) - Has a segment with terminal sequences of the NA segment for variable foreign gene expression (e.g. GFP)

Answer 9

All modules found in the heterotrimeric RdRp of Influenza virus can also be found in the monomeric bunyavirus polymerase Conclusion: Higher similarity than expected! Common drugs for all these RdRps?

Answer 10

Missing accuracy of RNA-polymerases - RdRp without proof-reading activity (missing 3 ́->5 ́exonuclease) - 1 mistake per 103-104 bases; accordingly 1-10 mistakes in each new genome - Formation of quasispecies - Influenza: „Antigenic drift“ Segmentation allows reassortment - Co-infection with two related segmented viruses allows for reassortment - Influenza: „Antigenic shift“

Answer 11

- Proliferation in insects and mammals (change of host!) - "Hemorrhagic fever with renal syndrome" (HFRS) Family: Bunyaviridae Genus: Hantavirus Nairovirus Orthobunyavirus Phlebovirus Tospovirus Type: Bunyamwera virus (BUNV) Hantaan virus (HTNV) Dugbe virus (DUGV) Rift valley fever virus (RVFV) Tomato spotted wilt virus (TSWV) Main: Crimean-Congo hemorrhagic fever virus (CCHFV)

Answer 12

Disease: Hemorrhagic fever with renal failure syndrome (HFRS) Puumala-Virus: 0.1 - 0.9% mortality South and western Germany: Puumala-Virus East and northern parts: Dobrava-Virus Zoonotic host: Rötelmaus

Answer 13

- Viral N-protein binds to cap of cellular mRNAs - After transport of mRNA into P-bodies mRNA is degraded - During mRNA degradation the 5 ́region and cap is protected by viral N protein - P-bodies serve as stock for N-Cap complexes - Protein in Hantavirus preparation (viral polymerase?) shortens 5 ́cap-RNA to ca. 17 bases - N-bound 5 ́fragment with Cap serves as primer for viral mRNA - N mediates already during transcription translation initiation - Role of N during packaging of 3-segment genome: Packaging in p-bodies?

Answer 14

(-)-strand RNA - Genome packaged in viral proteins „ribonucleoprotein(RNP)-complex“ with RdRp in the particle; „nucleocapsid RNA“ - stabilized against RNases - only recognized and amplified from Pol as RNP-complex „naked“ RNA non-infectious - serves as matrix for (+)-strand-RNA synthesis - nucleoproteins are cooperative ss-RNA binding proteins and are required in RNA synthesis to maintain the ss form (no stable doublestrand made of template and newly synthesised strand) (+)-strand RNA - Genome (except Retroviruses) not packaged in RNP; no RdRp within the particle - instable against RNases - „naked“ RNA is „infectious“ (formation of new virions after RNA transfection) - serves as matrix for protein synthesis and (-)-strand RNA synthesis - codes in most cases for a helicase (essential for replication, strand separation?) - RNA replication in the cytoplasm - dsRNA can be detected in infected cells