1. Virology 2015
RNA Virus RdRP Enzymes

2. General Comments
An RNA virus does not view replication of the genome and expression of the genome as separate events.
An RNA virus is an RNA replicon in a world full of DNA replicons.
For most RNA viruses, everything takes place in the same compartment.
A biological arms race has shaped the evolution of RNA viruses

3. Issues, problems and strategies related to the production of many copies of progeny viral RNA (replication)
The nature of the template (pp )
secondary structure
nucleocapsids
The nature of the enzymes (pp )
only viruses have RdRP (almost)
The nature of the reactions (pp )
initiation and priming
elongation/unwinding
balancing + and – strand synthesis

4. The Nature of the Template

5. RNA secondary structure is complex
Secondary structures help regulate RNA function Shape is usually the key Replication machinery must deal with the shape

6. Example: CSE4 of alphaviruses
Alphaviruses are members of a genus of the
Togavirus family
SS + RNA, T=4, envelope, wide host range
Replication initiates at 3’ CSE using
non-structural virus proteins and host factors
Shown for salmonid alphavirus 3 (SAV3)

7. Example for nucleocapsids: VSV Vesicular Stomatitis Virus: vesiculovirus genus of Rhabdoviridae (best-studied rhabdovirus)
Rhabdoviridae are – ssRNA with a “bullet” shape enveloped Wide host range among rhabdoviridae

8. Example: VSV Replication requiresor RNP nucleocapsid
“Transcription” of genomic RNA produces + sense messages and viral proteins Production of full-length + or – RNA requires coated template

9. The Nature of the Enzymes

10. RdRp-only viruses have it???
Increasing understanding of function of RNA in an uninfected cell

11. Common domains or “motifs”
RdRp Motifs:
A: nucleotide recognition/binding, phosphoryl transfer: “two-metal” mechanism
B: nucleotide recognition/binding C: active site, phosphoryl transfer: “two-metal” mechanism D: structure and shape of enzyme E: primer binding F: nucleotide entry tunnel (not shown above)
(A, B, C, D in all pols)

12. Common shape Polio 3Dpol (above), HCV (below) Shaped like a right hand
Key features: Palm, Thumb, Fingers
Closed configuration
Generic shape-no two are identical
Shape changes affect biological properties

13. Location of Domains
The conserved structural polymerase motifs. (a–c) The polymerase core of IBDV VP1 (a) compared with the equivalent cores in FMDV (b) and bacteriophage φ6 (c) RDRPs. The secondary structural elements containing the conserved motifs are colored as follows: A, red; B, green; C, yellow; D, purple; E, orange; F, blue. The N- and C-terminal domains are shown as thin ribbons.
FMDV = Foot and mouth disease virus IBDV = infectious bursal disease virus

14. RdRp with template Below: Polio Rdrp with RNA in substrate
channel, top removed
Right (A) WNV Rdrp with motifs and
position of substrate, template and
product
(B) 90 degree rotation of A

15. The Nature of the Reactions: Initiation and Priming

16. de novo Initiation Prime and realign Proposed mechanism for:
arenaviridae, bunyaviridae:
ss ambisense RNA
segmented genome
enveloped
wide host range-rodents
arena (Machupo)
bunya (hantaviruses)
De novo refers to lack of requirement for 3’ OH

17. Primer Dependent Initiation
Protein priming VPg of picornaviruses = best example ss + RNA, AAAAA, icosahedral, many species 22 aa covalently linked Virion RNA Removed upon infection to produce viral mRNA

18. Polio Gene Products 3AB = membrane anchor 3B = VPg
3C or 3CD = protease
3D = polymerase

19. Replication machine anchored to membrane
Addition of U to 3AB
Coupled to cleavage of 3B from 3AB
to produce 3A and 3B=VPg-pUpU
Uridylylated VPg transferred to 3’ end
5’ VPg-pUpU 
3’ ApApApApNNNNN 5’

20. Host proteins
PCBP
And
PABP
Are
Important

21. The Nature of the Reactions: Unwinding

22. Helicase activity Example:
Flaviviruses: Yellow Fever Virus, West Nile Virus,
Hepatitis C Virus
RNA helicase of flavivirus a member of helicase superfamily 2
Helicase a target for therapy?
ATP dependent
Mechanism?
See p 180
Not all helicases function in this way
Phage phi 6 is a dsRNA phage whose polymerase/helicase
works differently

23. The Nature of the Reactions: Balancing + and - Synthesis

24. +/- shift not well-characterized
Diverse mechanisms?
Differential stability of + and -?
Differential synthesis of + and -?
Deterioration of cells/buildup of virus factories
affects stoichiometry?