RNA VIRUS REPLICATION/ TRNSLATION STRATEGIES

1. RNA VIRUS REPLICATION/ TRNSLATION STRATEGIES SadiaAnjum Virology lecture 5

2. RNA -> RNA RNA-dependent RNA polymerase RNA -> DNA RNA-dependent DNA polymerase - reverse transcriptase Host cell DNA -> RNA DNA-dependent RNA polymerase RNA VIRUS STRATEGIES

3. AAA All animal RNA viruses code for a polymerase Need to make mRNA PLUS (POSITIVE) SENSE RNA GENOMES proteins (+ve) sense mRNA

4. AAA Need to make mRNA MINUS (NEGATIVE) SENSE RNA GENOMES proteins (+ve) sense mRNA (-ve) sense genomic RNA

5. AAA Need to make mRNA MINUS (NEGATIVE) SENSE RNA GENOMES RNA polymerase must be packaged in virion. proteins (+ve) sense mRNA If used, RNA modifying enzymes are packaged in virion. (-ve) sense genomic RNA

6. AAA DOUBLE-STRANDED RNA GENOMES RNA polymerase must be packaged in virion. proteins (+ve) sense mRNA If used, RNA modifying enzymes are packaged in virion. Double-stranded genomic RNA

7. RETROVIRUSES Reverse transcriptase must be packaged in virion. DS DNA DS DNA +VE RNA

8. RNA viruses that do NOT have a DNA phase

9. RETROVIRUSES

10. mRNA AAAAAAAA RIBOSOMES PROTEIN AAAAAAAA The monocistronic mRNA problem Eukaryotes Prokaryotes/viruses • Make one monocistronic mRNA per protein • Make a primary transcript and use alternative splicing • Make a large protein and then cut it into smaller proteins • Include special features in the mRNA which enable ribosomes to bind internally

12. PolycistronicvsMonocistronic

13. RNA GENOME SIZE • TAUTOMERIZATION • PROOF READING • GENOME SIZE • RNA VIRUSES HAVE SMALL GENOMES ~10,000 NUCLEOTIDES (herpesvirus DNA ~200,000)

14. GENOME SIZE • POLYMERASE • ATTACHMENT PROTEIN • CAPSID PROTEIN • RELATIVELY FEW OTHER PROTEINS • VIRION SIZE MAY BE QUITE LARGE BUT LIMITED NUMBER OF PROTEINS

15. POSITIVE-STRANDED RNA VIRUSES • EXAMPLES • PICORNAVIRUSES • TOGAVIRUSES • FLAVIVIRUSES

16. PICORNAVIRUS FAMILY (PICORNAVIRIDAE) • SMALL • ICOSAHEDRAL • POSITIVE SENSE RNA • NON-ENVELOPED

17. POLYPROTEIN stop codon for translation INTERNAL RIBOSOME ENTRY SITE (IRES) IRES GENOMIC (+ SENSE) RNA VPg AAAAA start codon for translation .

18. Vpg • VPg (viral protein genome-linked) is a protein that is covalently attached to the 5′ end of positive strand viral RNA and acts as a primer during RNA synthesis in a variety of virus families including Picornaviridae and Caliciviridae. • The primer activity of VPg occurs when the protein becomes uridylylated, providing a free hydroxyl that can be extended by the virally encoded RNA-dependent RNA polymerase. • Aside from functioning as a primer, VPg also has a role in translation initiation by acting like a 5' mRNA cap

19. HOST mRNA HOST mRNA CAP CAP AAAAA AAAAA Inhibition of host cell translation Uninfected cell Factors+40S ribosomal subunit Infected cell Altered factors+40S ribosomal subunit

20. IRES IRES GENOMIC (+ SENSE) RNA GENOMIC (+ SENSE) RNA VPg VPg AAAAA AAAAA Viral translation Uninfected cell Factors+40S ribosomal subunit Infected cell Altered factors+40S ribosomal subunit

21. GENOMIC (+ SENSE) RNA AAAAA translation

23. RNA REPLICATION GENOMIC (+ SENSE) RNA VPg 3’ (- SENSE) RNA VPg 3’ GENOMIC (+ SENSE) RNA VPg 3’

24. RNA REPLICATION • viral RNA polymerase (replicase) • host factors also involved as accessory proteins • new plus strands • packaged • templates for more replication • templates for more translation

25. VPg ASSEMBLY VP0, VP1, VP3 VPg-RNA enters, VP0 is cleaved VP2, VP4, VP1, VP3

26. NON-SEGMENTED NEGATIVE STRAND VIRUSES Examples: Rhabdovirus family (Rhabdoviridae) Paramyxovirus family (Paramyxoviridae) Filovirus family (Filoviridae)

27. polymerase complex RHABDOVIRUSES G glycoprotein SPIKES M protein lipid bilayer membrane helical nucleocapsid (RNA plus N protein)

28. AAA MINUS (NEGATIVE) SENSE RNA GENOME RNA polymerase must be packaged in virion. RNA modifying enzymes are packaged in virion. proteins (+ve) sense mRNA (-ve) sense genomic RNA

29. RHABDOVIRUSES • EXAMPLES • vesicular stomatitis virus (VSV) • rabies virus Dr FS Murphy http://www.vetnet.ucdavis.edu/fam_graphics/download.html

30. H+ ADSORPTION AND PENETRATION

31. CYTOPLASMIC REPLICATION • GENOMIC RNA REMAINS IN NUCLEOCAPSID FORM

32. replication (protein synthesis is a pre-requisite) full-length copy (+ve) 5’ 3’ (-ve) 3’ 5’ RNA SYNTHESIS genome (-ve) 3’ 5’ transcription mRNAs (+ve sense) AAA AAA AAA AAA AAA = cap

33. genome (-ve) 3’ 5’ (+ve) 5’ 3’ AA AA (-ve) AA AA 3’ 5’ AA NEW MINUS STRAND • PACKAGED • TEMPLATE FOR MORE REPLICATION • TEMPLATE FOR MORE TRANSCRIPTION

34. SOME POINTS TO NOTE ABOUT RHABDOVIRUSES • ENTIRE CYCLE OCCURS IN CYTOPLASM • RNA POLYMERASE AND RNA MODIFICATION ENZYMES ARE: • VIRALLY CODED • PRESENT IN THE VIRION • THERE IS NO EARLY/LATE DIVISION

35. PARAMYXOVIRUSES • MEMBERS INCLUDE • PARAINFLUENZA VIRUS • MUMPS VIRUS • MEASLES VIRUS • RESPIRATORY SYNCYTIAL VIRUS

36. PARAMYXOVIRUSES HN/H/G glycoprotein SPIKES pleomorphic F glycoprotein SPIKES helical nucleocapsid (RNA plus NP protein) lipid bilayer membrane polymerase complex M protein

37. PARAMYXOVIRIDAE • PARAMYXOVIRUS SUBFAMILY • PARAMYXOVIRUS • RUBULAVIRUS • MORBILLIVIRUS • PNEUMOVIRUS SUBFAMILY • PNEUMOVIRUS GENUS • METAPNEUMOVIRUS GENUS

38. PARAMYXOVIRUS FAMILYSURFACE GLYCOPROTEINS GENUS GLYCOPROTEINS TYPICAL MEMBERS Paramyxovirus HN, F HPIV1, HPIV3 Rubulavirus HN, F HPIV2, HPIV4, mumps virus Morbillivirus H, F measles virus Pneumovirus G, F respiratory syncytial virus Metapneumovirus G, F metapneumoviruses PARAMYXOVIRUS SUBFAMILY PNEUMOVIRUS SUBFAMILY

39. TRANSCRIPTION, TRANSLATION, REPLICATION genome (-ve) 3’ 5’ replication (protein synthesis is a pre-requisite) transcription mRNAs (+ve sense) full-length copy (+ve) AAA AAA 5’ 3’ AAA AAA AAA (-ve) AAA 3’ 5’ = cap

40. RHABDOVIRUSES PARAMYXOVIRUSES shape bullet bacilliform round pleomorphic glycoproteins one (has both attachment and fusion activities) two: one attachment one fusion fusion pH acidic neutral physiological Some differences between rhabdoviruses and paramyxoviruses

41. ORTHOMYXOVIRUSES HA - hemagglutinin NA - neuraminidase helical nucleocapsid (RNA and NP protein) lipid bilayer membrane polymerase complex M1 protein

42. H+ ADSORPTION AND PENETRATION NUCLEUS

43. viral endonuclease A A influenza virus nucleocapsid U viral polymerase influenza mRNA A U TRANSCRIPTION (mRNA SYNTHESIS) =cap host cell mRNA A AAA

44. RNA SYNTHESIS • Endonuclease is virally coded and packed in the virion • RNA polymerase is virally coded and packed in the virion • Poly(A) polymerase is virally coded and in the virion • Why doesn’t the virus use host cell enzymes?

45. RNA SPLICING Several segments give rise to transcripts which can have one of two fates AAAAAA AAAAAA

46. genomic (-ve) 3’ 5’ genomic (-ve) 3’ 5’ RNA REPLICATION • nuclear • authentic copies • replicase and transcriptase probably same enzyme • coated with protein as made • no clear early/late (+ve) 5’ 3’

47. H+ Flu Virus NUCLEUS Infection of next cell HA cleavage by host cell enzymes - HA conformational change, now active in membrane fusion acid pH inside endosome

49. REOVIRUS FAMILY double stranded RNA genome outer capsid inner capsid

50. AAA DOUBLE-STRANDED RNA GENOMES RNA polymerase must be packaged in virion. proteins (+ve) sense mRNA RNA modifying enzymes are packaged in virion. Double-stranded genomic RNA