1. The mechanism of RNA virus replication and regulation 刘广超 程在全 董兆勇 2002.11.11

2. Contents A Classification B Replication C Regulation D Summary

3. A The Baltimore Classification of Viruses A The Baltimore Classification of Viruses I: dsDNA virusesdsDNA viruses II: ssDNA virusesssDNA viruses III: dsRNA virusesdsRNA viruses IV: ssRNA viruses with (+)-sense genomesssRNA viruses with (+)-sense genomes V: ssRNA viruses with (-)sense genomesssRNA viruses with (-)sense genomes VI: "diploid" ssRNA viruses which replicate via greater-than- genome-length dsDNA"diploid" ssRNA viruses which replicate via greater-than- genome-length dsDNA VII: dsDNA viruses which replicate via greater-than-genome- length ssRNA.dsDNA viruses which replicate via greater-than-genome- length ssRNA. According to Their Genome Types and Their Replication Strategies David Baltimore, who originated the scheme, has given his name to the so-called "Baltimore Classification" of virus genomes.

4. III dsRNA Type III viruses include: enveloped phages (Cystoviridae, 囊状病毒 ), the animal- plant- and insect-infecting Reoviridae （ 呼肠孤病毒）,Reoviridae the vertebrate- and invertebrate-infecting Birnaviridae the Totiviridae （轮状病毒）, in primitive Eukarya (fungi and protozoa, and perhaps in insects) Partitiviridae （分病毒）, which mainly infect fungi, and the protozoa （原生动物） Cryptoviruses (family Partitiviridae 分病毒 ), which occur in plants, transmissible only via seed or pollen.

5. Rotavirus 轮状病毒

6. 一、 The viruses have : 1. single-component (Totiviridae 全病毒科 ) 2. two-component (Birna-, Crypto- and Partitiviruses) 3. three-component (Cystoviridae 囊状病毒 ) and multi- component (Reoviridae, 呼肠孤病毒科 10-12 segments) genomes All multiple component genomes are encapsidated in a single particle. 二、 Virus genome sizes : Partitiviridae 分病毒 : 4-9 kb ; Birnaviridae: about 6 kb Totiviridae 轮状病毒 : 4-7 kb ; Hypoviridae 亚病毒 : 10-13 kb Cystoviridae 囊状病毒 : 13 kb Reoviridae 呼肠孤病毒 : 20-27 kb All genomes apparently replicate by a conservative mechanism, wherein ds input RNA is transcribed by viral enzyme to mRNA, which both codes for protein, and acts as template for second strand synthesis

7. Type IV: (+)ve-sense ssRNA 一、 Class IV viruses include: Viruses infecting Eubacteria (Leviviridae) Viruses of insects (Noda-, Tetraviridae) Viruses of fungi (Barnaviridae) Viruses of plants (Bromo-, Como-, Poty-, Sequi- and Tombusviridae, and 19 unassigned genera) Viruses of vertebrates (Astro-, Calici-, Corona 冠状病毒 -, Flavi 黄病毒 -, Picorna 细小病毒 - Noda- 诺达病毒， and Togaviridae 披盖病毒, and genus Arterivirus)AstroCaliciCorona Picorna 二、 The viruses have : single-component genomes with single ORFs (Poty-, Picorna 细小病毒 -, Sequiviridae) single components with multiple ORFs (Toga-, Caliciviridae, Tobamovirus )

8. poliovirus 脊髓灰质炎病毒 350000X

9. two components with single ORFs (Como-, Noda-, Tetri- and Potyviridae genus Bymovirus) Potyviridae two components with multiple ORFs (Tobra-, Furo-, Enamovirus) three components with multiple ORFs (Hordeivirus, Bromoviridae) 三、 Genome sizes range from : Less than 5 kb (Levi-, Barna-, Tombusviridae), To 7-12 kb (Astro-, Como-, Picorna-, Calici-, Bromo-, Poty-, Sequi-, Toga-, Flaviviridae, Tobraviruses) To 13 kb (Arteriviruses) - 15.5 kb (Closteroviruses) To 20 - 30 kb (Coronaviridae) 四、 Genome expression strategies range from : Expressing a single polyprotein from each genome component and proteolytically processing it (Picorna-, Poty-, Sequi-, Como-, Nodaviridae)Picorna- Expressing proteins from 5'-proximal ORFs and from subgenomic messengers (Tobamoviruses, Bromoviridae)TobamovirusesBromoviridae A mixture of the two strategies (Togaviridae, Tymoviruses, Caliciviridae)

10. Type V: (-)ve-sense ssRNA 一、 Type V viruses include : viruses infecting vertebrates only (Arena-, Orthomyxo 棒状病毒 -, Paramyxoviridae 副粘病毒 );Orthomyxo Paramyxoviridae viruses infecting vertebrates and arthropods （节肢动物） (Bunya-, Rhabdoviridae 弹状病毒 );Bunya Rhabdoviridae viruses infecting plants and arthropds (Bunya-, Rhabdoviridae 弹状病毒 ); viruses infecting plants only (Tenuiviruses) 二、 The viruses have: single-component genomes with multiple ORFs (Filo-, Paramyxo-, Rhabdoviridae) two-component ambisense genomes (Arenaviruses 沙粒病毒 )Arenaviruses three-component, occasionally ambisense genomes (Bunyaviridae 布尼亚病毒, Tenuivirus (4))Bunyaviridae six to eight component genomes (Orthomyxoviridae 正粘病毒，流感病毒Orthomyxoviridae

11. Orthomyxoviridae 正粘病毒流感病毒 Orthomyxo Orthomyxo 棒状病毒 -, Paramyxoviridae Paramyxoviridae 副粘病毒

12. The families Paramyxo-, Rhabdo and Filoviridae are grouped together in the oder Mononegavirales, due to similarities in virion structure, gene order and ranscription. similarities 三、 Virus genome sizes: single component, 11-19 kb: Mononegavirales 6 - 8 component, 10-14 kb: Orthomyxoviridae 3 component, 11-20 kb: Bunyaviridae Class V viruses are probably a late evolutionary development, as they occur only among higher Eukarya (arthropods and vertebrates): viruses infecting plants probably do so as a result of close association of insects and host plants in recent evolutionary times.

13. VIa ssRNA retroid viruses Class VI viruses include : Only viruses of vertebrates in the family Retroviridae 逆转录病毒Retroviridae These are (by genus) : mammalian type B retroviruses (eg: mouse mammary tumour virus, MMTV) mammalian type C retroviruses (Moloney murine leukaemia virus, MuLV) avian type C viruses (avian leukaemia virus, ALV) type D viruses (Mason-Pfizer monkey virus, MPMV) BLV-HTLV viruses (human T-lymphotropic virus, HTLV) Lentivirus (HIV 人免疫缺陷病毒, SIV) Spumavirus (human spumavirus 泡沫病毒 )

14. HIV - Ⅰ

15. B The replication of various virial 1. Infection of a cell may be: Productive ; Abortive; Restrictive 2. Virus replication can be divided into (arbitrary) phases: ⑴ Initiation: attachment ; penetration ; uncoating ⑵ Replication: genome synthesis ; RNA production ; protein synthesis ⑶ Release: assembly ; maturation ; exit from cell

16. The replication of retroid viruses

17. (1.1) Initiation attachment ; penetration ; uncoating (1.1) Initiation attachment ; penetration ; uncoating HIV

18. The proteins associated with attachment. HIV

19. (1.2) Initiation attachment ; penetration ; uncoating (1.2) Initiation attachment ; penetration ; uncoating Influenza virus

20. (1.3) Initiation attachment ; penetration ; uncoating unenveloped Poliovirus(+)

21. ⑵ Replication: genome synthesis ; RNA production （ early,late ） ; protein synthesis

22. (2.1) Replication of dsRNA viruses

23. Replication complex of dsRNA virus TMV

24. (2.1)Replication of dsRNA viruses

25. Replication of (+) SSRNA

26. Transcriptonal and replication strategiesof (+)ssvRNA template virus protein (+)SSvRNA/mRNA (-)aRNA subgenomic RNA(mRNA) polyA cap polyA

27. Hypothetical model of the HCV replication cycle.

28. Replication of (+)ssRNA viruses

30. Replication of (-)ssRNA viruses

31. (-)ssRNA virus Replication complex of influenzavirus vRNA

32. Transcriptonal and replication strategiesof influenza virus vRNA template (-) vRNA 3`-UCG(U)UUUCGUCC GGAACAAAGAUA (+)aRNA 5`-ppAGC(A)AAAGCGG CCUUGUUUCUACU (-) vRNA 3`-UCG(U)UUUCGUCC GGAACAAAGAUGA (+)mRNA 5`m7GpppXmY AGC(A) AAAAA(n)3` 17~22nt virus special proteins

33. Replication of (-)ssRNA viruses

34. The replication of retroid viruses

35. 1. Retroviridae replicate via a dsDNA, longer- than-genome-length intermediate (provirus), which is integrated covalently into the host cell chromosomal DNA. 2. Conversion of RNA to DNA, and integration into host DNA, is done by the viral-coded RNA-dependent DNA polymerase, which also has RNAse H activity, and DNA- dependent DNA pol activity, as well as encoding the integrase function. 3. Transcription of viral mRNA - which is spliced to allow expression of 3'-proximal ORFs - is by host RNA pol II

36. HIV-1 replisome and tRNA primers

37. Double-stranded proviral DNA synthesis of HIV RNA

38. double-stranded proviral DNA Integration Of double-stranded proviral DNA into host DNA

39. The replication of (+)ssRNA retroid viruses

40. ⑶ Release of Virion Particles assembly ; maturation ; exit from cell

41. Virusis Assembly and Maturation （ TMV)

42. Poliovirus Assembly and maturation

43. Viral Particle Release ： Budding and cellular lysis Herpesvirus C

44. D Regulation of viral replication 1. Temporal regulation by Selecting temlate 2. The regulation of RNA replicase activity by viral and cellular factors 3. The regulation of viral replication by cis- - active elements and trans-active factors 4. Minimizing accessibility of their replicative intermediates to host defenses

45. 1.Temporal regulation by Selecting temlate virus protein (+)ssvRNA/mRNA (-)aRNA subgenomic RNA(mRNA) polyA cap polyA 1 preference

46. 2. The regulation of RNA replicase activity by viral and cellular cofactors replicase :some enzymic activties(many domains/motifs). : TMV,Sindbis virus RNA dependent RNA polymerase methyltranserase ; guanyltransferase helicase; protase

48. Cellular cofactors :eIF3 (TMV)

49. 3.The regulation of viral replication by cis- active elements and trans-active factors cis-acting elements primarily in 3` and 5` UTR : promoter, enhancer, silencer, et al (+)TMV 5` enhancer Promoter (+)CAAA (-)CAUA tRNA-like And UPD

50. influenza virus (-) The vRNA appears to be completely single-stranded in polymerase-free RNPs ; but a stable double-stranded panhandle structure exists In RNPs containing polymerase Polymerase 3` binding weakly; 5` strongly

51. 4. Minimizing accessibility of their replicative intermediates to host defenses  Efficiently inhibiting RNA Silencing Viral dsRNA forming RAPs or binding with membrane  Inhibiting dsRNA-stimulated pathways, such as the interferon and 2',5'-oligoadenylate/ribonuclease L pathways of animals makeing proteins which sequester the activating RNAs makeing a "suicide“ pseudosubstrate for PKR Expressing a protease which degrades it Inducing synthesis of a cellular repressor making RNAs which bind to PKR but do not activate it and prevent other RNAs from activating it

52. RNA induced Silencing complex RNase L ds/ssRNA dsRNA Degration

53. The genome structure of HIV The regulation of HIV transcription

54. The fuction of TAT A B TAR TAT

55. Expression of the entire complement of human immunodeficiency virus type 1 (HIV-1) viral proteins depends on the competing activities of viral RNA splicing and export into the cytoplasm by Rev. The effects of the SR proteins on Rev function are not mediated through interaction with these elements. The exon splicing enhancer (ESE) and/or silencer (ESS) does have significant effects on Rev function, with deletion of the ESS augmenting the magnitude of the response to Rev and deletion of the ESE significantly reducing it. The loss of Rev response upon deletion of the ESE was due to a failure of Rev to induce transport of the unspliced RNA into the cytoplasm. Cellular splicing factors and viral regulatory elements can have significant stimulatory and inhibitory effects on Rev function, raising the possibility that cells can be rendered permissive or nonpermissive for virus replication by modulation of splicing activities. Positive and Negative Modulation of HIV - Ⅰ Rev Function by cis and trans Regulators of Viral RNA Splicing

56. Unpliced RNA Completely Spliced RNA Unspliced RNA/ imcompletely Spliced RNA Structure Proteins DNA REV SF2/ASF TRa Cellular factors RRE ESS ESE Regulation Protein TAT REV NEF P17 p24 p9 p7 P66 p32 P22 gp120 p41

57. D Summary The diversity and complexity of viral replication and regulation

58. 谢 谢！

59. Effect of SR protein overexpression on Rev function. (A) Structural organization of SR proteins tested. Shaded areas indicate linker regions. (B) Schematic of Rev- dependent CAT reporter constructs. RRE, Rev- responsive element. (C) Effect of SR protein overexpression on Rev function. The level of CAT expression observed in the presence of Rev alone was set at 100%, and all other values are expressed relative to that level of CAT activity.

60. Effects of SR protein expression on HIV-1 env RNA splicing.

61. Effects of SR protein expression on Rev function are cell type dependent. The level of CAT expression observed in the presence of Rev alone was set at 100%, and all other values are expressed relative to that level of CAT activity.

62. Effect of SR protein overexpression on Rev abundance, subcellular distribution, and shuttling. (A) Effect on Rev expression. (B) Effect on Rev subcellular distribution in HeLa cells. (C) Effect on Rev shuttling in HeLa cells. Staining with antibody specific to human hnRNP C (MAb 4F4) identified human nuclei. Also shown is a DAPI-stained image of the heterokaryon to detect the nuclei.

63. Effect of ESE and/or ESS deletions on env RNA splicing.

64. Contribution of ESE and ESS to SR protein modulation of Rev function.

65. Subcellular distribution of viral RNAs in the absence of Rev unspliced env RNA(green), M10 (red) the DAPI staining (blue) of the nuclei

66. Effect of ESE and/or ESS deletions on viral RNA subcellular distribution in the presence of Rev. unspliced env RNA(green), REV (red) the DAPI staining (blue) of the nuclei the DAPI staining (blue) of the nuclei