The mechanism of RNA virus The mechanism of RNA virus replication and regulation replication and regulation

刘广超 程在全 董兆勇

2002.11.11

ContentsContentsA Classification

B Replication

C Regulation

D Summary

AA The Baltimore Classification of The Baltimore Classification of

VirusesViruses I: dsDNA viruses II: ssDNA viruses III: dsRNA viruses IV: ssRNA viruses with (+)-sense genomes V: ssRNA viruses with (-)sense genomes VI: "diploid" ssRNA viruses which replicate via greater-than-geno

me-length dsDNA VII: dsDNA viruses which replicate via greater-than-genome-lengt

h 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. 

III dsRNA 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） , 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.

Rotavirus 轮状病毒Reo

virus

呼 肠 孤 病 毒

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

Type IV: (+)ve-sense ssRNA 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)

二、 The viruses have: single-component genomes with single ORFs (Poty-, Picorna 细小病毒 -,

Sequiviridae) single components with multiple ORFs (Toga-, Caliciviridae, Tobamovir

us)

poliovirus 脊髓灰质炎病毒

350000X

two components with single ORFs (Como-, Noda-, Tetri- and Potyviridae genus Bymovirus) 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) Expressing proteins from 5'-proximal ORFs and from subgenomic messengers (Tobamoviruses, Bromoviridae) A mixture of the two strategies (Togaviridae, Tymoviruses, Caliciviridae)

Type V: (-)ve-sense ssRNA Type V: (-)ve-sense ssRNA

一、 Type V viruses include: viruses infecting vertebrates only (Arena-, Orthomyxo 棒状病毒 -, Paramyxo

viridae 副粘病毒 ); viruses infecting vertebrates and arthropods （节肢动物） (Bunya-, Rhabdo

viridae 弹状病毒 ); 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-, Rhabdovi

ridae) two-component ambisense genomes (Arenaviruses 沙粒病毒 ) three-component, occasionally ambisense genomes (Bunyaviridae 布尼亚病

毒 , Tenuivirus (4)) six to eight component genomes (Orthomyxoviridae 正粘病毒，流感病毒

Orthomyxoviridae 正粘病毒流感病毒Orthomyxo 棒状病毒 -,

Paramyxoviridae 副粘病毒

The families Paramyxo-, Rhabdo and Filoviridae are grouped together in the oder Mononegavirales, due to similarities in virion structure, gene order and ranscription.

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.  

VIa ssRNA retroid virusesVIa ssRNA retroid viruses

Class VI viruses include: Only viruses of vertebrates in the family Retroviridae 逆转录病毒

These are (by genus): mammalian type B retroviruses (eg: mouse mammary tumour vir

us, 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 )

HIV -ⅠHIV -Ⅰ

B B The replication of various virialThe replication of various virial

1. Infection of a cell may be:

Productive ; Abortive; Restrictive

2. Virus replication can be divided into (arbitrary) phases:

⑴Initiation: attachment ; penetration ; uncoating

⑵Replication:genome synthesis ; RNA production ; protein synthesis

⑶Release:assembly ; maturation ; exit from cell

The replication of The replication of retroidretroid viruse virusess

(1.1) (1.1) InitiationInitiationattachment ; penetration ; uncoatingattachment ; penetration ; uncoating

HIV

The proteins associated with The proteins associated with attachmentattachment

.

HIV

(1.2) Initiation(1.2) Initiationattachment ; penetration ; uncoatingattachment ; penetration ; uncoating

Influenza virus

(1.3) Initiation(1.3) Initiationattachment ; penetration ; uncoatingattachment ; penetration ; uncoating

unenvelopedPoliovirus(+)

⑵Replication:

genome synthesis ; RNA production （ early,late ） ; protein synthesis

(2.1) Replication of dsRNA viruses(2.1) Replication of dsRNA viruses

Replication complex of dsRNA virus

TMV

(2.1)Replication of dsRNA viruses (2.1)Replication of dsRNA viruses

Replication of (+) SSRNAReplication of (+) SSRNA

Transcriptonal and replication strTranscriptonal and replication strategiesof (+)ssvRNA templateategiesof (+)ssvRNA template

virus protein

(+)SSvRNA/mRNA

(-)aRNA

subgenomic RNA(mRNA)

polyAcap

cap polyA

Hypothetical model of the HCV replication cycle.

Replication of (+)ssRNA virusesReplication of (+)ssRNA viruses

Replication of (+)ssRNA virusesReplication of (+)ssRNA viruses

Replication of (-)ssRNA virusesReplication of (-)ssRNA viruses

(-)ssRNAvirus

Replication complex of Replication complex of influenzavirus vRNA influenzavirus vRNA

Transcriptonal and replication strTranscriptonal and replication strategiesof influenza virus vRNA teategiesof influenza virus vRNA te

mplatemplate (-) vRNA 3`-UCG(U)UUUCGUCC GGAACAAAGAUA

(+)aRNA5`-ppAGC(A)AAAGCGG CCUUGUUUCUACU (-) vRNA3`-UCG(U)UUUCGUCC GGAACAAAGAUGA

(+)mRNA 5`m7GpppXmY AGC(A) AAAAA(n)3` 17~22nt

virus special proteins

Replication of (-)ssRNA virusesReplication of (-)ssRNA viruses

The replication of retroid virThe replication of retroid virusesuses

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

HIV-1 replisome and tRNA primers

Double-stranded proviral DNADouble-stranded proviral DNAsynthesis of HIV RNAsynthesis of HIV RNA

Integration Of double-stranded prodouble-stranded proviral DNAviral DNA into host DNA

The replication of (+)ssRNA retroid virusesThe replication of (+)ssRNA retroid viruses

⑶ Release of 　 Virion Particles

assembly ; maturation ; exit from cell

Virusis Assembly and Maturation（ TMV)

Poliovirus Assembly and maturation

Viral Particle Release ：Budding and cellular lysis

Herpesvirus C

D Regulation of viral replicationD 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

1.Temporal regulation by Sel1.Temporal regulation by Selecting temlateecting temlate

virus protein

(+)ssvRNA/mRNA

(-)aRNA

subgenomic RNA(mRNA)

polyAcap

cap polyA

1

preference preference

2. The regulation of RNA replicase activityby viral and cellular cofactors

replicase :some enzymic activties(many domains/motifs). : TMV ,Sindbis virus

RNA dependent RNA polymerase

methyltranserase ; guanyltransferase

helicase; protase

Cellular cofactors :eIF3 (TMV)

3.The regulation of viral replication by cis-a3.The regulation of viral replication by cis-active elements and trans-active factorsctive 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

influenza virusinfluenza 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

Polymerase3` binding weakly; 5` strongly

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

RNA induced Silencing complex

RNase L

ds/ssRNA

dsRNA

Degration

The genome structure of HIVThe genome structure of HIV

The regulation of HIV transcription

The fuction of TAT

A

B

TAR

TAT

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 -ⅠPositive and Negative Modulation of HIV -ⅠRev FunctionRev Function by by ciscis and and transtrans Regulators Regulators

of Viral of Viral RNARNA Splicing Splicing

Unpliced RNA

Completely

Spliced RNA

Unspliced RNA/imcompletely

Spliced RNA

Structure Proteins

DNA

REV

SF2/ASF

TRaCellularfactors

RRE

ESS

ESE

Regulation Protein

TAT REV NEF

P17 p24 p9 p7P66 p32

P22gp120 p41

D SummaryD Summary

The diversity and complexity of viral replication and regulation

谢 谢！谢 谢！

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 over

expression 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.

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

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.

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 nucl

ei.

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

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

Subcellular distribution of viSubcellular distribution of viral ral RNARNAs in the absence of s in the absence of RevRevunspliced unspliced envenv RNA(green), RNA(green), M10 (red) M10 (red)

the DAPI staining (blue) of tthe DAPI staining (blue) of the nucleihe nuclei

Effect of ESE and/or ESS deletions on viral RNA subcellular distribution in the presence of Rev.

unspliced unspliced envenv RNA(green), R RNA(green), REV (red)EV (red)

the DAPI staining (blue) of ththe DAPI staining (blue) of the nucleie nuclei