Sadia Anjum Virology lecture 5 1 RNA VIRUS STRATEGIES 2 RNA gt RNA RNAdependent RNA polymerase RNA gt DNA RNAdependent DNA polymerase reverse transcriptase Host cell DNA gt RNA ID: 751712
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Slide1
RNA VIRUS
REPLICATION/ TRNSLATION STRATEGIES
Sadia AnjumVirology lecture 5
1Slide2
RNA VIRUS STRATEGIES2
RNA -> RNA
RNA-dependent RNA polymerase RNA -> DNA
RNA-dependent DNA polymerase
- reverse transcriptase
Host cell DNA -> RNA
DNA-dependent RNA polymerase Slide3
3All animal RNA viruses code for a polymerase
Need to make mRNA
PLUS (POSITIVE) SENSE RNA GENOMES
AAA
proteins
(+
ve
) sense mRNASlide4
Need to make mRNA4
MINUS (NEGATIVE) SENSE RNA GENOMES
AAA
proteins
(+ve) sense mRNA
(-ve) sense genomic RNASlide5
Need to make mRNA5
MINUS (NEGATIVE) SENSE RNA GENOMESRNA polymerase must be packaged in virion
.
AAA
proteins
(+ve) sense mRNA
(-ve) sense genomic RNA
If used, RNA modifying enzymes are
packaged in virion
.Slide6
6DOUBLE-STRANDED RNA GENOMES
AAA
proteins
(+ve) sense mRNA
Double-stranded genomic RNA
If used, RNA modifying enzymes are
packaged in virion
.
RNA polymerase must
be packaged
in
virion
.Slide7
7RETROVIRUSES
DSDNA
+VE
RNA
DS
DNA
Reverse transcriptase
must be
packaged in
virion
.Slide8
RNA viruses that do NOT have a DNA phase8Slide9
RETROVIRUSES9
Genome
RNA-dependent DNA polymerase
(=reverse transcriptase) in virion
Infectivity of RNA
Initial event in cell
Plus-stranded RNA
Yes
Non-infectious
Reverse transcriptionSlide10
The monocistronic mRNA problemMake one monocistronic mRNA per protein
Make a primary transcript and use alternative splicingMake a large protein and then cut it into smaller proteinsInclude special features in the mRNA which enable ribosomes to bind internally
10
AAAAAAAA
RIBOSOMES
mRNA
PROTEIN
AAAAAAAA
Eukaryotes
Prokaryotes/virusesSlide11
11Slide12
Polycistronic vs Monocistronic
12Slide13
RNA GENOME SIZETAUTOMERIZATION
PROOF READINGGENOME SIZERNA VIRUSES HAVE SMALL GENOMES
~10,000 NUCLEOTIDES (herpesvirus DNA ~200,000)13Slide14
GENOME SIZEPOLYMERASEATTACHMENT PROTEINCAPSID PROTEINRELATIVELY FEW OTHER PROTEINS
VIRION SIZE MAY BE QUITE LARGE BUT LIMITED NUMBER OF PROTEINS14Slide15
POSITIVE-STRANDED RNA VIRUSESEXAMPLES
PICORNAVIRUSESTOGAVIRUSESFLAVIVIRUSES
15Slide16
PICORNAVIRUS FAMILY (PICORNAVIRIDAE)16
SMALLICOSAHEDRALPOSITIVE SENSE RNANON-ENVELOPEDSlide17
INTERNAL RIBOSOME ENTRY SITE (IRES)17
GENOMIC (+ SENSE) RNA
AAAAAVPg
IRES
start codon
for translation
stop codon
for translation
POLYPROTEIN
.Slide18
VpgVPg (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
18Slide19
Inhibition of host cell translation19
HOST mRNA
AAAAA
CAP
Uninfected cell
Factors+40S ribosomal subunit
HOST mRNA
AAAAA
CAP
Altered factors+40S ribosomal subunit
Infected cellSlide20
Viral translation20
GENOMIC (+ SENSE) RNA
AAAAA
IRES
VPg
Factors+40S ribosomal subunit
Altered factors+40S ribosomal subunit
GENOMIC (+ SENSE) RNA
AAAAA
IRES
VPg
Uninfected cell
Infected cellSlide21
21
GENOMIC (+ SENSE) RNA
AAAAA
translationSlide22
22Slide23
RNA REPLICATION23
GENOMIC (+ SENSE) RNA
3’VPg
(- SENSE) RNA
3’
VPg
GENOMIC (+ SENSE) RNA
3’
VPgSlide24
RNA REPLICATIONviral RNA polymerase (replicase)host factors also involved as accessory proteinsnew plus strandspackaged
templates for more replicationtemplates for more translation24Slide25
ASSEMBLY25
VP0
, VP1, VP3
VP2, VP4, VP1, VP3
VPg
VPg-RNA enters, VP0 is cleavedSlide26
NON-SEGMENTED NEGATIVE STRAND VIRUSES26
Examples:Rhabdovirus family (Rhabdoviridae)Paramyxovirus family (Paramyxoviridae)
Filovirus family (Filoviridae)Slide27
RHABDOVIRUSES27
helical nucleocapsid (RNA plus
N protein)
G glycoprotein
SPIKES
lipid bilayer membrane
polymerase complex
M proteinSlide28
28MINUS (NEGATIVE) SENSE RNA GENOME
RNA polymerase must be packaged in virion.
AAA
proteins
(+ve) sense mRNA
(-ve) sense genomic RNA
RNA modifying enzymes
are packaged
in
virion
.Slide29
RHABDOVIRUSESEXAMPLESvesicular stomatitis virus (VSV)
rabies virus29
Dr FS Murphyhttp://www.vetnet.ucdavis.edu/fam_graphics/download.htmlSlide30
ADSORPTION AND PENETRATION30
H+Slide31
CYTOPLASMIC REPLICATIONGENOMIC RNA REMAINS IN NUCLEOCAPSID FORM
31Slide32
RNA SYNTHESIS32
3’
5’genome (-ve)
3’
5’
(-ve)
full-length copy
5’
3’
(+ve)
replication (protein
synthesis is a pre-requisite)
transcription
mRNAs (+ve sense)
AAA
AAA
AAA
AAA
AAA
= capSlide33
NEW MINUS STRANDPACKAGEDTEMPLATE FOR MORE REPLICATION
TEMPLATE FOR MORE TRANSCRIPTION33
3’
5’
genome (-ve)
3’
5’
(-ve)
5’
3’
(+ve)
AA
AA
AA
AA
AASlide34
SOME POINTS TO NOTE ABOUT RHABDOVIRUSESENTIRE CYCLE OCCURS IN CYTOPLASM
RNA POLYMERASE AND RNA MODIFICATION ENZYMES ARE: VIRALLY CODEDPRESENT IN THE VIRIONTHERE IS NO EARLY/LATE DIVISION
34Slide35
MEMBERS INCLUDEPARAINFLUENZA VIRUSMUMPS VIRUS
MEASLES VIRUSRESPIRATORY SYNCYTIAL VIRUS35
PARAMYXOVIRUSESSlide36
PARAMYXOVIRUSES36
M protein
helical nucleocapsid (RNA plusNP protein)
HN/H/G glycoprotein
SPIKES
polymerase complex
lipid bilayer membrane
F glycoprotein
SPIKES
pleomorphicSlide37
PARAMYXOVIRIDAEPARAMYXOVIRUS SUBFAMILYPARAMYXOVIRUS
RUBULAVIRUSMORBILLIVIRUSPNEUMOVIRUS SUBFAMILYPNEUMOVIRUS GENUSMETAPNEUMOVIRUS GENUS
37Slide38
38
GENUS
GLYCOPROTEINSTYPICAL 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 FAMILY
SURFACE GLYCOPROTEINS
PARAMYXOVIRUS SUBFAMILY
PNEUMOVIRUS SUBFAMILYSlide39
TRANSCRIPTION, TRANSLATION, REPLICATION
39
3’5’
genome (-ve)
full-length copy
5’
3’
(+ve)
3’
5’
(-ve)
replication (protein
synthesis is a pre-requisite)
transcription
mRNAs (+ve sense)
AAA
AAA
AAA
AAA
AAA
= cap
AAASlide40
Some differences between rhabdoviruses and paramyxoviruses
40
two:one attachmentone fusionone (has both attachment and fusion activities)
glycoproteins
neutral
physiological
acidic
fusion pH
round
pleomorphic
bullet
bacilliform
shape
PARAMYXOVIRUSES
RHABDOVIRUSESSlide41
ORTHOMYXOVIRUSES41
M1 protein
helical nucleocapsid (RNA and NP protein)
HA - hemagglutinin
polymerase
complex
lipid bilayer membrane
NA - neuraminidaseSlide42
ADSORPTION AND PENETRATION42
H+
NUCLEUSSlide43
TRANSCRIPTION (mRNA SYNTHESIS)
43
AAA
A
viral endonuclease
A
host cell mRNA
influenza mRNA
=cap
influenza virus nucleocapsid
A
U
A
U
viral polymeraseSlide44
RNA SYNTHESISEndonuclease is virally coded
and packed in the virionRNA polymerase is virally coded and packed in
the virionPoly(A) polymerase is virally coded and in the virionWhy doesn’t the virus use host cell enzymes?
44Slide45
RNA SPLICING45
Several segments give rise to transcripts which can have one of two fates
AAAAAA
AAAAAASlide46
RNA REPLICATION
nuclearauthentic copiesreplicase and transcriptase probably same enzymecoated with protein as made
no clear early/late46
3’
5’
genomic (-ve)
3’
5’
genomic (-ve)
5’
3’
(+ve)Slide47
47
H+
NUCLEUS
acid pH inside endosome
- HA conformational change, now active in membrane fusion
Infection of next cell
HA cleavage by host cell enzymes
Flu Virus Slide48
48Slide49
REOVIRUS FAMILY49
double stranded RNA genome
outer capsid
inner capsidSlide50
50DOUBLE-STRANDED RNA GENOMES
AAA
proteins
(+ve) sense mRNA
Double-stranded genomic RNA
RNA modifying enzymes are
packaged in virion.
RNA polymerase must be
packaged in virion.Slide51
REOVIRUS FAMILY51
double stranded RNA genome
outer capsid
inner capsid
RNA polymerase plus 5’ end
modification enzymesSlide52
REOVIRUS FAMILYINCLUDESREOVIRUSESROTAVIRUSESIMPORTANT HUMAN PATHOGENSORBIVIRUSES
INCLUDE COLORADO TICK FEVER VIRUS52Slide53
53
Annual Review of Microbiology 51:227 RF Ramig