RNA VIRUS REPLICATION STRATEGIES

(1)

RNA VIRUS REPLICATION

STRATEGIES

(2)

RNA VIRUS STRATEGIES

RNA -> RNA

RNA-dependent RNA polymerase

RNA -> DNA

RNA-dependent DNA polymerase

- reverse transcriptase

(3)

ALL ANIMAL RNA VIRUSES

CODE FOR A POLYMERASE

(4)

Need to make mRNA

PLUS (POSITIVE) SENSE RNA GENOMES

(5)

Need to make mRNA

MINUS (NEGATIVE) SENSE RNA GENOMES

AAA

(+ve) sense mRNA

(6)

Need to make mRNA

MINUS (NEGATIVE) SENSE RNA GENOMES

RNA polymerase must be

packaged in virion.

AAA

(+ve) sense mRNA

(-ve) sense genomic RNA

(7)

Need to make mRNA

DOUBLE-STRANDED RNA GENOMES

AAA

(+ve) sense mRNA

Double-stranded genomic RNA

If used, RNA modifying

RNA polymerase must be

packaged in virion.

(8)

Need to make mRNA

RETROVIRUSES

DS DNA +VE RNA DS DNA

Reverse transcriptase must

be packaged in virion.

(9)

RNA viruses that do NOT have a DNA

phase

Transcription Non-infectious Yes Double-stranded RNA Transcription Non-infectious Yes Negative-stranded RNA Translation Infectious No Plus-stranded RNA Initial event in cell Infectivity of RNA RNA-dependent RNA polymerase (=transcriptase) IN VIRION Genome Transcription Non-infectious Yes Double-stranded RNA Transcription Non-infectious Yes Negative-stranded RNA Translation Infectious No Plus-stranded RNA Initial event in cell Infectivity of RNA RNA-dependent RNA polymerase (=transcriptase) IN VIRION Genome

(10)

Genome RNA-dependent DNA polymerase (=reverse transcriptase) in virion Infectivity of RNA Initial event in cell Plus-stranded

RNA Yes Non-infectious

Reverse transcription

(11)

The monocistronic mRNA problem

• 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

AAAAAAAA RIBOSOMES mRNA PROTEIN AAAAAAAA

(12)

GENOME SIZE

• TAUTOMERIZATION

• PROOF READING

• RNA VIRUSES HAVE SMALL

GENOMES

~10,000 NUCLEOTIDES

(13)

GENOME SIZE

• POLYMERASE

• ATTACHMENT PROTEIN

• CAPSID PROTEIN

• RELATIVELY FEW OTHER PROTEINS

• VIRION SIZE MAY BE QUITE LARGE

BUT LIMITED NUMBER OF

PROTEINS

(14)

POSITIVE-STRANDED RNA

VIRUSES

• EXAMPLES

• PICORNAVIRUSES

• TOGAVIRUSES

(15)

PICORNAVIRUS FAMILY

(PICORNAVIRIDAE)

• SMALL

• ICOSAHEDRAL

• POSITIVE SENSE

RNA

• NON-ENVELOPED

(16)

ADSORPTION AND

PENETRATION

AAA

(17)

INTERNAL RIBOSOME

ENTRY SITE (IRES)

GENOMIC (+ SENSE) RNA

AAAAA

VPg

IRES

start codon

for translation

stop codon

for translation

POLYPROTEIN

(18)

Inhibition of host cell translation

HOST mRNA

AAAAA

CAP

Uninfected cell

Factors+40S ribosomal subunit

HOST mRNA

AAAAA

CAP

Altered factors+40S ribosomal subunit

(19)

Viral translation

AAAAA

IRES

VPg

Factors+40S ribosomal subunit

Altered factors+40S ribosomal subunit

AAAAA

IRES

VPg

Uninfected cell

(20)

AAAAA

(21)

RNA REPLICATION

3’

VPg

(- SENSE) RNA

3’

VPg

3’

VPg

(22)

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

(23)

ASSEMBLY

VP0

, VP1, VP3

VP2, VP4, VP1, VP3

VPg

(24)

RELEASE

(25)

NON-SEGMENTED NEGATIVE

STRAND VIRUSES

Examples:

Rhabdovirus family (Rhabdoviridae)

Paramyxovirus family (Paramyxoviridae)

Filovirus family (Filoviridae)

(26)

helical nucleocapsid (RNA plus N protein)

G glycoprotein SPIKES

lipid bilayer membrane M protein

(27)

Need to make mRNA

MINUS (NEGATIVE) SENSE RNA GENOME

RNA polymerase must be

packaged in virion.

AAA

(+ve) sense mRNA

(-ve) sense genomic RNA

(28)

helical nucleocapsid (RNA plus N protein)

G glycoprotein SPIKES

lipid bilayer membrane

polymerase complex

M protein

(29)

RHABDOVIRUSES

• EXAMPLES

– vesicular stomatitis

virus (VSV)

(30)

ADSORPTION AND

PENETRATION

(31)

ADSORPTION AND

PENETRATION

• CYTOPLASMIC REPLICATION

• GENOMIC RNA REMAINS IN

NUCLEOCAPSID FORM

(32)

3’ genome (-ve) 5’ (-ve)

full-length copy

5’ (+ve) 3’ replication (protein synthesis is a pre-requisite) transcription

mRNAs (+ve sense)

AAA AAA AAA AAA AAA

RNA SYNTHESIS

(33)

NEW MINUS STRAND

• PACKAGED

• TEMPLATE FOR

MORE

REPLICATION

• TEMPLATE FOR

MORE

TRANSCRIPTION

3’ genome (-ve) 5’ 3’ (-ve) 5’ 5’ (+ve) 3’ AA AA AA AA AA

(34)

endoplasmic reticulum

Golgi plasma membrane

(35)

polymerase inside outside cell

ASSEMBLY

M protein

(36)

SOME POINTS TO NOTE

ABOUT RHABDOVIRUSES

• ENTIRE CYCLE OCCURS IN

CYTOPLASM

• RNA POLYMERASE AND RNA

MODIFICATION ENZYMES ARE:

– VIRALLY CODED

– PRESENT IN THE VIRION

(37)

PARAMYXOVIRUSES

• MEMBERS INCLUDE

– PARAINFLUENZA VIRUS

– MUMPS VIRUS

– MEASLES VIRUS

(38)

helical nucleocapsid (RNA plus NP protein)

HN/H/G glycoprotein SPIKES

polymerase complex

lipid bilayer membrane F glycoprotein

SPIKES

PARAMYXOVIRUSES

pleomorphic

(39)

PARAMYXOVIRIDAE

• PARAMYXOVIRUS SUBFAMILY

– PARAMYXOVIRUS

– RUBULAVIRUS

– MORBILLIVIRUS

• PNEUMOVIRUS SUBFAMILY

– PNEUMOVIRUS GENUS

– METAPNEUMOVIRUS GENUS

(40)

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 FAMILY SURFACE GLYCOPROTEINS

PARAMYXOVIRUS SUBFAMILY

(41)

(42)

(43)

(44)

HEMAGGLUTINATION

• Quick, easy, cheap

• Can use to detect virus or antibody to

virus (hemagglutination inhibition)

• A measure of total virus particles

(45)

(46)

GENUS ATTACHMENT GLYCOPROTEIN TYPICAL MEMBERS

Paramyxovirus HN HPIV1, HPIV3

Rubulavirus HN HPIV2, HPIV4, mumps virus Morbillivirus H measles virus

Pneumovirus G respiratory syncytial virus Metapneumovirus G metapneumoviruses

PARAMYXOVIRUS FAMILY

PROPERTIES OF THE ATTACHMENT PROTEIN

(47)

Neuraminidase activity

(48)

GENUS ATTACHMENT GLYCOPROTEIN TYPICAL MEMBERS

Paramyxovirus HN HPIV1, HPIV3

Rubulavirus HN HPIV2, HPIV4, mumps virus Morbillivirus H measles virus

Pneumovirus G respiratory syncytial virus Metapneumovirus G metapneumoviruses

PARAMYXOVIRUS FAMILY

PROPERTIES OF THE ATTACHMENT PROTEIN

(49)

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 FAMILY

PROPERTIES OF THE SURFACE GLYCOPROTEINS

(50)

F PROTEIN

• Fusion

(51)

(52)

3’ genome (-ve) 5’

full-length copy

5’ (+ve) 3’ (-ve) replication (protein synthesis is a pre-requisite) transcription

mRNAs (+ve sense)

AAA AAA AAA AAA AAA

TRANSCRIPTION,

TRANSLATION, REPLICATION

(53)

ASSEMBLY

Golgi plasma membrane

(54)

ASSEMBLY

polymerase

outside cell

(55)

(56)

ROLE OF NEURAMINIDASE

(57)

(58)

(59)

ACTIVATION OF THE F PROTEIN

lipid membrane

S

CYTOPLASM

S

(60)

ACTIVATION OF THE F

PROTEIN

(61)

Some differences between

rhabdoviruses and paramyxoviruses

two:

one attachment

one fusion

one (has both

attachment and

fusion activities)

glycoproteins

neutral

acidic

fusion pH

round

pleomorphic

bullet

bacilliform

shape

PARAMYXOVIRUSES

RHABDOVIRUSES

(62)

ORTHOMYXOVIRUSES

helical nucleocapsid (RNA and NP protein)

HA - hemagglutinin

polymerase complex

lipid bilayer membrane NA - neuraminidase

(63)

ORTHOMYXOVIRUSES

(64)

(65)

ADSORPTION AND

PENETRATION

(66)

TRANSCRIPTION

(mRNA SYNTHESIS)

AAA

A

viral endonuclease

A

host cell mRNA

influenza mRNA

=cap

influenza virus nucleocapsid

A

U

A

(67)

RNA SYNTHESIS

• Endonuclease is virally coded and in the

virion

• RNA polymerase is virally coded and in

the virion

• Poly(A) polymerase is virally coded and

in the virion

• Why doesn’t the virus use host cell

enzymes?

(68)

RNA SPLICING

SEVERAL SEGMENTS GIVE RISE TO TRANSCRIPTS

WHICH CAN HAVE ONE OF TWO FATES

AAAAAA

(69)

RNA REPLICATION

• nuclear

• authentic copies

• replicase and

transcriptase

probably same

enzyme

• coated with protein

as made

• no clear early/late

3’ 5’ genomic (-ve) 3’ 5’ genomic (-ve) 5’ 3’ (+ve)

(70)

ASSEMBLY

• NUCLEOCAPSIDS TRANSPORTED TO

CYTOPLASM

• GLYCOPROTEINS INSERTED IN PLASMA

MEMBRANE

• M PROTEINS ASSOCIATE WITH

MEMBRANE

• BUD THROUGH PLASMA MEMBRANE

• HA CLEAVAGE

(71)

flu HA protein and fusion

S

cleavage by

host cell

enzymes

S

acid pH inside

endosome

virus

(72)

Infection of next cell

HA cleavage by host cell enzymes

(73)

PARAMYXOVIRIDAE ORTHOMYXOVIRIDAE genome nonsegmented segmented

RNA synthesis cytoplasmic nuclear

need for RNA primer

no yes

hemagglutinin, neuraminidase

if both, part of same protein (HN)

Influenza A and B viruses have both, but on different proteins (HA and NA)

syncytial formation

yes (F functions at physiol. pH)

no (HA functions at acid pH)

(74)

REOVIRUS FAMILY

double stranded RNA genome

outer capsid

(75)

Need to make mRNA

DOUBLE-STRANDED RNA GENOMES

AAA

(+ve) sense mRNA

Double-stranded genomic RNA

RNA modifying

RNA polymerase must be

packaged in virion.

(76)

REOVIRUS FAMILY

double stranded RNA genome

outer capsid

inner capsid

RNA polymerase plus 5’ end modification enzymes

(77)

(78)

REOVIRUS FAMILY

• INCLUDES

– REOVIRUSES

– ROTAVIRUSES

• IMPORTANT HUMAN PATHOGENS

– ORBIVIRUSES

(79)

ADSORPTION, PENETRATION,

UNCOATING

(80)

ADSORPTION, PENETRATION,

UNCOATING

• proteases in GI tract

• attachment proteins at vertices

(81)

(82)