Replication of Positive-Replication of Positive-Sense RNA VirusesSense RNA Viruses

Replication of Positive-Replication of Positive-Sense RNA VirusesSense RNA Viruses

Virus Replication

RNA Virus Replication

• Replicative Intermediate 1– Genome template– Makes Antigenome

(opposite sense)• Replicative

Intermediate 2– Antigenome

template– Makes new

Genome for progeny virus

Family: Picornaviridae• “small”• (+)RNA virus• Nonenveloped• Icosahedral capsid, 30 nm

Genus: Enterovirus• “intestine”• Large group of related viruses:

– Poliovirus (poliomyelitis -“gray”, “marrow”)– Echovirus (enteric cytopathic human

orphan)– Coxsackievirus– Enterovirus

• Infect URT, GI, CNS, heart, muscle, skin• pH stable

Genus: Rhinovirus• “nose”• Human rhinovirus (>100

serotypes)• Common cold viruses• Acid pH labile

Genus: Aphthovirus• “ulcer”• Foot-and-mouth disease virus

(FMDV) of livestock• Infect cattle, sheep, pigs, goats

Genus: Cardiovirus• “heart”• Encephalomyocarditis virus (EMC)

of mice• Infect brain, muscle, heart

Genus: Heparnavirus• “liver”• Hepatitis A virus• “infectious” hepatitis• Transmission via contaminated

food or water

Poliovirus: (+)RNA Genome

• 7.7 kb• Single open

translational reading frame (ORF)

• 5’ end has small viral protein (VPg)

• 3’ polyA tail• Serves directly as

mRNA (“infectious”)

Poliovirus: RNA Genome Expression

• Attachment and entry by receptor-mediated endocytosis

• Uncoat and release of RNA into cytoplasm

• RNA attaches to cell ribosome - recognition by Internal ribosome entry site (IRES) sequence on 5’ end of genome RNA

• Viral mRNA translates for one large polyprotein

Poliovirus Polyprotein• Single polyprotein cleaved by viral

protease (2A, 3C) into 3 precursor proteins (P-1, P-2, P-3)

• Precursor proteins cleaved further by viral protease

Poliovirus Proteins

• P-1: capsid proteins (VP4, VP2, VP3, VP1)• P-2: protease (2A), regulatory proteins (2B, 2C)

• P-3: regulatory protein (3A), VPg, protease (3C), RNA polymerase (3D)

Poliovirus: Transcription of Complimentary RNA (RI-1)

• Antigenome copied from released (+)RNA genome

• Transcription complex:– (+)RNA genome– VPg serves as

primer (attaches to 3’ end of viral RNA)

– Host proteins required

– Viral RNA Replicase (viral protein 3D)

Poliovirus: Replication of Genome RNA (RI-2)

• (+)RNA genome copied from Antigenome template

• Transcription complex: antigenome, VPg, host proteins, viral RNA replicase

• New (+)RNA serves as:– mRNA (more viral proteins)– genome RNA (for new

virus)

Poliovirus RNA

Synthesis

• Each cell makes ~ 4 x 105 RNA molecules

• (-)RNA ~ 10%• (+)RNA ~

90%

Poliovirus: Assembly

• Protomer (VP0, VP1, VP3)

• Pentamer (five protomers)

• Procapsid (twelve pentamers)

• Provirion (sixty protomers, “head-full” insertion of viral RNA genome)

• Virion (cleavage of VP0 into VP2, VP4)

Poliovirus: Cell Cytopathic Effects

(CPE)• Host cell translation stopped shortly (30

min.) after infection• Viral protease activates host cell

protease to cleave a 220 kd cell ribosome protein of eIF-4F

• No cap-binding complex of mRNA• No attachment of host mRNA to

ribosome• No cell protein synthesis, leads to cell

death

Poliovirus: Release By Cell Lysis

• Occurs within 5-10 hours in cell culture

• Each cell releases ~ 25 – 100,000 virus particles

• Not all virus particles are infectious

• Virus spreads to other cells

“The fight against infantile paralysis is a fight to the finish, and the terms

are unconditional surrender.”• Franklin D. Roosvelt, 32nd U.S.

President and polio survivor, 1944

Family: Flaviviridae• “yellow”• (+)RNA, 10 kb

– Single ORF– 5’ cap, 3’ polyA

tail– Translates for

single polyprotein, cleaved into structual (capsid, M, E) and non-structual (NS) proteins

• Enveloped• Icosahedral, 40-

50 nm• Replicate in

arthropods, animals, humans

Genus: Flavivirus

• Former “Group B Arboviruses” (animal to human transmission by arthropod bite)

• Yellow fever virus (monkeys)

• West Nile virus (birds)• St. Louis encephalitis

virus (birds)• Hepatitis C virus

(transmission by blood and body secretions)

Family: Togaviridae• “coat”• (+)RNA virus• Enveloped• Icosahedral capsid, 60-70 nm• Replicate in arthropods, animals,

humans

Genus: Alphavirus

• Former “Group A Arboviruses”• Sindbis virus (bird, monkey, rare

mild disease in human)• Equine encephalitis virus (horse,

bird, rodent)

Genus: Rubivirus• “red”• Rubella virus• “3 day measles”• Transmission by direct contact, aerosols• Mild skin rash disease, but infected

moms may transmit to fetus (congenital defects, death)

Sindbis Virus: (+)RNA Genome

• Genome (11 kb), two ORF, 5’ cap, 3’ polyA tail

• Genome RNA (11 kb) translates ORF-1

• Synthesis of a subgenomic mRNA (4 kb) to translate ORF-2

Sindbis Virus: “Early” Gene Expression

• Attachment, endocytosis, uncoat and release of (+)RNA into cytoplasm

• RNA binds to ribosome and translates ORF-1 for a polyprotein

• Polyprotein cleaved into four nonstructural proteins

Sindbis Virus: Transcription

Complementry RNA (RI-1)

• Antigenome copied from released viral Genome RNA

• By “early” viral replication enzymes

Sindbis Virus: Replication

Genome RNA(RI-2)

• Genome (+)RNA (49s) copied from Antigenome

• By viral replication enzymes

• New RNA serves as:– mRNA– Genome RNA

Sindbis Virus: “Late” Gene Expression

• Viral RNA replicase binds to Interior replicase start site of antigenome

• Synthesis of a subgenomic mRNA (26s), 5’ Cap, 3’ PolyA

• Translation of ORF-2 for structural (capsid, envelope) proteins

Sindbis Virus RNA• Temporal regulation of viral RNA

synthesis• 1–3 hours: both (+)RNA and (-)RNA• >3 hours: mainly (+)RNA• 10x more subgenomic mRNA (26s)

than genomic mRNA (49s)

Sindbis Virus: Assembly and Release By Budding

• Envelope proteins glycosylated in ER/Golgi, transported and inserted into plasma membrane

• Capsid forms in cytoplasm

• Bud through the virus modified plasma membrane, picks up envelope, and released from cell

Family: Coronaviridae

• Corona - “crown”• Envelope, large petal

spikes, 80-120 nm• Flexible helical

nucelocapsid, 10-20 nm• (+)RNA, 30 kb, 5’ cap,

3’ polyA, five ORFs• Synthesis of

subgenomic mRNAs

Genus: Coronavirus• Human coronavirus (common cold,

LRTI, gastroenteritis)• SARS-CoV (severe acute

respiratory syndrome, fatal pneumonia)

• Also infections of mammals and birds

Reading & Questions• Chapter 14: Replication of

Positive-Sense RNA Viruses

QUESTIONS???

Class Discussion – Lecture 5

• 1. Why is the RNA genome of poliovirus by itself “infectious” when transfected into a host cell?

• 2. How does (+)RNA viruses replicate their genome via two replicative intermediates? Why is this necessary?

• 3. Is the replication strategy of Sindbis virus similar/different from poliovirus?

MICR 401 FIRST EXAM• Tuesday, Oct. 16, 2012• History through Positive-sense RNA

virus: Coronavirus• Lecture, Discussion Questions, Reading,

Chapter Questions• Test Format:

– Objective questions – Multiple Choice, True/False Statement, Identification (term, figure, diagram)

– Short essay questions (similar to Class Discussion Questions and Basic Virology Chapter Questions)