http://www.bioteach.ubc.ca/MolecularBiology/AMonksFlourishingGarden/

The DNA -> RNA -> Protein Pathway

RNA Polymerase = enzyme that

makes mRNA from the DNA gene template

• Plus-strand RNA viruses have genomes comprised of RNA. They have no DNA in their replication cycles.

• In most cases, the replication cycle takes place in the cytoplasm of the cells.

• No involvement of the transcription machinery in the nucleus; so, the plus-strand RNA viruses produce their own enzymes for RNA transcription and replication, which recognize RNA as the template.

• The plus-strand RNA viruses do use the host cell’s translation machinery to generate viral proteins.

• Many plus-strand RNA viruses produce numerous viral proteins from a single “gene.”

Plus-strand RNA Viruses

Types of Plus-Strand RNA Viruses with single-stranded, non-segmented plus-strand genomes

Genome Polarity

Polymerase in

Virions?

RNA by itself infectious?

Types of mRNAs

Types of protein products

Example virus family

I +(mRNA) No Yes One

Long polyprotein; subsequently

cleaved

Picornaviruses (ex. Rhinovirus)

Flaviviruses (ex. West Nile Virus)

II +(mRNA) No Yes Multipl

eOne for each

mRNA Coronaviruses

Class

Picornavirus Diseases

ENTEROVIRUSES (stable in GI tract)

• Poliomyelitis• Hepatitis A Virus• Coxsackieviruses• Enteroviruses Types 68-72• Echoviruses

RHINOVIRUSES (not stable in GI tract)

• Common Cold• Numerous subtypes

Fecal-Oral Transmission

Enterovirus Pathogenesis: Target Tissues

Poliovirus Pathogenesis

Only a fraction of patients develop paralytic disease

• Asymptomatic infection – 90%

• Abortive/minor illness – 5%

• Non-paralytic progression to the CNS – 1%-2%

• Paralytic poliovirus – 0.1%-2%

• 3-4 days after minor illness

• Virus infects motor neurons in anterior horn of spinal cord and the motor cortex

Poliovirus Infection: Progression to CNS

Disease

http://www.vadscorner.com/outbreak.html

Protection by Antibodies

• Secretory antibodies can prevent primary infection

• Serum antibodies prevent viremic spread to target tissues

Poliovirus Vaccination

• Salk Vaccine• Killed Virus (formalin inactivated)• IPV

• Sabin Vaccine• Live, attenuated viruses• OPV

• Attenuated strains can revert to virulent forms

• Vaccinees shed the attenuated viruses in feces

• Plusses• Minuses

WHO Eradication of Poliovirus

Eradication campaign started in 1988Current target date for eradication: Beyond 2009

http://www.polioeradication.org/content/general/casemap.shtml

Is Eradication Possible?• Oral Poliovirus Vaccine

• Preferred for Third World Vaccination• Cheaper, No Sterile Needles Required• Campaign of usage has drastically reduced wild poliovirus transmission

• Major issue is REVERSION of OPV strains to virulent forms

• Laboratory Stocks• Poliovirus present in many laboratories around the World

• Poliovirus contaminants in stocks of other viruses, i.e. Coxsackievirus, Rhinovirus

• Bioterrorism• cDNA copies of poliovirus genome exist in many laboratories; transfection into tissue culture cells results in virus production

• The poliovirus genome has been generated on a gene synthesizer (Plus-strand RNA viral genomes alone are infectious!)

Picornavirus Diseases

ENTEROVIRUSES (stable in GI tract)

• Poliomyelitis• Hepatitis A Virus• Coxsackieviruses• Enteroviruses Types 68-72• Echoviruses

RHINOVIRUSES (not stable in GI tract)

• Common Cold• Numerous subtypes

Human Rhinovirus Structure

AAAAAAAAVPg

5’-NTROpen Reading Frame Encoding Polyprotein

~7200 bases

~630 nts

http://www.rcsb.org/pdb/molecules/pdb20_3.html

Human Rhinovirus Structure

Virus exterior = protein shell

Icosahedral shape

Cross-section

Viral RNA genome inside

Protein capsid layer outside

http://cumicro2.cpmc.columbia.edu/PICO/Chapters/Cellular.html#Receptor

Rhinovirus Life Cycle

http://www.rcsb.org/pdb/molecules/pdb20_3.html

Rhinovirus Binding to Receptor and Antibodies

ICAM-1 RECEPTORBOUND TO RHINOVIRUS

ANTIBODIES BOUNDTO RHINOVIRUS

http://rhino.bocklabs.wisc.edu/virusworld/images/r14_rhino+receptor.jpg

ICAM-1 Receptor Binds to Canyon on Rhinovirus Surface

http://cumicro2.cpmc.columbia.edu/PICO/Chapters/Cellular.html#Receptor

Rhinovirus Life Cycle

http://www.ag.uidaho.edu/mmbb/p_gustin_k.htm

Rhinovirus Polyprotein Processing: Method for Generation of Individual Viral

Proteins

http://cumicro2.cpmc.columbia.edu/PICO/Chapters/Cellular.html#Receptor

Rhinovirus Life Cycle

Rhinovirus Pathogenesis• Entry of virus into upper respiratory tract

• Hands and fomites• Inhalation of droplets that contain virus

• Unable to replicate in the GI tract (not stable to acid)

• Receptor is ICAM-1 (Intercellular Adhesion Molecule 1)

• Preferentially replicates at 33oC

• Over 100 serotypes• Reason for repeat infections throughout lifetime• Antigenic sites change, but receptor-binding site is protected

Rhinovirus Pathogenesis

Targets for Rhinovirus Therapy

New Rhinovirus Therapies62 million cases of Rhinovirus infection

in U.S. each yearCause more than 50% of respiratory tract

infections• Traditional vaccine approaches as with poliovirus not possible (too many serotypes), so antiviral therapies must exploit unique properties of the virus

• Soluble ICAM-1: Blocks virus binding to its receptor

• AG7088: 3C protease inhibitor (being formulated for intranasal delivery)

• Pleconaril: Binds a pocket in the capsid; interferes with attachment and uncoating• Promising early clinical studies – reduction in disease length

• Not approved due to possible drug-drug interactions

• Reformulations being developed

West Nile Virus

Types of Plus-Strand RNA Viruses with single-stranded, non-segmented plus-strand genomes

Genome Polarity

Polymerase in

Virions?

RNA by itself infectious?

Types of mRNAs

Types of protein products

Example virus family

I +(mRNA) No Yes One

Long polyprotein; subsequently

cleaved

Picornaviruses (ex. Rhinovirus)

Flaviviruses (ex. West Nile Virus)

II +(mRNA) No Yes Multipl

eOne for each

mRNA Coronaviruses

Class

Figure 1. Genomic structure of flaviviruses. The flavivirus genome is 11,000 to 12,000 nucleotides long. Both the 5'- and 3'- ends contain noncoding (NC) regions. The genome encodes 10 proteins, 3 of which are structural proteins (C, M, and E), and 7 of which are nonstructural proteins (NS1, NS2a, NS2b, NS3, NS4a, NS4b, and NS5). The M protein is synthesized as a precursor (prM) protein. The prM protein is processed to pr + M protein late in the virus maturation by a convertase enzyme (furin).

http://www.cdc.gov/ncidod/eid/vol7no4/petersenG1.htm

Flavivirus Gene Structure(West Nile Virus = member of

Flavivirus family)

www.med.sc.edu:85/ virol/flavi1.jpg

West Nile Virus Genetic Structure and Protein

Expression

http://www.cdc.gov/ncidod/eid/vol7no4/petersenG2.htmhttp://news.uns.purdue.edu/UNS/html4ever/031009.Kuhn.westnile.html

West Nile Virus Structure

Figure 2 The WNV replication cycle. A. Attachment and entry of the virion. B. Uncoating and translation of the virion RNA. C. Proteolytic processing of the polyprotein. D. Synthesis of the minus-strand RNA from the virion RNA. E. Synthesis of nascent genome RNA from the minus-strand RNA. F. Transport of structural proteins to cytoplasmic vesicle membranes. G. Encapsidation of nascent genome RNA and budding of nascent virions. H. Movement of nascent virions to the cell surface. I. Release of nascent virions. SHA, slowly sedimenting hemagglutinin, a subviral particle that is also sometimes released.

http://arjournals.annualreviews.org/doi/full/10.1146/annurev.micro.56.012302.160654;jsessionid=i8FGn3DIgEu8

West Nile Virus Replication Cycle

*The WNV receptor in vertebrate cells is V3 integrin, which is highly conserved in vertebrates

Coronaviruses

• Another virus group with members responsible for the “common cold”

• Coronavirus member in the news = SARS Virus

http://www.lapublichealth.org/acd/images/SARS_CoV_Picture.gif

Types of Plus-Strand RNA Viruses with single-stranded, non-segmented plus-strand genomes

Genome Polarity

Polymerase in

Virions?

RNA by itself infectious?

Types of mRNAs

Types of protein products

Example virus family

I +(mRNA) No Yes One

Long polyprotein; subsequently

cleaved

Picornaviruses (ex. Rhinovirus)

Flaviviruses (ex. West Nile Virus)

II +(mRNA) No Yes Multipl

eOne for each

mRNA Coronaviruses

Class

Coronaviruses

http://www-micro.msb.le.ac.uk/335/Coronaviruses.html

http://www.csic.es/hispano/charlas/2002/m1/m1abr-02/m1abr-02.htm

http://www.cmlab.csie.ntu.edu.tw/~jsyeh/SARS/images/coronavirus-Alankann.gif

Coronaviruses

http://www.learn-line.nrw.de/angebote/agenda21/archiv/03/05/coronavirus.jpg

Coronaviruses

Coronavirus Receptor = ACE-2

http://www.rndsystems.com/DAM_public/5531.gif

ACE-2 = angiotensin-converting enzyme 2

Expressed in heart, lung, kidney, GI tract

http://www1.lf1.cuni.cz/~hrozs/sarsu0403.htm

SARS Virus Budding

Targets for SARS Therapy

http://www.nyas.columbia.edu/sars/web/slide_presentations/s3_2/18.html