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

The DNA -> RNA -> Protein Pathway

RNA Polymerase =enzyme that makes

mRNA from theDNA gene template

•Plus-strand RNA viruses have genomes comprised of RNA. They haveno DNA in their replication cycles.

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

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

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

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

Plus-strand RNA Viruses

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

Picornaviruses(ex. Rhinovirus)

Flaviviruses(ex. West Nile Virus)

Longpolyprotein;subsequently

cleaved

OneYesNo+

(mRNA)I

CoronavirusesOne for eachmRNAMultipleYesNo

+(mRNA)

II

Example virus familyTypes of proteinproducts

Types ofmRNAs

RNA byitself

infectious?

Polymerasein Virions?

GenomePolarity

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 themotor cortex

Poliovirus Infection:Progression to CNS Disease

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

Enterovirus Replication and DiseaseProgression

Primary InfectionMucosa and lymphoid tissues

Tonsils, pharynx, Peyer’s Patches

Viremia

Infection of secondary target tissue

Secondary replicationPhase

SYMPTOMS

Release of virus to environment

via fecal shedding

Protection by Antibodies

•Secretory antibodies can prevent primaryinfection

•Serum antibodies prevent viremic spread totarget 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

Total cases in 2006 Compared to same period in 2005

Globally 1403 1349

-in endemic countries: 1300 552

-in non-endemic countries: 103 797

WHO Eradication of Poliovirus:

2006 vs. 2005

http://www.polioeradication.org/casecount.asp

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; transfectioninto 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

AAAAAAAA

VPg

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 onRhinovirus 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 forGeneration 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 year

Cause more than 50% of respiratory tract infections

•Traditional vaccine approaches as with poliovirus not possible (toomany serotypes), so antiviral therapies must exploit unique properties ofthe virus

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

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

•Pleconaril: Binds a pocket in the capsid; interferes with attachment anduncoating

•Promising early clinical studies – reduction in disease length•Not approved due to possible drug-drug interactions•Reformulations being developed

West Nile Virus

Figure 1. Genomic structure of flaviviruses. The flavivirus genome is 11,000 to 12,000 nucleotides long. Both the 5'- and 3'- ends containnoncoding (NC) regions. The genome encodes 10 proteins, 3 of which are structural proteins (C, M, and E), and 7 of which arenonstructural proteins (NS1, NS2a, NS2b, NS3, NS4a, NS4b, and NS5). The M protein is synthesized as a precursor (prM) protein. The prMprotein 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 ofFlavivirus family)

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

West Nile Virus Genetic Structureand 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 nascentgenome 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 receptorin vertebrate cells isαVβ3 integrin, whichis highly conserved invertebrates

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

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

Coronavirus Spike Proteins:Virulence Factors

http://www.nyas.columbia.edu/sars/web/slide_presentations/s3_2/04.html

Targets for SARS Therapy

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