The DNA -> RNA -> Protein Pathway
RNA Polymerase =
enzyme that makes
mRNA from the
DNA gene template
http://www.bioteach.ubc.ca/MolecularBiology/AMonksFlourishingGarden/
Plus-strand RNA Viruses
•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.”
Types of Plus-Strand RNA Viruses with single-stranded, non-segmented
plus-strand genomes
Class
I
II
Genome
Polarity
Polymerase
in Virions?
+
(mRNA)
No
+
(mRNA)
No
RNA by
itself
infectious?
Yes
Yes
Types of
mRNAs
One
Multiple
Types of protein
products
Long
polyprotein;
subsequently
cleaved
One for each
mRNA
Example virus family
Picornaviruses
(ex. Rhinovirus)
Flaviviruses
(ex. West Nile Virus)
Coronaviruses
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 1988
Current 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 (Plusstrand 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
5’-NTR
VPg
~630 nts
Open Reading Frame Encoding Polyprotein
~7200 bases
AAAAAAAA
Human Rhinovirus Structure
Virus exterior = protein shell
Icosahedral shape
Cross-section
Viral RNA genome inside
Protein capsid layer outside
http://www.rcsb.org/pdb/molecules/pdb20_3.html
Rhinovirus Life Cycle
http://cumicro2.cpmc.columbia.edu/PICO/Chapters/Cellular.html#Receptor
Rhinovirus Binding to Receptor and Antibodies
ICAM-1 RECEPTOR
BOUND TO RHINOVIRUS
ANTIBODIES BOUND
TO RHINOVIRUS
http://www.rcsb.org/pdb/molecules/pdb20_3.html
ICAM-1 Receptor Binds to Canyon on
Rhinovirus Surface
http://rhino.bocklabs.wisc.edu/virusworld/images/r14_rhino+receptor.jpg
Rhinovirus Life Cycle
http://cumicro2.cpmc.columbia.edu/PICO/Chapters/Cellular.html#Receptor
Rhinovirus Polyprotein Processing: Method for
Generation of Individual Viral Proteins
http://www.ag.uidaho.edu/mmbb/p_gustin_k.htm
Rhinovirus Life Cycle
http://cumicro2.cpmc.columbia.edu/PICO/Chapters/Cellular.html#Receptor
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 Therapies
62 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 (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
Class
I
II
Genome
Polarity
Polymerase
in Virions?
+
(mRNA)
No
+
(mRNA)
No
RNA by
itself
infectious?
Yes
Yes
Types of
mRNAs
One
Multiple
Types of protein
products
Long
polyprotein;
subsequently
cleaved
One for each
mRNA
Example virus family
Picornaviruses
(ex. Rhinovirus)
Flaviviruses
(ex. West Nile Virus)
Coronaviruses
Flavivirus Gene Structure
(West Nile Virus = member of
Flavivirus family)
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
West Nile Virus Genetic Structure
and Protein Expression
www.med.sc.edu:85/ virol/flavi1.jpg
West Nile Virus Structure
http://www.cdc.gov/ncidod/eid/vol7no4/petersenG2.htm
http://news.uns.purdue.edu/UNS/html4ever/031009.Kuhn.westnile.html
West Nile Virus Replication Cycle
*The WNV receptor
in vertebrate cells is
V3 integrin, which
is highly conserved in
vertebrates
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
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
Class
I
II
Genome
Polarity
Polymerase
in Virions?
+
(mRNA)
No
+
(mRNA)
No
RNA by
itself
infectious?
Yes
Yes
Types of
mRNAs
One
Multiple
Types of protein
products
Long
polyprotein;
subsequently
cleaved
One for each
mRNA
Example virus family
Picornaviruses
(ex. Rhinovirus)
Flaviviruses
(ex. West Nile Virus)
Coronaviruses
Coronaviruses
http://www-micro.msb.le.ac.uk/335/Coronaviruses.html
Coronaviruses
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
Coronavirus Receptor = ACE-2
ACE-2 = angiotensin-converting enzyme 2
Expressed in heart, lung, kidney, GI tract
http://www.rndsystems.com/DAM_public/5531.gif
SARS Virus Budding
http://www1.lf1.cuni.cz/~hrozs/sarsu0403.htm
Targets for SARS Therapy
http://www.nyas.columbia.edu/sars/web/slide_presentations/s3_2/18.html