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Genetic recombination during reverse transcription
• All recombination occurs between coencapsidated
genomes at the time of reverse transcription
• The copy choice model postulates a mechanism during
(-) strand DNA synthesis
Strand displacement assimilation model proposes that
recombination occurs during (+) strand DNA synthesis
Domain and subunit relationships of RTs
from different retroviruses
•Integrase is encoded at the 3’ end of the pol gene
•Mature protein is made by protease mediated
processing of the Gag-Pol precursor
Retroviral DNA Integration
Viral DNA is
shortened by 2
bp at each end
6 bp target site
is duplicated on
either side of
proviral DNA
Retroviral DNA Integration
• Integrase (IN) is the enzyme that catalyzes integration of
the reverse transcribed viral DNA into the host genome
• Viral DNA is shortened by 2 bp from each end and a short
(4-6 bp) duplication of host DNA flanks the provirus at
either end.
• Proviral ends of all retroviruses comprise the same
dinucleotide: 5’-TG----CA-3’
• This dinucleotide is found in an inverted repeat
characteristic of the virus
• The inverted repeat, conserved terminal dinucleotide
sequence and flanking direct repeats of host DNA are
characteristic of features of bacterial insertion sequences
• These similarities suggest common mechanisms of
retroviral DNA integration and DNA transposition
Steps in retroviral DNA integration
The two ends of viral DNA are
recognized, nicked and then joined
covalently to host DNA in random
locations at staggered nicks also
introduced by Integrase
Endonucleolytic nicking and
removal of 2 nt and formation of a
new 3’ recessed end
Joining of 3’ ends to
phosphates at the target site
Gapped intermediate
Gaps are
repaired
• Most retrotransposons are distinguished from retroviruses
by lack of an extracellular phase
• They have no env gene, thus virus like particles formed in
vivo are noninfectious
Retrovirus assembly
• Association of Gag molecules with the plasma
membrane and with the RNA molecules initiates
assembly at the inner surface of the plasma membrane
• A minor fraction of Gag translation products carry the
retroviral enzymes, PR, RT and IN at their C-termini
• Assembly continues by incorporation of additional
molecules of Gag
• Fusion of the membrane around the budding particle
releases the immature noninfectious particle
• Cleavage of Gag and Gag-Pol polyproteins by the viral
protease produces infectious particles
Retrovirus assembly
Phylogenetic relationships among
retroviruses
The Origin of HIV
“HIV-1 probably originated from
SIVcpz in chimpanzees less than
100 years ago. HIV-2, a virus still
largely confined in Western Africa,
probably originated from SIVsm in
Mangabes”.
http://www.cdc.gov/ncidod/EID/index.htm
•
Similarities and differences between
Lentiviruses
Similarities
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Nucleic acid homology, organization
Transmission (sex, blood, milk)
Macrophage often a target cell
Lentivirus never cleared
Slow pathogenesis / Long time to disease
Vaccines hard
Diseases : no cancers; not endogenous; immune system
dysregulation; immune complexes; neurological
• Differences
– Many silent infections vs. high penetrance of AIDS
– CD4 lymphocytes in addition to macrophages
• Examples of lentiviruses infecting a different host,
under different conditions, and producing a new
disease.
Oncogenic retroviruses
• Cancer is a genetic disease- oncogenesis consists of
the processes that result in growth of cells in which
mutations have accumulated
• Viruses are a contributing factor in about 20% of all
human cancers
• Growth properties and morphologies of cultured cells
could be changed upon infection with certain virusescells become transformed
• Cells become immortal in an early step in oncogenesisthey continue to grow and divide even though the body
has sufficient numbers of these cells
• They lose contact inhibition and the need to adhere to a
surface
• They look different, more rounded
Avian cells transformed with two strains of the Rous
sarcoma virus:
• Transformed cells grow to high densities, they grow on
top of untransformed cells forming clumps or foci
• They lose the need to adhere to a surface, can grow in
agar
• They are more rounded and look different from normal
cells
The phases of a eukaryotic cell cycle:
Errors in the signaling pathways that regulate cell cycle
progression can lead to cancer
Oncogenic viruses
• Oncogenesis is the result of
genetic changes that alter the
expression or function of proteins
that play critical roles in the control
of cell growth and division
•Oncogenic viruses cause cancer
by inducing changes that affect
cell growth and division
• Cancer arises from a
combination of dominant gain of
function mutations in protooncogenes and recessive loss of
function mutations in tumor
suppressor genes
• Oncogenic viruses were discovered by Ellerman and
Bang in 1908 who showed that avian leukemia can be
transmitted by filtered extracts of leukemic cells
• In 1911 Peyton Rous showed that solid tumors can be
produced in chicken using cell free extracts
Oncogenic retroviruses are classified into two groups:
1) Transducing oncogenic retroviruses:
- highly carcinogenic, cause malignancies in 100% of the
infected animals in a matter of days
- cause cancer because their genomes contain transduced
cellular genes that become oncogenes
-virally transduced versions of cellular genes are called voncogenes, their cellular counterparts are called concogenes or proto-oncogenes
2) Nontransducing oncogenic retroviruses:
-less carcinogenic
-do not encode cell derived oncogenes
-activate transcription of proto-oncogenes by integration of
the provirus close to these genes in the host genome
Properties of viral transforming genes
• Results with Rous sarcoma virus showed that
transformation and viral replication are distinct
processes
• With the exception of RSV, these viruses are all
replication defective
• Defective transducing viruses can be propagated in
mixed infections with replication-competent helper
viruses
• In many transducing retroviruses, the viral and
cellular protein coding sequences are fused
• In most cases, the captured oncogenes have
undergone additional changes that contribute to their
transforming potential
Possible mechanisms of oncogene capture by retroviruses
Mechanisms of transformation by oncogenes
• The discovery that the transforming gene of RSV
was a transduced cellular gene led to identification
of cellular proto-oncogenes and the pathways in
which they function
• Mutations introduced into these genes during or
following capture into retroviral genomes lead to
constitutive activation of signaling
• Viral transformation can be the result of either
constitutive activation of cytoplasmic signal
transduction cascades or disruption of nuclear
pathways that negatively regulate cell cycle
progression
Family Flaviviridae
Family Flaviviridae
Hepatitis C virus
IRES
3’ ends of flavivirus RNAs are not polyadenylated
HCV Infection
• HCV first identified in 1989
• HCV first identified in 1989
• Blood-borne infection is often subclinical, despite
• Blood-borne infection is often subclinical, despite
persistent and progressive inflammation and fibrosis
persistent and progressive inflammation and fibrosis
of the liver, resulting in liver cirrhosis, hepatic failure
of the liver, resulting in liver cirrhosis, hepatic failure
and hepatocellular carcinoma
and hepatocellular carcinoma
• It is estimated that HCV has infected more than 170
• It is estimated that HCV has infected more than 170
million people globally -- nearly five times more than
million people globally -- nearly five times more than
HIV-infected individuals
HIV-infected individuals
HCV Infection
• HCV establishes a chronic infection in ~85% of cases
• HCV infection has become the most common cause
of liver cancer and the primary reason for liver
transplants among adults in western countries
• Currently no broadly effective anti-HCV therapies are
available
Difficulties in treatment
• Persistence of virus
• Genetic diversity during replication in the host
• Development of drug-resistant viral mutants
• Lack of reproducible infectious culture systems and
small-animal models for HCV replication and
pathogenesis
HCV Genome and Lifecycle
• Enveloped single-stranded RNA Hepacivirus in the
Flaviviridae family
• Closely related human viruses include GB virusC
(GBV-C), hepatitis H virus, yellow fever virus and
dengue virus
Cap-dependent vs.
cap-independent translation
initiation
40S
40S
4B
Cap-Dependent
Cap-Independent
Where internal ribosome entry sites
(IRESs) are found
• Picornavirus
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Poliovirus
Hepatitis A
Apthovirus
Rhinovirus
Encephalomyocarditis
virus
• Flavivirus
– Yellow Fever Virus
– GBV-B
– HCV
• Pestivirus
– BVDV
– CSFV
• Eukaryotic mRNA
– Fibroblast growth factor 2
– Insulin-like growth factor 2
– Platelet derived growth
factor 2
– c-myc
– BiP
– Ornithine decarboxylase
The 40S ribosomal subunit is part of
the 43S particle
HCV 5’
untranslated
region
• One of the most
conserved
regions of
genome
• Extensive
secondary
structure
Complex of the 40S subunit with the
hepatitis C virus IRES
• HCV IRES binds
directly to the 40S
subunit
• Binding of HCV
IRES induces a
conformational
change in the 40S
subunit of the
ribosome