Retroviruses 205

8/14/2019 Retroviruses 205

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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


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Strand displacement assimilation model proposes that

recombination occurs during (+) strand DNA synthesis


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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


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Retroviral DNA Integration

6 bp target site

is duplicated on

either side of

proviral DNA

Viral DNA is

shortened by 2

bp at each end


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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 samedinucleotide: 5-TG----CA-3

This dinucleotide is found in an inverted repeat

characteristic of the virus

The inverted repeat, conserved terminal dinucleotidesequence 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

Retroviral DNA Integration


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Endonucleolytic nicking andremoval of 2 nt and formation of a

new 3 recessed end

Joining of 3 ends tophosphates at the target site Gapped intermediate Gaps arerepaired

The two ends of viral DNA are

recognized, nicked and then joined

covalently to host DNA in randomlocations at staggered nicks also

introduced by Integrase

Steps in retroviral DNA integration


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Most retrotransposons are distinguished from retroviruses

by lack of an extracellular phase

They have no envgene, thus virus like particles formed in

vivo are noninfectious


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Retrovirus assembly

Association of Gag molecules with the plasmamembrane 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


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Retrovirus assembly


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Phylogenetic relationships among

retroviruses


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The Origin of HIV

HIV-1 probably originated from

SIVcpz in chimpanzees less than

100 years ago. HIV-2, a virus stilllargely confined in Western Africa,

probably originated from SIVsm inMangabes.


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http://www.cdc.gov/ncidod/EID/index.htm


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Similarities and differences between

Lentiviruses Similarities 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 systemdysregulation; 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, underdifferent conditions, and producing a new disease.


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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

Avian cells transformed with two strains of the Rous

sarcoma virus:


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The phases of a eukaryotic cell cycle:

Errors in the signaling pathways that regulate cell cycle

progression can lead to cancer


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Oncogenesis is the result of

genetic changes that alter the

expression or function of proteinsthat play critical roles in the

control of cell growth and division

Oncogenic viruses cause cancer

by inducing changes that affectcell growth and division

Cancer arises from a

combination of dominant gain of

function mutations in proto-

oncogenes and recessive loss of

function mutations in tumor

suppressor genes

Oncogenic viruses


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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


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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 v-oncogenes, their cellular counterparts are called c-

oncogenes 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


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Properties of viral transforming genes

Results with Rous sarcoma virus showed that

transformation and viral replication are distinctprocesses

With the exception of RSV, these viruses are all

replication defective

Defective transducing viruses can be propagated inmixed 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

ibl h i f b i


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Possible mechanisms of oncogene capture by retroviruses


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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 nuclearpathways that negatively regulate cell cycle

progression

Mechanisms of transformation by oncogenes


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Family Flaviviridae


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Family Flaviviridae

IRES

Hepatitis C virus

3 d f fl i i RNA t l d l t d


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3 ends of flavivirus RNAs are not polyadenylated


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HCV first identified in 1989

Blood-borne infection is often subclinical, despitepersistent and progressive inflammation and fibrosisof the liver, resulting in liver cirrhosis, hepatic failureand hepatocellular carcinoma

It is estimated that HCV has infected more than 170million people globally -- nearly five times more thanHIV-infected individuals

HCVfirst identifiedin 1989

Blood -borneinfectionis often subclinical , despite

persistent andprogressive inflammationandfibrosisof theliver, resulting inliver cirrhosis, hepatic failureand hepatocellular carcinoma

It is estimatedthat HCVhas infected morethan 170

million people globally -- nearly fivetimes morethanHIV-infected individuals

HCVInfection


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HCV Infection

HCV establishes a chronic infection in ~85% of cases

HCV infection has become the most common causeof liver cancer and the primary reason for livertransplants among adults in western countries

Currently no broadly effective anti -HCV therapies are

available


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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 andsmall-animal models for HCV replication andpathogenesis


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HCV Genome and Lifecycle

Enveloped single-stranded RNA Hepacivirus in theFlaviviridae family

Closely related human viruses include GB virusC(GBV-C), hepatitis H virus, yellow fever virus anddengue virus


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Cap-dependent vs.

cap-independent translationinitiation

Cap-Dependent Cap-Independent

40S40S40S

4B

40S40S


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Where internal ribosome entry sites

(IRESs) are found

Picornavirus Poliovirus

Hepatitis A

Apthovirus

Rhinovirus

Encephalomyocarditisvirus

Flavivirus

Yellow Fever Virus

GBV-B

HCV

Pestivirus BVDV

CSFV

Eukaryotic mRNA

Fibroblast growth factor 2

Insulin-like growth factor 2

Platelet derived growthfactor 2

c-myc

BiP

Ornithine decarboxylase


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The 40S ribosomal subunit is part of

the 43S particle


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HCV 5

untranslated

region

One of the most

conserved

regions of

genome

Extensive

secondarystructure

Complex of the 40S subunit with the


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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 40Ssubunit of the

ribosome

Retroviruses 205

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