Retro viruses

Dr. Anita Devi K Ravindran

MBBS, MD, Dip Cert Med Edn

Learning Outcomes
At the end of the lecture, the student should be able to:
Describe the structure and elaborate on the important
structural and functional genes associated with HIV.
Discuss the lab diagnostic tests commonly used to diagnose HIV
infection.
List and discuss briefly the clinical stages in the progression of
HIV AIDS.
Recount and explain the drugs used in the treatment and
methods of prevention of HIV.
Describe the epidemiological features of HIV.
Discuss the structure, patho physiology , lab diagnosis of HTLV
induced diseases & discuss the application of retroviruses as
viral vectors in research and therapy.

Interest in Retro viruses

Human Immunodeficiency Virus
Tumorogenic/ oncogenic viruses
Viral vectors

Retro viruses
RNA viruses
positive sense, enveloped, icosahedral.
Distinguished from all other RNA viruses by
presence of an unusual enzyme, reverse
transcriptase. Retro = reversal
Reverse transcriptase - RNA dependant DNA
polymerase
RNA is serving as a template for DNA synthesis.
The reverse transcriptase is highly error
prone and rapid genetic variation is a feature
of this group.

Retro viruses
Viral RNA

Viral DNA
(integrated into host genome)

Viral mRNA,
genome

Reverse (retro) transfer of genetic information

Reverse transcriptase - is an enzyme that

functions as a RNA-dependent DNA
polymerase.
Reverse transcriptases are encoded by
retroviruses, where they copy the viral RNA
genome into DNA prior to its integration into host
cells.

The viral genome

The Retrovirus is made of 3 main genes gag, pol
& env that are required for virus replication.
Long terminal
repeat
LTR

RNA dependant DNA

polymerase
GAG

POL

Group specific antigens

(nucleocapsid)

ENV

LTR

Envelope proteins
(type-specific antigens)

All three genes - GAG, POL, ENV - required for replication

Retroviruses
Retroviruses can also cause cancer in variety of
animals and humans.
A retrovirus can transform cells from normal to
cancer if they include a specific gene that is
capable of inducing cell transformation. This gene
is known as Oncogene.

Retrovirus
Cancerous
Retrovirus

Oncogene

Retroviridae
Feline leukemiavirus
Human T lymphocyte virus HTLV
Oncovirinae

Lentivirinae

Avian leukosis viruses

Feline, bovine, human
immunodeficiency viruses
Equine infectious anemia virus

Spumavirinae

Foamy agents

Human retroviruses
Four human retroviruses have been identified.
All infect CD4 bearing cells.
These were only identified in the 1980's when it
became possible to culture T-cells in vitro.
HTLV1 - T-cell leukaemias/lymphoma.
Tropical spastic paraparesis
HTLV2 - No known pathology
HIV 1 & 2 - AIDS

Replicative cycle
receptor

uncoating
RNA
viral dsDNA integrated
into host genome

blocked
receptors

ds DNA

mRNA

proteins

pro-virus
genomic RNA

assembly

HIV

Acquired immunodeficiency syndrome

(AIDS) was first reported in US in 1981.

By 1984, AIDS was recognized as an

infectious disease caused by a virus.

Origin of HIV
New York & Canada Sudden increase in 2 rare
disease Pneumocystis carinii pneumonia &
Kaposis sarcoma
Homosexuals & heroin addicts
Hemophiliacs on factor VIII injections, vertical
transmission & sexual transmission in families .
Luc Montagnier & co 1983 isolated a
retrovirus from a West african patient with
Persistent Generalized Lymphadenopathy
LAV
Robert Gallo 1984 isolated retrovirus in AIDS
HTLV -III

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

Structure

Electron microscopic structure

Structure
The viral envelope formed from host cell membrane;
contains 72 spiked knobs.
These consist of a transmembrane protein TM (gp 41),
which is
linked to surface protein SU (gp 120) that binds to a cell
receptor during infection.
The virion has cone-shaped, icosahedral core,
containing the major capsid protein CA (p24).
Between capsid and envelope is an outer matrix protein,
MA (p17).
Two identical copies of positive sense ssRNA genome
(retroviruses are diploid).
Enzymes: reverse transcriptase, integrase and
protease.

Main antigens of HIV

A. Envelope antigens

B. Shell antigen

1. Spike antigen gp 120

2. Transmembrane pedicle
protein gp 41
1. Nucleocapsid protein p 17

C. Core antigens

1. Principal core Ag p 24
2. Other core Ags p 15, p 55

D. Polymerase
antigens

1.
2.
3.

p 31
p 51
p 64

Genes of HIV

Gene

Function

gag

(structural)

Core proteins

pol

(structural)

enzymes

env

(structural)

Envelope protein

tat

(functional)

Positive regulator

nef

(functional)

Negative regulator

rev

(functional)

Regulator of viral protein

synthesis

vif

(accessory )

Viral infectivity factor

vpr

(accessory)

Hold cell in G2 phase, aids

attaching to nuclues

vpu/ vpx (accessory )

HIV 1 / HIV 2
Aids virion relase; degrades
CD4

HIV Replication
The first phase of viral entry, reverse transcription
and integration into host genome is accomplished
by viral protein.
The second phase of synthesis and processing of
viral genomes, mRNAs and structural proteins,
uses host cell machinery.
Attachment to specific cell surface receptor:
gp120 binds CD4 molecule on the helper T cells,
monocytes ,dendritic cell and macrophages
This triggers binding of gp 40 to co-receptors
Viral entry.

HIV Replication
Reverse transcription of viral RNA into DNA.

The resulting double stranded DNA is

called provirus.
Integration of provirus into host cell DNA.

The viral integrase cleaves the chromosomal

DNA and covalently inserts the provirus.
Transcription and translation of viral DNA
sequences.
Assembly and maturation of progeny virus.

Role of Viral Receptors and CoReceptors in HIV Pathogenesis

HIV-1 utilizes two major co-receptors along with
CD4 to bind, fuse and enter CD4 cells.
CCR5 and CXCR4 ( endogenous chemokine
receptors)
Strains of HIV that utilize CCR5 R5 viruses
Strains of HIV that utilize CXCR4 X4 viruses.
Dual tropic ( utilize both CCR5 and CXCR4)
R5X4 viruses

conformational change in
the gp120 molecule allows
it to bind to the co-receptor
CCR5

virus then firmly attaches to the

host cell membrane in a coiledspring fashion via the newly
exposed gp41 molecule
Harrison's Principles of
Internal Medicine, 18e, 2012
Copyright 2012 McGraw-Hill Medical. All rights reserved.

Transmission of HIV
Sexual contact: HIV is present in semen and
vaginal secretions; either homosexual or
heterosexual contact

Transfusions: whole blood, plasma, clotting

factors or cellular fractions of blood.
Contaminated needles: accidentally or sharing
needles by drug users.
Perinatal: Transplacental, during delivery or via
breast milk.

AIDS(Transmission)

Pathogenesis and clinical significance

Initial infection:
genital tract macrophages
HIV disseminates via blood
Dendritic cells in lymphoid tissue
CD4+ lymphocytes

Events that transpire from primary HIV infection through the

establishment of chronic persistent infection to the ultimate
destruction of the immune system.

Clinical course of HIV disease

Primary Infection
Acute HIV syndrome ( acute retroviral syndrome)
Clinical latency / asymptomatic stage
Acquired Immunodeficiency syndrome AIDS
(Opportunistic disease)

Pathogenesis and clinical significance

Acute phase viremia( Acute HIV infection):
Several weeks after the initial infection
1/3 2/3 of individuals experience an acute
disease syndrome fever, lymph node
enlargement (similar to infectious mononucleosis).
Circulating antibody appears in 1 10 weeks after
the initial infection (seroconversion).

Pathogenesis and clinical significance

Latent period ( Asymptomatic infection):
lasts from months to many years (average 10
years).
90% of HIV proviruses are transcriptionally silent.
Although there is continous loss of CD4+ cells in
which HIV is replicating, active replacement through
stem cell multiplication is occurring.

The infection remains clinically asymptomatic as

long as the immune system is functional.

Pathogenesis and clinical significance

Persistent Generalized Lymphadenopathy :
-Enlarged lymph nodes in 2 or more extra inguinal
sites, persists for more than 3 months.
AIDS Related Complex (ARC)
The CD4+ cell count remains normal or
gradually declines but is greater than 200 / ul.
Diarrhea, chronic fevers, night sweats and weight
loss.
Common opportunistic infections (herpes zoster
and candidiasis) may occur repeatedly during
this period.
Hairy cell leucoplakia, TB, salmomellosis

Progression to AIDS in few months.

Pathogenesis and clinical significance

AIDS:

appearance of serious diseases and

opportunistic infections.
CD4+ count falling below 200 / ul
Coinfection with HHV-6 can transactivate
transcription from the silent HIV provirus,
increasing HIV replication.
Any stimulation of an immune response causing
activation of resting T cells also activates HIV
replication.
Appearance of HIV mutants with altered
antigenic specificity which are not recognized
by the existing antibody or cytotoxic T
lymphocytes.

Opportunistic Infections (1)

Bacteria:
Mycobacterium avium complex
Disseminated miliary disease
Chronic bronchitis, pneumonia
Chronic osteomyelitis, renal infection
Mycobacterium tuberculosis
Chronic pneumonitis, osteomyelitis
Meningitis, miliary disease
Streptococcus pneumoniae
Salmonella spp.
Haemophilus influenza (pneumonia)
Campylobacter spp. (diarrhea)
Shigella spp. (diarrhea)

Opportunistic Infections (2)

Fungi:
Candida spp. Oral, vaginal or systemic
candidiasis
Histoplasma capsulatum (disseminated
disease)
Cryptococcus neoformans (meningitis)
Pneumocystis jirovecii
Unicellular eukaryote
Taxonomic status is uncertain
Most common opportunistic pathogen in
AIDS patients
Fatal pneumonia

Parasites:
Toxoplasma gondii (focal encephalitits)
Isospora belli
Gay bowel syndrome c/c colitis ( giardia, ameba)
Viruses:
HHV-8 (Kaposis sarcoma-associated herpesvirus)
HSV (oral, genital ulcers)
JCV (progressive multifocal leukoencephalopathy,
affect the white matter of the brain)
CMV (Chorioretinitis, encephalitis, enterocolitis,
gastritis)
Cutaneous:
Xeroderma, folliculitis, seborrhoeic dermatitis
Dementia :
Direct cytopathogenic damage to CNS

Opportunistic Infections

Malignancies associated with AIDS

Kaposis sarcoma
HHV-8
Lymphomas
EBV
Kaposis sarcoma:
Characteristic lesion in male homosexuals.
Indolent, mucosal/ cutaneous tumor.
Non metastasizing ; endothelial in origin.

Kaposi Sarcoma

Laboratory Diagnosis
Antigen / antibody detection

ELISA using serum

HIV-1 & -2 antibodies, HIV-1 CA (p24) antigen
Screening of blood donors
Western Blotting

PCR
Viral RNA or DNA provirus
Blood or tissue specimens
Quantitative PCR (viral load): to determine disease
stage and treatment follow up.

Primary HIV infection

precedes seroconversion,
and therefore testing limited
to viral antibodies may
initially convey negative or
borderline results.
The infection is
characterised by a high
plasma virus load, and
direct testing for viral antigen
(p24 antigen) or nucleic acid
(HIV RNA) usually confirms
the diagnosis.
Although p24 antigen
testing is less sensitive
than HIV RNA (88.7% v
100%), it is usually preferred
in non-specialist settings
because it is less expensive
and associated with fewer HIV RNA testing
p24 antigen testing
false positive results.
Viral load

WINDOW PERIOD

ELISA for HIV antibody

The window period is
the time from infection
until a test can detect
any change.
The average window
period with HIV-1
antibody tests is 25
days for subtype B.

Microplate ELISA for HIV antibody: coloured wells

indicate reactivity

Antigen testing (p24)

cuts the window period
to approximately 16
days and NAT (Nucleic
Acid Testing) further
reduces this period to
12 days.

Western blot for HIV antibody

The most important
antibodies are those
against the envelope
glycoproteins gp120,
and gp41

Is the confirmatory test

for HIV. ( More specific)
Done once ELISA test
is positive ( More
sensitive).

Viral Load & CD4 count in HIV

Treatment
Anti-retroviral drugs
Reverse transcriptase inhibitors
Protease inhibitors

Multidrug therapy
RT has no proofreading activity, resulting in production of many
errors during viral DNA synthesis which leads to mutations in all
HIV genes and accumulation of mutant viral strains. In presence
of an antiviral drug, there is strong selection for mutations
that confer resistance to that drug.

Early therapy
Viral load is a prognostic indicator of the rate of progression to
AIDS. Infection should be treated as aggressively and as early
as possible to minimize initial spread of the virus and give a
lower chance for mutants to arise.

Highly active antiretroviral therapy (HAART)

Nucleoside analog reverse transcriptase inhibitors

Act by serving as a chain terminator

Zidovudine (AZT)
Didanosine (ddi)
Lamivudine (3TC)

Non-nucleoside reverse transcriptase inhibitors

Act by targeting the enzyme itself

Efavirenz
Delaviridine
Nevirapine

Protease inhibitors

Interfere with the processing of polyproteins in the budding

virion, resulting in non-infectious particle.

Ritonavir
Amprenavir
Indinavir
Lopinavir

Prevention
Vaccine: not yet available
Blood supply screening
Perinatal transmission: AZT therapy
Sexual preference/ practices

ONCOVIRINAE
Tumor viruses and those with similar
morphology
First member of this group to be discovered was
Rous Sarcoma Virus (RSV) - which causes a
slow neoplasm in chickens.
Viruses in this group that cause tumors in
humans are:
HTLV -1
HTLV -2

Retroviruses and cancerMechanisms

Transduction of oncogenes
GAG

POL

LTR

LTR

src

Activation of oncogenes
GAG

POL

ENV

LTR

myc

LTR

Inactivation of
tumour-suppressors
GAG

Rb

LTR

POL

ENV
LTR

Rb

HTLV
Genetically and biologically similar
HTLV-1
Adult T-cell leukemia ( Sezarys T cell
leukemia)
HTLV-associated myelopathy/tropical
spastic paraparesis

HTLV-2
Hairy cell leukemia

HTLV -1
Originally isolated by Gallo from an African
American patient.
This disease is found in

some Japanese islands,

the Caribbean,
Latin America
Africa.

Transmission
Sexually transmitted
- common among sex workers.
Mother to child transmission rate of 10%
Intravenous drug users
Prevalence of HTLV-1 infection in HIV-infected
patients is about five times higher

HTLV-1 (human T-cell lymphotropic

virus-1)
causes Adult T-cell Leukemia (Sezary T-cell
Leukemia).
The virus is also associated with
- tropical spastic paraparesis
( neuromyelopathy )
- infective dermatitis.
- arthritis and polymyositis

Opportunistic Infections in HTLV-1

Staphylococcus species,
Klebsiella species or
Bordetella pertussis
which can cause bronchiectasis, bronchial
inflammation and necrosis., leading to pneumonia

Staphylococcus aureus leading to sepsis

Helminth infections

HTLV-2 (human T-cell lymphotropic

virus-2)
causes Hairy Cell Leukemia
virus is endemic to very specific regions of the
Americas, particularly in native American
populations.

Hairy cell leukemia

a rare lymphocytic
leukemia,
of B cell origin;
caused by HTLV-2.
it is characterized
by malignant cells
that look ciliated.

Laboratory diagnosis

Screening of blood donors using ELISA

Confirmation by western blotting
PCR

Retro/ Lenti viruses are Viral

Vectors

Infectious Viruses: A Genetic Syringe

Viruses are composed of genetic
material encapsulated in a
protein coat.

DNA
Loaded
Syringe

Viruses inject their genetic

material into target cells.
Viruses infect target cells with their genetic material.
The viral DNA can be altered to contain a gene of interest (rDNA)
to infect that gene into the target cell.
Viral DNA
Gene of Interest

Target Cell

Virus

Cells DNA

Target Cell Infected With

Viral DNA Containing The
Gene of Interest

Replication Deficient Viral Vectors: Genetically

Engineered So The Viral Infection Cannot Spread
The viral DNA does not contain the viral genes needed to make
more viruses.
Viral DNA
Gene of Interest

Target Cell

Virus

Target Cell Infected With

Viral DNA Containing The
Gene of Interest

Cells DNA

No New Viral Particles are

Created
Infection dose not spread

Problems : Insertional Mutagenesis

Oncogenesis

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