Retroviruses, HIV/AIDS

By the end of today’s class, you should be able to:

1. Explain what is a retrovirus and identify members associated with the
retrovirus family
2. Discuss the structure and mode of replication of HIV and HTLV
3. Discuss the mode of transmission and mechanisms involved in the
pathogenesis of HIV and HTLV
4. Discuss the immunopathogenesis of HTLV and HIV infections
5. Explain the clinical syndromes associated with HIV and HTLV and progression of
HIV infection to AIDS
6. Discuss the role of various laboratory techniques in the diagnosis of HTLV and
HIV/AIDS
7. Discuss the mechanism of action of various antiviral compounds utilized in the
treatment and prevention of HIV/AIDS
8. Discuss the role of various opportunistic infections in HIV/AIDS

March 26 - 27, 2018 ♦SUSOM ♦ Dr. Rachel L. Robson

ame Retroviruses.

RNA VIRUSES
Nonenveloped Enveloped

Single stranded Single stranded Single stranded

Double stranded Retrovirus
Positive sense Positive sense Negative sense
Reoviruses Lenti (slow) vir
Astroviruses Togaviruses Linear HIV
Rhabdoviruses Oncoviruses
Rotaviruses Flaviviruses HTLV-1
Caliciviruses
Filoviruses

Coronaviruses Bornaviruses
Picornaviruses
Paramyxoviruses

Segmented
Arenaviruses

Bunyaviruses

Orthomyxoviruses
 What characterizes the retroviruses?

 The name is derived from the fact that the

virus particle contains an RNA-dependent
DNA polymerase, a.k.a reverse transcriptase.

 This enzyme converts the RNA genome into

DNA, which then integrates into the host
chromosomal DNA.
 Retrovirus-Classification
Subfamily Characteristics Example

Oncovirinae Oncogenic, are associated with

cancer and neurologic disorders.
 Transducing viruses, normally
replication defective (exception RSV).
 Non-transducing viruses: Replication
competent.
 Non-transducing, long-latency
viruses: Replication competent.
Rous sarcoma virus (RSV)
Mouse mammary tumor virus
(MMTV)
Human T-cell leukemia virus
(HTLV)

Lentivirinae Slow progressive Human

neurologic and immunodeficiency
immunosupressive virus (HIV)
diseases
Spumavirinae No pathology known Human foamy virus (HFV)
 Retroviruses can be either “simple” or
“complex,” depending on what genes they
have
 Simple retrovirus:

LTR-Gag-Pol-Env-LTR
bird viruses
LTR-Gag-Pol-Env-Oncogene-LTR

 Complex retrovirus:

Also encode regulatory proteins and accessory proteins that require

more complex transcriptional processing (splicing) in their genome
e.g. HIV, HTLV and HFV
  Transducing viruses
Host cell genome
LTR-Gag-Pol-Env-v-Oncogene-LTR

Ex:Tyrosine kinase: src (Rous sarcoma virus)

re not in humans Transcription factor: Myc (Avian myelocytomatosis virus)
lecular biology  Growth factor receptor: erb-B (Avian erythroblastosis virus)
G protein: ras (Harvey murine sarcoma virus)
Rapidly transform the cells
 Non-transducing viruses (days), efficiency high

LTR-Gag-Pol-Env-LTR c-Oncogene
Insertion upstream, downstream or
Transform the cells
In the oncogene gene itself.
Ex: Avian leukosis viruses, insertional activation of c-Myc but less rapidly
(weeks, months),
efficiency high to
 Non-transducing, long-latency viruses
intermediate
ses LTR-Gag-Pol-Env-pX-LTR
move
Transform the cells
ell to another cell.
but very slow
Viral protein with
(months, years),
use cancer because oncogenic properties
efficiency very low
on of these genes. Ex: Tax from HTLV-1
 Retrovirus structure
(2) Size of a virion: 80-120
nm in diameter.
A typical, "minimal"
retrovirus consists of:
 an outer envelope (1)
that is derived from the
(3) plasma membrane of its
host.
 many copies of
(1) envelope proteins (2)
embedded in the lipid
(4) bilayer of its envelope
 a capsid (3); a
protein shell containing
two molecules of RNA
(5) (4) (single stranded),
molecules of the
enzyme reverse
transcriptase (5).
 Nomenclature for common retrovirus proteins
env
5’ LTR gag pol 3’ LTR
U3 R U5 U3 R U5
PR

Gene Name, Protein Function

gag
MA, Matrix matrix protein; lines envelope

ed capsid protein; protects the core; most abundant protein in virus

CA, Capsid
particle

NC, Nucleocapsid nucleocapsid protein; protects the genome; forms the core

pol PR, Protease Essential for gag protein cleavage during maturation
Reverse transcribes the RNA genome; also has RNAseH
RT, Reverse transcriptase
activity

d IN, Integrase Needed for integration of the provirus

SU, Surface glycoprotein The outer envelope glycoprotein; major virus antigen
env
TM, Transmembrane protein The inner component of the mature envelope glycoprotein
Contains control regions that bind host transcription factors
LTR Long Terminal Repeat
Required for the initiation of transcription
es
Retrovirus-Infectious cycle

Goff SP, Nature Reviews Microbiology 2007

Transition from the RNA genome to the proviral DNA genome

Genomic RNA + tRNA primer

Reverse Transcriptase
DNA

Degradation of gRNA (RNAseH activity of RT)

ds DNA synthesis
Integrase
Permanent integration into the host cell genome: provirus
Cellular RNA polymerase II

RNA gRNA

Viral proteins

Virion
Retroviral genome before and after integration
Viral RNA genome
5’ gag pol env 3’
RNA (+) CAP AAAAAAAA
R-U5 U3-R

Reverse transcription
Chromosomal integration (random)

U3 R U5 gag pol env U3 R U5

5’ LTR 3’ LTR
Integrated provirus DNA
 All retrovirus genomes consist of two molecules of
ssRNA that are (+)sense and have a 5' cap and a 3' poly-
(A) (equivalent to mRNA).

 These vary in size from ~8-11kb.

 Human T-cell Leukemia Virus,
type 1 and type 2 (HTLV-1 and HTLV-2)
 Endemic: SW Japan, W Africa, Caribbean basin, S America,
Southern US
 Infects mature CD4+ T-cells
 Associated with two major diseases:
-Adult T-cell leukemia (ATL)
-HTLV-1 associated myelopathy or tropical spastic
paraparesis (HAM/TSP)
 20 million people infected worldwide
 Only 5% of this population will develop a disease associated with
HTLV-1
 Transmission occurs by breast feeding, blood transfusion and sexual
transmission
 Low genetic variability (<10%)
 HTLV-2 is not linked to any disease (HTLV-3 and 4)
How does HTLV-1 transform the cell?
• The HTLV-1 genome does not encode for an oncogene similar to
a known cellular proto-oncogene

• HTLV-1 does not systematically deregulate a cellular gene by

integrating into a specific site

• Tax exhibits oncogenic properties (Tax is unique to HTLV-1):

– transforms rodent fibroblast

– immortalizes human peripheral T lymphocytes
– Tax transgenic mice develop diverse tumors
Adult T-cell leukemia/lymphoma (ATL)
Characteristics Healthy Smoldering Chronic Acute Lymphoma
carrier (5%) (19%) (57%) (19%)
Serology + + + + +

Lymphocytose Normal Normal High High Normal

Abnormal cells (%) none <5% >10% >10% <1%

Hypercalcemia - - - + +
LDH Normal Normal Normal High High

Skin and lung - + + + +

lesions

Impaired hepatic - - + + +
functions

Bones and brains - - - + +

lesions
Adult T-cell leukemia/lymphoma (ATL)
• Characterized by an abnormal proliferation of
mature CD4+ T-cells
• Develops ~40 years following infection
• Lytic bone lesions in the skull and long bones
• Skin lesions due to infiltration of leukemic cells
• Unresponsive to aggressive chemotherapy
• Median survival ~6 months

From Manns et al., Lancet. 1999

• Abnormal T-lymphocytes (flower cells) in ATL

From Dr. John Krause’s Hematopathology Homepage

HTLV-1 associated myelopathy/tropical spastic
paraparesis (HAM/TSP)
Clinical features of HAM/TSP:
1. Leg weakness and spasticity; difficulty walking
2. Backache, leg aches
3. Urinary frequency or incontinence
4. Women 3X more likely to develop (unknown reason)
5. Develops much more rapidly following initial infection than ATL

The lifetime risk for development of HAM/TSP is ~5%. Caused by severe

demyelination of the nerves of the spinal cord.

Evidence suggests that HAM/TSP is an autoimmune disease.

HAM/TSP patients make antibodies against the viral protein Tax that cross
react with heterogeneous nuclear ribonuclear protein-A1 (hnRNP-A1) -
expressed in neurons of the spinal cord.

Is almost indistinguishable from multiple sclerosis.

 HTLV-Laboratory diagnosis
 Detect antibody: detection of anti-HTLV antibodies (ELISA and
Western blot),
 Identify antigen: detection of HTLV p19 antigen (ELISA or Western
blot)
 Detect or monitor viral nucleic acids: detection of proviral DNA (real-
time PCR) or detection of viral RNA (RT-PCR)

HTLV-Treatment
No effective treatments
Currently, aggressive chemotherapy-CHOP (Cyclophosphamide, an alkylating agent
which damages DNA by binding to it and causing the formation of cross-links.
Hydroxydaunorubicin (also called doxorubicin or Adriamycin), an intercalating agent
which damages DNA by inserting itself between DNA bases. Oncovin (vincristine),
which prevents cells from duplicating by binding to the protein tubulin. Prednisone or
prednisolone, which are corticosteroids.
Allogenic stem cell/bone marrow transplantation, interferon a and arsenic trioxide,
block NF-kB, monoclonals antibodies, antiretroviral treatment etc…
HIV/AIDS
HIV genes
 HIV proteins

Retrovirus
 HIV proteins

Polyprotein gp160 gp120 and gp41

ENV gp120-is present as surface knob- attachment

protein
gene gp41 is transmembrane protein - fusion

Enzymes
GAG gene • POL gene
• Polymerase (reverse transcriptase – RNA
p17: inner surface
dependent DNA polymerase)- p66/ 51

p24: nucleocapsid • Integrase- p32

p7: nucleocapsid • Protease (cuts polyproteins) -p10

associated with RNA
 HIV infection is initiated by virus using CCR5 (M-tropism, HIV-R5)
 strains that can infect CXCR4 cells emerge in the course of infection (T-
tropism, HIV-X4). Involved in syncytia formation.
 Interestingly, a 32 bp deletion within the exon of the CCR5 gene results in
almost complete protection against HIV infection and a slower progression
to AIDS in individuals homozygous for the allele.
 HIV-Attachment and Entry

CCR5
CXCR4

 CD4: receptor of HIV (necessary but not sufficient for HIV infection)

 CCR5 and CXCR4 (chemokine receptors) are the co-receptors of HIV

HIV
replication
cycle
 HIV-Clinical syndromes and disease progression
Infection by HIV produces three phases of disease:

 an early phase that immediately follows the infection (acute): characterized by a rapid viral
replication

Has few symptoms: flu-like/mononucleosis-like symptoms with fever, malaise,

lymphoadenopathy, pharyngitis, headache, myalgia and sometimes a rash

CD4 count

Anti-HIV-1
antibody

Viral
protein
  a middle phase: 2 weeks - 20 years (chronic)

produces few symptoms

A strong immune response will reduce the number of viral particles in the
blood stream, therefore the patient's blood contains few viruses. Detection of
antibodies to the virus possible.

CD4 count

Anti-HIV-1
antibody

Viral
protein
 The late phase, AIDS-related complex (ARC)/ and acquired immune deficiency syndrome
(AIDS)
- Decline in the number of CD4 T-cells: ~400 per ul the patients cell-mediated immunity is
lost and opportunistic infections appear. ARC.
viruses: CMV, herpes simplex, herpes varicella-zoster, EBV
fungi: Candida albicans (the cause of "thrush"), Pneumocystis jiroveci
protozoans: Toxoplasma gondii
- When the CD4 count drops below 200 per ul: AIDS. Infections become more severe,
cancers (Kaposi’s sarcoma [HHV-8] or lymphoma [Burkitt’s lymphoma – EBV]) and
dementia may develop.

CD4 count

Anti-HIV-1
antibody

Viral
protein
HIV-Infection and Immunity
Some characteristics of HIV-1 & HIV-2

 HIV-1 and -2 are the etiologic agents of AIDS

HIV-1: common worldwide

HIV-2: is more prevalent in West Africa, less virulent, but more
transmissable

 Both infect CD4+ T-cells and cells of the macrophage lineage

e.g. monocytes, alveolar macrophages of the lung, denditric cells

of the skin, microglial cells of the brain

HIV-1 has a higher tropism for CD4+ T-cells

HIV-2 has a higher tropism for macrophages/monocytes
 Some characteristics of HIV-1 & HIV-2

 Persistent low-level productive infection of macrophage lineage cells

(reservoir), resting CD4+ T-cells, CD34+ cells (multipotent hematopoietic
progenitor cells in bone marrow)

Virus causes reduction of CD4+ T-cells level

- impaired production of T-cells
- apoptosis
- CD8 cytotoxicity
- [lytic infection]

 High genetic variability due to RT (error prone, lacks the proofreading

capabilities of other polymerases)
Up to 10 mutations per replication cycle
 HIV-Laboratory
diagnosis
Tests can be classified into those that:
 Detect antibody: Detection of anti-
HIV antibodies (ELISA and
Western blot), may not be positive
for several weeks following
symptoms. The most widely
used and most effective way to
identify HIV infection.
 Identify antigen: Detection of HIV
p24 antigen (ELISA or Western
blot)
 Detect or monitor viral nucleic
acids: Detection of viral RNA (RT-
PCR)
 Provide an estimate of T
lymphocyte numbers (cell
phenotyping), CD4 and CD8 ratio
(2:1 normally, less than 1 in
infected patients).
Pattern and incidence of opportunistic infections

You will not get

cryptococcosis,
toxoplasmosis,
CMV retinitis, etc
unless you are
immunocompromised.
CD4 cell count <200
 HIV-Treatment
Treatment actually used: Highly Active Anti-retroviral Therapy or (HAART)
Combined therapy usually with three drugs: two that target the reverse
transcriptase and one that targets the viral protease.

Reverse transcriptase inhibitors: Nucleoside analogs that the reverse

transcriptase incorporates into the growing DNA strand but then halts further DNA
synthesis. Example: Azidothymidine (AZT) or Zidovudine (ZDV, Retrovir®).

Non-nucleoside reverse transcriptase inhibitors inhibit the enzyme by other

mechanisms (alter the binding site).

Protease Inhibitors: These block the viral protease so that the proteins needed
for assembly of new viruses cannot be cleaved from the large protein precursor
(Ritonavir®).

Fusion Inhibitors: These inhibit the allosteric change that enables gp41 to
penetrate the host plasma membrane. You can also block CCR5.
Azidothymidine (AZT) is a reverse transcriptase-
inhibiting nucleotide analog. It was among the first
drugs used to treat HIV, and is still one of the most
commonly used drugs for HIV.
 HAART has dramatically improved
outcomes for HIV+ patients, such that life
expectancy for HIV+ patients on HAART is
* almost the same as for HIV- people.
HIV+ patients can now have viral loads
* * reduced to undetectable and likely
untransmissable levels because of HAART.
Steps of HIV replication
targeted by different anti-
retrovirals
 Problems with HAART:
• Cannot clear the virus from an infected individuals
• Cannot stop taking the drugs because of the reservoirs of the virus
• Drug resistant viruses evolve
• Drugs are expensive, limiting access and reducing compliance
Currently, about 53% of HIV-infected people are
receiving treatment with highly-active anti-retroviral
therapy (HAART)
In the USA, HIV infections are more common
among MSM than women or MSW. Worldwide,
however, heterosexual sexual intercourse is the
most common means of HIV transmission.
 HIV-Transmission
Blood and  Blood transfusions. This transmission practically disappeared in
blood United States, however remains a problem in countries were
products screening is not stringent
 Intravenous drug abusers (sharing of needles)
 Health care workers: needle stick injuries (risk approximately
0.36%), open wound and mucocutaneous exposure (depends on
the injury)
Sexual Anal and vaginal intercourse exposure
intercourse Prevention: abstinence, monogamy, latex/nitrile condoms, and pre-
or post-exposure prophylaxis (antiretroviral treatment); male
circumcision reduces risk of transmission.

Perinatal Infection may occur:

transmission  In utero
 During birth
 Post-natal, through breast feeding
Prevention: antiretroviral treatment to the mother, Caesarean
section
Pre-exposure prophylaxis (PrEP) for HIV refers to the
administration of a low dose of HAART drugs to HIV-
negative people who are at increased risk of
contracting HIV (e.g., sexual partners of HIV+ people;
injecting drug users). Trade name = Truvada