Retroviridae
• The Retroviridae are a family of enveloped (+) sense ssRNA
viruses that have been intensely studied because of their
association with cancers, leukemias and the AIDS syndrome
• The first association of viruses with cancer was in early
1900’s with the discovery by Ellerman and Bang that
leukemia could be transmitted from one chicken to another
by injecting leukemia cell extracts
• In 1911 Peyton Rous showed that a bacterial free filtrate
from solid tumors of chickens could cause an identical
cancer in chickens inoculated with the filtrate
• The virus causing the leukemia was subsequently shown to
be avian leukosis virus and the virus causing tumors was
designated Rous sarcoma virus
Retroviridae
• Although the discoveries by Ellerman, Bang and Rous
were not well accepted at the time, 60 years later these
viruses were designated retroviruses and Rous won the
Nobel Prize for his work in 1963 at the age of 83
• In early 1970, Baltimore and Temin independently
identified the unusual enzyme, reverse transcriptase and
won the Nobel Prize in 1975 for their work
• Their discovery shattered the central dogma of molecular
biology which stated the flow of genetic information was
from DNA to RNA
• In 1989, Bishop and Varmus won the Nobel Prize for
elucidating that retroviral oncogenes are derived from
cellular genes and brought us closer to understanding
cancer
History
• Vallee and Carre (1904) - Filterable virus is cause of “Swamp
Fever” of horses (EIAV)
• Ellerman and Bang (1908) - chicken leukemia transferred by
bacteria-free filtrate (virus)
• Rouse (1911) - Rous sarcoma in chicken (RSV)
• Work in chicken and mice 1930s - 1970s: RNA tumor viruses
• Temin (1962) - Hypothesized “Provirus state”
• Huebner and Todaro (1969) - the “Viral Oncogene Hypothesis”
• Temin and Baltimore (1970) - discovery of Reverse
Transcriptase
• Bishop and Varmus (1978) - oncogenes are derived from
cellular genes involved in growth (src product is cellular
phosphokinase)
• Gallo (1981) - human retrovirus (HTLV-Human T Cell Leukemia
Virus)
• Barre-Sinoussi (1983) - HIV isolated / shown to be the cause of
AIDS
Retroviridae
• Retroviruses (family Retroviridae) are
enveloped, single stranded (+) RNA viruses
that replicate through a DNA intermediate
using reverse transcriptase.
• This large and diverse family includes
members that are oncogenic, are associated
with a variety of immune system disorders,
and cause degenerative and neurological
syndromes.
Retrovirus Classification
Derivation of Names
•
•
•
•
Retro (Latin) - backwards
Onco (Greek, oncos) - tumor
Spuma (Latin)-foam
Lenti (Latin, lentus) - slow
Retrovirus Classification
Family: Retroviridae
Genus
Features
Examples
1. Alpharetrovirus
Simple,
Onco
Avian leucosis virus, RSV
2. Betaretrovirus
Simple,
Onco
Mouse Mammary Tumor
Virus
3. Gammaretrovirus
Simple,
Onco
Murine leukemia virus
(Moloney, Harvey)
4. Deltaretrovirus
Complex,
Onco
Bovine Leukemia, Human
T Cell Leukemia (HTLV)
5. Epsilonretrovirus
Complex,
Onco
Walleye Dermal Sarcoma
6. Lentivirus
Complex
HIV, Visna, EIAV
7. Spumavirus
Complex
Simian Foamy Virus
Retrovirus Virions
Thin Section EM of Some Retroviruses
Type A (donut)
Type B (eccentric)
MMTV
Type C (central)
ALV,RSV
Type D (bar)
Lentivirus (cone)
HIV
Retrovirus structure
• Retrovirus virions are 80-120 nm in diameter, have
spherical morphology, a phospholipid envelope with knobs
• Contain around 2000 molecules of nucleocapsid (NC)
protein that bind to the two copies of (+) strand RNA genome
•Retroviral ribonucleoproteins are encased within a protein
shell built from the capsid protein to form an internal core,
which can have different shapes and has a conical shape in
HIV
Rous sarcoma virus
Avian leukosis virus
Retroviruses are enveloped, and contain: two copies of RNA;
internal structural proteins (MA,CA,NC); enzymes (PR,RT,IN);
and exterior proteins (SU,TM). There are several different
complex morphological types. ”
HIV Structure
surface
transmembrane
matrix protein
capsid protein
nucleocapsid protein
RT
Integrase
protease
HIV Virions
Cryoelectron micrograph
of mature human
immunodeficiency virus
type 1 (HIV-1) showing
the elongated internal
cores
HIV genome organization
• Single standed (+) sense RNA genome of about 10 kb
• 5’ cap, 3’ poly(A) tail
• Stop codon between gag and pol, suppressed by
readthrough or frameshifting
• Looks like mRNA, but does not serve as mRNA immediately
after infection
• Has a direct repeat (R) and unique (U) regions at both ends
• All retroviruses encode Gag, Pol and Env
• Lentiviral genomes encode a number of additional auxiliary
proteins, Tat, Rev, Nef, Vif, Vpr and Vpu
HIV integration:
Genome of Simple vs. Complex Retroviruses
• ALV and MLV are
“Simple” retroviruses
• HTLV, HIV, HFV, and
WDSV are “Complex”
retroviruses that
contain accessory
genes
• Notice gag-pol-env in
both simple and
complex
Genome of Simple vs. Complex Retroviruses
Retroviral Proteins
• gag, pol, and env
– Gag protein proteolytically processed into
• MA (matrix)
• CA (capsid)
• NC (nucleocapsid)
– Pol protein encodes enzymes
• PR (protease)
• RT (Reverse Transcriptase which has both DNA polymerase and
RNase H activities)
• IN (Integrase)
– Env protein encodes
• SU surface glycoprotein
• TM transmembrane protein
• “Accessory” genes (in Complex Retroviruses) - regulate
and coordinate virus expression; function in immune
escape
• Oncogene products (v-Onc, in Acutely Transforming
Retroviruses) - produce transformed phenotype
Gag Proteins
MA, Matrix, p17
CA, Capsid, p24
NC, Nucleocapsid, p7
– Binds to RNA
– Zinc fingers
• Note: retroviral proteins are
sometimes designated by their
apparent molecular weight.
This varies from virus to virus
Enzymatic Proteins: Protease
• 10 kd, dimer
• Cuts Gag
polyprotein to
MA,CA,NC
• Aspartyl protease
• Exquisite cleavage
specificity
• Major class of antiHIV drugs are
Protease Inhibitors
Enzymatic Proteins: Reverse
Transcriptase
• DNA Polymerase Activity
– Requires primer with 3’ OH termination
– Template either RNA or DNA
– Requires Mg++ (or Mn++)
– Lacks proof-reading function; high error rate
(10-4 errors per base)
• RNase H Activity (Nuclease specific for RNA
in RNA:DNA hybrids)
– Activity encoded in different domain than
polymerase
Enzymatic Proteins: Integrase
• Integrates retroviral
DNA into host
genome
• Endonuclease
activity
• Drugs being
developed
Env Proteins: Transmembrane (TM)
• Holds SU to
retroviral envelope
• Involved in
membrane
fusion/penetration
of virus into cell
Env Proteins: Surface (SU)
• Glycoprotein (gp, followed by apparent molecular
weight)
• Attaches to a specific receptor on cell surface
• Major neutralizing antigen on retrovirus, also
often highly variable (EIAV, HIV). Hard to make
vaccines.
SU (gp120)
Lipid Bilayer
(derived from cell)
TM (gp 41)
Regulatory proteins: Tat
• HIV LTR functions as a promoter in a variety of cell
types in vitro
• It includes an enhancer sequence that binds a number
of cell type specific transcription activators
Mechanism of Tat activation
• Downstream of the site of initiation of transcription is the
TAR RNA sequence which forms a stem loop that binds
the 14 kd viral regulatory protein Tat
• In the absence of Tat, viral transcription terminates
prematurely
• Tat facilitates efficient elongation
• Tat protein is cytotoxic to cells in culture
• Causes depolarization and degeneration of cell
membranes
• Transgenic expression in mice causes a disease that
resembles Kaposi’s sarcoma
Stimulation of transcription
by HIV-1 Tat protein:
• Before Tat is made proviral
transcripts are terminated within
60 bp of the initiation site
• Production of the Tat protein
allows transcription complexes to
synthesize full length RNA
• Binding of Tat to TAR together
with the cyclin T subunit of Tak
leads to stimulation of
phosphorylation of the largest
subunit of RNA polymerase II
• As a result, the transcriptional
complexes become competent to
carry out transcription
Regulatory proteins: Rev
• Rev Protein is an RNA binding protein that recognizes a
specific sequence within the structural element in env
called the Rev-responsive element (RRE)
Rev protein:
• Rev activates the nuclear export of any RRE
containing RNA
• As the Rev concentration increases, unspliced or
singly spliced transcripts containing the RRE are
exported from the nucleus
• Rev facilitates synthesis of the viral structural
proteins and enzymes and ensures availability of full
length genomic RNA to be incorporated into new virus
particles
• The accessory proteins, Vif, Vpr and Vpu are also
expressed later in infection from singly spliced
mRNAs and their export to the cytoplasm is Rev
dependent
HIV accessory proteins
Nef protein:
• Translated from multiply
spliced early transcripts
• myristylated at its N-terminus
and anchored to the inner
surface of the plasma membrane
• Nef deleted HIV and SIV are
much less pathogenic in vivo
• Nef downregulates expression
of CD4 by enhancing
endocytosis
• Can activate CD4+ T
lymphocytes by modulating
signaling pathways
HIV accessory proteins
Vif Protein:
• Viral infectivity factor
• Accumulates in the cytoplasm and at the plasma
membrane of infected cells
• Mutant viruses lacking the vif gene were less
infectious and defective in some way
• vif-defective virions enter cells, initiate reverse
transcription, but do not produce full-length double
stranded DNA
• vif inhibits antiviral action of a cytidine deaminase,
which is synthesized in nonpermissive cells
•This enzyme deaminates deoxycytidine to
deoxyuridine and leads to endonucleolytic digestion
or G to A transitions
Retrovirus replication cycle
1. Attachment of the virion to a specific cell surface receptor
2. Penetration of the virion core into the cell
3. Reverse transcription within the core structure to copy the
genome RNA into DNA
4. Transit of the DNA to the nucleus
5. Integration of the viral DNA into random sites in cellular
DNA to form the provirus
6. Synthesis of viral RNA by cellular RNA polymerase II
using the integrated provirus as a template
7. Processing of the transcripts to genome and mRNAs
8. Synthesis of virion proteins
9. Assembly and budding of virions
10. Proteolytic processing of capsid proteins
Retrovirus
life cycle:
HIV binding and entry
•The gp120 has a specific domain that binds to the
CD4 molecule present on susceptible cells
•Upon binding to CD4, the gp120 undergoes a
conformational change that allows binding to
specific subset of chemokine receptors on the cell
surface, the CCr5 receptor and the CXCr4 receptor
Coreceptors for macrophage and T-celltropic Strains of HIV
CXCr4 is the major coreceptor for T-cell-tropic strains
CCr5 is the major coreceptor for macrophage-tropic strains
HIV binding and entry
• The use of each coreceptor corresponds to viruses with
different biological properties and pathogenicity.
• Viruses isolated at the beginning of infection use the CCr5
co-receptor, which is the major coreceptor for macrophagetropic strains, these viruses are not cytophatic
• In full-blown AIDS cases, new viral species appear with
high level of replication, cytophatic effects and they use the
coreceptor CXCr4, which is the major receptor for T-cell
tropic strains
• There are also dual tropic viruses that can use both CXCr4
and CCr5 coreceptors or alternative chemokine coreceptors
• The fusion between the viral membrane and the cellular
membrane involves a change in conformation of gp41, which
enables it to insert into the cellular phospholipid bilayer
Retroviral genome
•Retroviruses contain two
copies of the RNA genome held
together by multiple regions of
base pairing
• This RNA complex also
includes two molecules of a
specific cellular RNA (tRNAlys)
that serves as a primer for the
initiation of reverse transcription
• The primer tRNA is partially
unwound and hydrogen bonded
near the 5’ end of each RNA
genome in a region called the
primer binding site
Reverse transcription
1) (-) strand synthesis starts near the 5’ end of the (+)
strand RNA genome with a specific host tRNA as a primer
and runs out of template after ~100 nt
2) Synthesis proceeds to the 5’ end of the RNA genome
through the u5 region ending after the r region, forming the
(-) strand strong stop DNA (-ssDNA)
Reverse transcription cont.
2) RNA portion of the RNA-DNA hybrid is digested by
the RNase H activity of RT, resulting in a singlestranded DNA product
3) This facilitates hybridization with the r region at the
3’ end of the same or second RNA genome, resulting
in the first template exchange for RT
Reverse transcription cont.
4) (-) strand DNA terminates at the primer binding site
5) When (-) strand elongation passes the polypurine
tract (ppt) region, the RNA template escapes digestion
by RNase H and serves as a primer for (+) strand
synthesis by DNA dependent DNA polymerization
(DDDP)
Reverse transcription cont.
6) (+) strand synthesis then continues back to the U5
region with the (-) strand DNA as the template and
terminates after copying the first 18 nt of the primer tRNA
and stops, forming the (+) strand strong stop product
(+ssDNA)
Reverse transcription cont.
7) The tRNA is then removed by RNase H activity of RT
8) The exposed PBS anneals to the PBS sequence at the 3’
end of the (-) strand DNA, allowing the second template
exchange.
Product of the second template exchange is a circular DNA
molecule with overlapping 5’ ends.
Reverse transcription cont.
9) DNA synthesis is terminated at the breaks in the
template strands at the PBS and PPT ends,
producing a linear molecule with long terminal
repeats (LTRs).