Structure of retrovirus

Virions of retroviruses consist of enveloped particles about 100 nm in diameter. The virions also contain two
identical single-stranded RNA molecules 710 kilobases in length. Although virions of different retroviruses do not
have the same morphology or biology, all the virion components are very similar.[2]
The main virion components are:

Envelope: composed of lipids (obtained from the host plasma membrane during budding process) as well as
glycoprotein encoded by the env gene. The retroviral envelope serves three distinct functions: protection from
the extracellular environment via the lipid bilayer, enabling the retrovirus to enter/exit host cells through
endosomal membrane trafficking, and the ability to directly enter cells by fusing with their membranes.

RNA: consists of a dimer RNA. It has a cap at the 5' end and a poly(A) tail at the 3' end. The RNA genome
also has terminal noncoding regions, which are important in replication, and internal regions that encode virion
proteins for gene expression. The 5' end includes four regions, which are R, U5, PBS, and L. The R region is a
short repeated sequence at each end of the genome used during the reverse transcription to ensure correct
end-to-end transfer in the growing chain. U5, on the other hand, is a short unique sequence between R and
PBS. PBS (primer binding site) consists of 18 bases complementary to 3' end of tRNA primer. L region is an
untranslated leader region that gives the signal for packaging of the genome RNA. The 3' end includes 3
regions, which are PPT (polypurine tract), U3, and R. The PPT is a primer for plus-strand DNA synthesis
during reverse transcription. U3 is a sequence between PPT and R, which serves as a signal that the provirus
can use in transcription. R is the terminal repeated sequence at 3' end.

Proteins: consisting of gag proteins, protease (PR), pol proteins, and env proteins.

Group-specific antigen (gag) proteins are major components of the viral capsid, which are about
20004000 copies per virion.

Protease is expressed differently in different viruses. It functions in proteolytic cleavages during

virion maturation to make mature gag and pol proteins.

Pol proteins are responsible for synthesis of viral DNA and integration into host DNA after infection.

Env proteins play a role in association and entry of virions into the host cell. [3] Possessing a
functional copy of an env gene is what makes retroviruses distinct from retroelements. [4] The ability of the
retrovirus to bind to its target host cell using specific cell-surface receptors is given by the surface
component (SU) of the Env protein, while the ability of the retrovirus to enter the cell via membrane fusion is
imparted by the membrane-anchored trans-membrane component (TM). Thus the Env protein is what
enables the retrovirus to be infectious.

Retroviral RNA is arranged in 5 terminus to 3 terminus. The site where the primer is annealed to viral RNA
is called the primer-binding site (PBS). The RNA 5end to the PBS site is called U5, and the RNA 3 end to
the PBS is called the leader. The tRNA primer is unwound between 14 and 22 nucleotides and forms a

base-paired duplex with the viral RNA at PBS. The fact that the PBS is located near the 5 terminus of viral
RNA is unusual because reverse transcriptase synthesize DNA from 3 end of the primer in the 5 to 3
direction (with respect to the RNA template).Therefore, the primer and reverse transcriptase must be
relocated to 3 end of viral RNA. In order to accomplish this reposition, multiple steps and various enzymes
including DNA polymerase, ribonuclease H(RNase H) and polynucleotide unwinding are needed. [9]

The HIV reverse transcriptase also has ribonuclease activity that degrades the viral RNA during the
synthesis of cDNA, as well as DNA-dependent DNA polymerase activity that copies the sense cDNA strand
into an antisense DNA to form a double-stranded viral DNA intermediate (vDNA).[

Retroviral reverse transcription[edit]

Retroviruses, also referred to as class VI ssRNA-RT viruses, are RNA reverse transcribing viruses with a DNA
intermediate. Their genomes consist of two molecules of positive-sense single stranded RNA with a 5' cap and 3'
polyadenylated tail. Examples of retroviruses include the human immunodeficiency virus (HIV) and the human Tlymphotropic virus (HTLV). Creation of double-stranded DNA occurs in the cytosol[7] as a series of these steps:
1. A specific cellular tRNA acts as a primer and hybridizes to a complementary part of the virus RNA genome
called the primer binding site or PBS

2. Complementary DNA then binds to the U5 (non-coding region) and R region (a direct repeat found at both
ends of the RNA molecule) of the viral RNA
3. A domain on the reverse transcriptase enzyme called RNAse H degrades the 5 end of the RNA which
removes the U5 and R region
4. The primer then jumps to the 3 end of the viral genome and the newly synthesised DNA strands hybridizes
to the complementary R region on the RNA
5. The first strand of complementary DNA (cDNA) is extended and the majority of viral RNA is degraded by
RNAse H
6. Once the strand is completed, second strand synthesis is initiated from the viral RNA
7. There is then another jump where the PBS from the second strand hybridizes with the complementary PBS
on the first strand
8. Both strands are extended further and can be incorporated into the hosts genome by the enzyme integrase
Creation of double-stranded DNA also involves strand transfer, in which there is a translocation of short DNA
product from initial RNA dependent DNA synthesis to acceptor template regions at the other end of the genome,
which are later reached and processed by the reverse transcriptase for its DNA-dependent DNA activity
A Reverse transcriptase (RT) is an enzyme used to generate complementary DNA (cDNA) from an RNA template,
a process termed reverse transcription. It is mainly associated with retroviruses. It should be noted however that
also non-retroviruses use RT (for example, the hepatitis B virus, a member of the Hepadnaviridae, which
are dsDNA-RT viruses, while retroviruses are ssRNA viruses). RT inhibitors are widely used as antiretroviral drugs.
RT activities are also associated with the replication of chromosome ends (telomerase) and some mobile genetic
elements (retrotransposons).
Retroviral RT has three sequential biochemical activities:

(a) RNA-dependent DNA polymerase activity,

(b) ribonuclease H, and

(c) DNA-dependent DNA polymerase activity.

These activities are used by the retrovirus to convert single-stranded genomic RNA into double-stranded cDNA
which can integrate into the host genome, potentially generating a long-term infection that can be very difficult to
eradicate. The same sequence of reactions is widely used in the laboratory to convert RNA to DNA for use
in molecular cloning, RNA sequencing, polymerase chain reaction (PCR), or genome analysis.
Reverse transcriptases were discovered by Howard Temin at the University of WisconsinMadison in RSV virions,
[4]

and independently isolated by David Baltimore in 1970 at MIT from two RNA tumour viruses: R-MLV and

again RSV.[5] For their achievements, both shared the 1975 Nobel Prize in Physiology or Medicine (with Renato
Dulbecco).
The idea of reverse transcription was very unpopular at first as it contradicted the central dogma of molecular
biology which states that DNA is transcribed into RNA which is then translated into proteins. However, in 1970 when
the scientists Howard Temin and David Baltimore both independently discovered the enzyme responsible for
reverse transcription, named reverse transcriptase, the possibility that genetic information could be passed on in this
manner was finally accepted

HIV GENOME STRUCTURE

HIV has several major genes coding for structural proteins that are found in all retroviruses as well as several
nonstructural ("accessory") genes unique to HIV. The HIV genome contains three major genes, 5'gag-pol-env-3',
encoding major structural proteins as well as essential enzymes.[15] These are synthesized as polyproteins which
produce proteins for virion interior, called Gag, group specific antigen; the viral enzymes (Pol, polymerase) or the
glycoproteins of the virion env (envelope).[16] In addition to these, HIV encodes for proteins which have certain
regulatory and auxiliary functions as well.[16] HIV-1 has two important regulatory elements: Tat and Rev and few
important accessory proteins such as Nef, Vpr, Vif and Vpu which are not essential for replication in certain tissues.
[17]

The gag gene provides the basic physical infrastructure of the virus, and pol provides the basic mechanism by

which retroviruses reproduce, while the others help HIV to enter the host cell and enhance its reproduction. Though
they may be altered by mutation, all of these genes except tev exist in all known variants of HIV;
HIV employs a sophisticated system of differential RNA splicing to obtain nine different gene products from a less
than 10kb genome.[18] HIV has a 9.2kb unspliced genomic transcript which encodes for gag and pol precursors; a
singly spliced, 4.5 kb encoding for env, Vif, Vpr and Vpu and a multiply spliced, 2 kb mRNA encoding for Tat, Rev
and Nef.[18]

Proteins encoded by the HIV genome

Class

Viral structural proteins

Gene name

gag

Primary protein products

Gag polyprotein

Processed protein products

MA, CA, SP1, NC, SP2, P6

Essential regulatory elements

Accessory regulatory proteins

pol

Pol polyprotein

RT, RNase H, IN, PR

env

gp160

gp120, gp41

tat

Tat

rev

Rev

nef

Nef

vpr

Vpr

vif

Vif

vpu

Vpu

Viral structural proteins

gag (group-specific antigen) codes for the precursor gag polyprotein which is processed by viral protease
during maturation to MA (matrix protein, p17); CA (capsid protein,p24); SP1 (spacer peptide 1, p2); NC
(nucleocapsid protein, p7); SP2 (spacer peptide 2, p1) and P6 protein. [19]

pol codes for viral enzymes reverse transcriptase (RT) and RNase H, integrase (IN), and HIV protease (PR).
[16]

HIV protease is required to cleave the precursor Gag polyprotein to produce structural proteins, RT is

required to transcribe DNA from RNA template, and IN is necessary to integrate the double-stranded viral DNA
into the host genome.[15]

env (for "envelope") codes for gp160, which is cleaved by a host protease, furin, within the endoplasmic
reticulum of the host cell. The post-translational processing produces a surface glycoprotein, gp120 or SU,
which attaches to the CD4 receptors present on lymphocytes, and gp41 or TM, which embeds in the viral
envelope to enable the virus to attach to and fuse with target cells.[15][19]

Essential regulatory elements

tat (HIV trans-activator) plays an important role in regulating the reverse transcription of viral genome RNA,
ensuring efficient synthesis of viral mRNAs and regulating the release of virions from infected cells. [16] Tat is
expressed as 72-amino acid one-exon Tat as well as the 86-101 amino-acid two-exon Tat, and plays an

important role early in HIV infection. Tat (14-15kDa) binds to the bulged genomic RNA stem-loop secondary
structure near the 5' LTR region forming the trans-activation response element (TAR).[5][16]

rev (regulator of expression of virion proteins): The Rev protein binds to the viral genome via an arginine-rich
RNA-binding motif that also acts as a NLS (nuclear localization signals), required for the transport of Rev to the
nucleus from cytosol during viral replication.[16] Rev recognizes a complex stem-loop structure of the
mRNA env located in the intron separating coding exon of Tat and Rev, known as the HIV Rev response
element (RRE).[5][16] Rev is important for the synthesis of major viral proteins and is hence essential for viral
replication.

Accessory regulatory proteins[edit]

vpr (lentivirus protein R): Vpr is a virion-associated, nucleocytoplasmic shuttling regulatory protein. [16] It is
believed to play an important role in replication of the virus, specifically, nuclear import of the preintegration
complex. Vpr also appears to cause its host cells to arrest their cell cycle in the G2 phase. This arrest activates
the host DNA repair machinery which may enable integration of the viral DNA. [5] HIV-2 and SIV encode an
additional Vpr related protein called Vpx which functions in association with Vpr.[16]

vif - Vif is a highly conserved, 23 kDa phosphoprotein important for the infectivity of HIV-1 virions depending
on the cell type.[5] HIV-1 has been found to require Vif to synthesize infectious viruses in lymphocytes,
macrophages, and certain human cell lines. It does not appear to require Vif for the same process in HeLa cells
or COS cells, among others.[16]

nef- Nef, negative factor, is a N-terminal myristoylated membrane-associated phosphoprotein. It is involved

in multiple functions during the replication cycle of the virus. It is believed to play an important role in cell
apoptosis and increase in virus infectivity.[16]

vpu (Virus protein U) - Vpu is specific to HIV-1. It is a class I oligomeric integral membrane phosphoprotein
with numerous biological functions. Vpu is involved in CD4degradation involving the
ubiquitin proteasome pathway as well as in the successful release of virions from infected cells. [5][16]

tev: This gene is only present in a few HIV-1 isolates. It is a fusion of parts of the tat, env, and rev genes,
and codes for a protein with some of the properties of tat, but little or none of the properties of rev

he human immunodeficiency virus (HIV):

HIV belongs to a group of retroviruses called lentiviruses. The genome of retroviruses is made
of RNA (ribonucleic acid), and each virus has two single chains of RNA; for replication, the
virus needs a host cell, and the RNA must first be transcribed into DNA (deoxyribonucleic
acid), which is done with the enzyme reverse transcriptase.
HIV infects mainly the CD4+ lymphocytes (T cells), but also to a lesser degree monocytes,
macrophages, and dendritic cells (these cells are also CD4+ cells). Once infected, the cell
turns into an HIV-replicating cell and loses its function in the human immune system.

HIV structure:
An HIV virus particle is spherical and
has a diameter of about 1/10,000
mm.
Like other viruses, HIV does not
have a cell wall or a nucleus.

The basic structure of the virus is as follows:

- The viral envelope, the outer coat of the virus, consists of two layers of lipids; different
proteins are embedded in the viral envelope, forming "spikes" consisting of the
outer glycoprotein (gp) 120 and the transmembrane gp41. The lipid membrane is borrowed
from the host cell during the budding process (formation of new particles). gp120 is needed to
attach to the host cell, and gp41 is critical for the cell fusion process.
- The HIV matrix proteins (consisting of the p17 protein), lie between the envelope and
core.
- The viral core, contains the viral capsule protein p24 which surrounds two single strands of
HIV RNA and the enzymes needed for HIV replication, such as reverse transcriptase,
protease, ribonuclease, and integrase; out of the nine virus genes, there are three, namely
gag, pol and env, that contain the information needed to make structural proteins for new virus
particles.

HIV sequence database

DATABASES
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HIV-1 Gene Map

Landmarks of the HIV-1 genome, HXB2 (K03455). Open reading frames are shown as rectangles.
The gene start, indicated by the small number in the upper left corner of each rectangle, normally
records the position of the a in the ATG start codon for that gene, while the number in the lower
right records the last position of the stop codon. For pol, the start is taken to be the first T in the
sequence TTTTTTAG, which forms part of the stem loop that potentiates ribosomal slippage on the
RNA and a resulting -1 frameshift and the translation of the Gag-Pol polyprotein. The tat and rev
spliced exons are shown as shaded rectangles. In HXB2, *5772 marks position of frameshift in the
vpr gene caused by an "extra" T relative to most other subtype B viruses; !6062 indicates a
defective ACG start codon in vpu; 8424, and 9168 mark premature stop codons in tat and nef. See
Korber et al., Numbering Positions in HIV Relative to HXB2CG, in the database compendium, Human
Retroviruses and AIDS, 1998.

Annotation Resources
Spreadsheets with in-depth annotation of genome features and coordinates are available: In-depth
Annotation Resources.

Genes and Gene Products

GAG The genomic region encoding the capsid proteins (group specific antigens). The precursor is
the p55 myristoylated protein, which is processed to p17 (MAtrix), p24 (CApsid), p7 (NucleoCapsid),
and p6 proteins, by the viral protease. Gag associates with the plasma membrane, where virus
assembly takes place. The 55-kDa Gag precursor is called assemblin to indicate its role in viral
assembly. Read review article: HTML PDF

POL The genomic region encoding the viral enzymes protease, reverse transcriptase, and integrase.
These enzymes are produced as a Gag-Pol precursor polyprotein, which is processed by the viral
protease; the Gag-Pol precursor is produced by ribosome frameshifting near the 3' end of gag.
ENV Viral glycoproteins produced as a precursor (gp160), which is processed to give a noncovalent
complex of the external glycoprotein gp120 and the transmembrane glycoprotein gp41. The mature
gp120-gp41 proteins are bound by non-covalent interactions and are associated as a trimer on the
cell surface. A substantial amount of gp120 can be found released in the medium. gp120 contains
the binding site for the CD4 receptor, and the seven transmembrane domain chemokine receptors
that serve as co-receptors for HIV-1. Read review article: HTML PDF
TAT Transactivator of HIV gene expression. One of two essential viral regulatory factors (Tat and
Rev) for HIV gene expression. Two forms are known, Tat-1 exon (minor form) of 72 amino acids and
Tat-2 exon (major form) of 86 amino acids. Low levels of both proteins are found in persistently
infected cells. Tat has been localized primarily in the nucleolus/nucleus by immunofluorescence. It
acts by binding to the TAR RNA element and activating transcription initiation and elongation from
the LTR promoter, preventing the 5' LTR AATAAA polyadenylation signal from causing premature
termination of transcription and polyadenylation. It is the first eukaryotic transcription factor
known to interact with RNA rather than DNA and may have similarities with prokaryotic antitermination factors. Extracellular Tat can be found and can be taken up by cells in culture. Read
review article: HTML PDF
REV The second necessary regulatory factor for HIV expression. A 19-kD phosphoprotein, localized
primarily in the nucleolus/nucleus, Rev acts by binding to RRE and promoting the nuclear export,
stabilization, and utilization of the viral mRNAs containing RRE. Rev is considered the most
functionally conserved regulatory protein of lentiviruses. Rev cycles rapidly between the nucleus
and the cytoplasm.
VIF Viral infectivity factor, a basic protein typically 23 kD. Promotes the infectivity but not the
production of viral particles. In the absence of Vif, the produced viral particles are defective, while
the cell-to-cell transmission of virus is not affected significantly. Found in almost all lentiviruses,
Vif is a cytoplasmic protein, existing in both a soluble cytosolic form and a membrane-associated
form. The latter form of Vif is a peripheral membrane protein that is tightly associated with the
cytoplasmic side of cellular membranes. In 2003, it was discovered that Vif prevents the action of
the cellular APOBEC-3G protein, which deaminates DNA:RNA heteroduplexes in the cytoplasm. Read
review article: PDF
VPR Vpr (viral protein R) is a 96-amino acid (14-kD) protein, which is incorporated into the virion.
It interacts with the p6 Gag part of the Pr55 Gag precursor. Vpr detected in the cell is localized to
the nucleus. Proposed functions for Vpr include the targeting the nuclear import of preintegration
complexes, cell growth arrest, transactivation of cellular genes, and induction of cellular
differentiation. In HIV-2, SIV-SMM, SIV-RCM, SIV-MND-2, and SIV-DRL the Vpx gene is apparently the
result of a Vpr gene duplication event, possibly by recombination.
VPU Vpu (viral protein U) is unique to HIV-1, SIVcpz (the closest SIV relative of HIV-1), SIV-GSN, SIVMUS, SIV-MON and SIV-DEN. There is no similar gene in HIV-2, SIV-SMM, or other SIVs. Vpu is a 16-kd
(81-amino acid) type I integral membrane protein with at least two different biological functions:
(a) degradation of CD4 in the endoplasmic reticulum, and (b) enhancement of virion release from
the plasma membrane of HIV-1-infected cells. Env and Vpu are expressed from a bicistronic mRNA.

Vpu probably possesses an N-terminal hydrophobic membrane anchor and a hydrophilic moiety. It is
phosphorylated by casein kinase II at positions Ser52 and Ser56. Vpu is involved in Env maturation
and is not found in the virion. Vpu has been found to increase susceptibility of HIV-1 infected cells
to Fas killing.
NEF A multifunctional 27-kd myristoylated protein produced by an ORF located at the 3' end of the
primate lentiviruses. Other forms of Nef are known, including nonmyristoylated variants. Nef is
predominantly cytoplasmic and associated with the plasma membrane via the myristoyl residue
linked to the conserved second amino acid (Gly). Nef has also been identified in the nucleus and
found associated with the cytoskeleton in some experiments. One of the first HIV proteins to be
produced in infected cells, it is the most immunogenic of the accessory proteins. The nef genes of
HIV and SIV are dispensable in vitro, but are essential for efficient viral spread and disease
progression in vivo. Nef is necessary for the maintenance of high viral loads and for the
development of AIDS in macaques, and viruses with defective Nef have been detected in some HIV1 infected long term survivors. Nef downregulates CD4, the primary viral receptor, and MHC class I
molecules, and these functions map to different parts of the protein. Nef interacts with
components of host cell signal transduction and clathrin-dependent protein sorting pathways. It
increases viral infectivity. Nef contains PxxP motifs that bind to SH3 domains of a subset of Src
kinases and are required for the enhanced growth of HIV, but not for the downregulation of CD4.
Read review article: HTML PDF
VPX A virion protein of 12 kD found in HIV-2, SIV-SMM, SIV-RCM, SIV-MND-2, and SIV-DRL and not in
HIV-1 or other SIVs. This accessory gene is a homolog of HIV-1 vpr, and viruses with vpx carry both
vpr and vpx. Vpx function in relation to Vpr is not fully elucidated; both are incorporated into
virions at levels comparable to Gag proteins through interactions with Gag p6. Vpx is necessary for
efficient replication of SIV-SMM in PBMCs. Progression to AIDS and death in SIV-infected animals can
occur in the absence of Vpr or Vpx. Double mutant virus lacking both vpr and vpx was attenuated,
whereas the single mutants were not, suggesting a redundancy in the function of Vpr and Vpx
related to virus pathogenicity.

HIV Genomic Structural Elements

LTR Long terminal repeat, the DNA sequence flanking the genome of integrated proviruses. It
contains important regulatory regions, especially those for transcription initiation and
polyadenylation. Read review article: HTML PDF
TAR Target sequence for viral transactivation, the binding site for Tat protein and for cellular
proteins; consists of approximately the first 45 nucleotides of the viral mRNAs in HIV-1 (or the first
100 nucleotides in HIV-2 and SIV.) TAR RNA forms a hairpin stem-loop structure with a side bulge;
the bulge is necessary for Tat binding and function.
RRE Rev responsive element, an RNA element encoded within the env region of HIV-1. It consists of
approximately 200 nucleotides (positions 7710 to 8061 from the start of transcription in HIV-1,
spanning the border of gp120 and gp41). The RRE is necessary for Rev function; it contains a high
affinity site for Rev; in all, approximately 7 binding sites for Rev exist within the RRE RNA. Other
lentiviruses (HIV-2, SIV, visna, CAEV) have similar RRE elements in similar locations within env,
while HTLVs have an analogous RNA element (RXRE) serving the same purpose within their LTR; RRE

is the binding site for Rev protein, while RXRE is the binding site for Rex protein. RRE (and RXRE)
form complex secondary structures, necessary for specific protein binding. See Mishra et al.
2006 for structural information about RRE.
PE Psi elements, a set of 4 stem-loop structures preceding and overlapping the Gag start codon. PE
are the sites recognized by the cysteine histidine box, a conserved motif with the canonical
sequence CysX2CysX4HisX4Cys, present in the Gag p7 NC protein. The Psi Elements are present in
unspliced genomic transcripts, but absent from spliced viral mRNAs.
SLIP A TTTTTT slippery site, followed by a stem-loop structure, is responsible for regulating the -1
ribosomal frameshift out of the Gag reading frame into the Pol reading frame.
CRS Cis-acting repressive sequences postulated to inhibit structural protein expression in the
absence of Rev. One such site was mapped within the pol region of HIV-1. The exact function has
not been defined; splice sites have been postulated to act as CRS sequences.
INS Inhibitory/Instability RNA sequences found within the structural genes of HIV-1 and of other
complex retroviruses. Multiple INS elements exist within the genome and can act independently;
one of the best characterized elements spans nucleotides 414 to 631 in the gag region of HIV-1. The
INS elements have been defined by functional assays as elements that inhibit expression
posttranscriptionally. Mutation of the RNA elements was shown to lead to INS inactivation and upregulation of gene expression.

STRUCTURAL PROTEINS/VIRAL ENZYMES The products of gag, pol, and env genes, which are
essential components of the retroviral particle.
REGULATORY PROTEINS Tat and Rev proteins of HIV/SIV and Tax and Rex proteins of HTLVs. They
modulate transcriptional and posttranscriptional steps of virus gene expression and are essential
for virus propagation.
ACCESSORY OR AUXILIARY PROTEINS Additional virion and non-virion-associated proteins produced
by HIV/SIV retroviruses: Vif, Vpr, Vpu, Vpx, Nef. Although the accessory proteins are in general not
necessary for viral propagation in tissue culture, they have been conserved in the different
isolates; this conservation and experimental observations suggest that their role in vivo is very
important. Their functional importance continues to be elucidated.
COMPLEX RETROVIRUSES Retroviruses regulating their expression via viral factors and expressing
additional proteins (regulatory and accessory) essential for their life cycle.