THE GENOME OF HIV

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THE GENOME OF HIV

• Since HIV has a more complex life cycle that
  simple retroviruses such as RSV and it appears
  that HIV can control its replication in a more
  complex fashion, we might expect more genetic
  information but this is not so.
  

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• HIV genome is 9749 nucleotides-- about the same
  size as any other retrovirus, for example Rous
  sarcoma virus (RSV).

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• The genome of HIV is more complex than RSV,
  however, since it has extra open reading frames
  that clearly code for small proteins. Some of
  these are protein synthesis-controlling proteins.
• The HIV genome has nine open reading frames but
  15 proteins are made in all
• The GAG gene and the GAG and POL genes together
  are translated into large polyproteins which are
  then cleaved by a virus-encoded protease that is
  part of the POL polyprotein.

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IMAGES
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• GAG polyprotein  is cleaved to into four proteins
  that are found in the mature virus  MA (matrix),
  CA (capsid), NC (nucleocapsid), p6
• POL polyprotein is cleaved to PR (protease), RT
  (reverse transcriptase), IN (integrase)
• ENV gene is translated to a polyprotein (Gp160)
  which is then cleaved by a host cell protease
  that is found in the Golgi Body.
• Gp160 is cleaved to SU (Gp120) and TM (Gp41) 

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Frameshift
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????? pol ????? ?gag.
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• In a addition to the nine protein derived from 
  GAG, POL and ENV, there are six other proteins
  made by HIV. Three of these are incorporated into
  the virus (Vif,  Vpr and Nef) while the others
  are not found in the mature virus Tat and Rev
  are regulatory proteins and Vpu indirectly
  assists in assembly. The genes that encode these
  proteins are known by three letter names that are
  derived as follows

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• TAT Trans-Activator of Transcription
• REV Regulator of Virion protein expression
• NEF Negative Regulatory Factor
• VIF Virion Infectivity Factor
• VPU Viral Protein U
• VPR Viral Protein R

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• These genes encode small proteins. They overlap
  with the structural genes (especially ENV) but
  are in different reading frames. From the above
  diagram of the organization of the HIV genome, it
  can be seen  that some are encoded in two exons
  (unlike the structural genes) and therefore their
  mRNAs can be derived by alternative splicing of
  structural gene mRNAs. This is rather important
  to the way in which the levels of these are
  controlled. Mutants in the TAT and REV genes show
  that their proteins are both necessary for virus
  production.

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TAT

• TAT gene product binds to a sequence in all of
  the genes of HIV and positively stimulates
  transcription. It is thus a positive regulator of
  protein synthesis, including its own synthesis.

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????? ?????? Tat

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• 86 residues

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Rev ???? ???? ?????? ???? ???? RRE (Rev
Responsive Element). ???? ???? ???? ?? env ?????
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REV

• REV binds to an element only in the mRNA for
  structural proteins (GAG/POL/ENV) and regulates
  the ratio of GAG/POL/ENV to non-structural,
  controlling protein (TAT/REV) synthesis. When REV
  levels are high, structural protein synthesis
  rises and controlling protein synthesis falls.
  Thus REV inhibits its own production and that of
  TAT.
• The normal result is homeostasis, low or
  non-existent virus production and latency in the
  resting CD4 cell.
• There is an inherent problem in HIV's lifestyle.
  It uses genomic RNA as its messenger RNA. This
  RNA is unspliced and the nucleus has a mechanism
  to prevent unspliced mRNAs from leaving the
  nucleus and being translated. It is the function
  of Rev to overcome this problem.

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???? ???? ????? Negative factor (Nef)
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• c) The name, NEF, comes from negative factor.
  Originally, it seemed that virions that lacked
  NEF grew better than wild type. Now the consensus
  is for the opposite, that is that virus produced
  in the presence of NEF is a little more
  infectious than virus produced in its absence.

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NEF

• Nef protein is synthesized early in infection.
  Despite its small size NEF has several functions.
• a) Homeostasis leads to problems for the
  parasitic provirus 
• i) Super-infection by other HIV particles which
  bind to surface CD4 antigen of the infected cell
  may kill the cell. ii) Probably more
  importantly, virus bound via CD4 antigen at the
  cell surface or free Gp120 bound to CD4 antigen
  at the cell surface may result in the cell being
  subject to an immune attack and the infected cell
  may be destroyed.

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• The translation of the NEF gene as a result of
  the first infecting virus causes the
  internalization of CD4 antigen from the cell
  surface and its destruction in lysosomes. Thus no
  more HIV or gp120 can bind to the surface of an
  infected cell! 
• b) By a different mechanism from its down
  regulation of CD4 antigen, NEF reduces surface
  expression of MHC class I molecules. This alters
  antigen presentation by the infected cell and is
  proposed to protect the infected cell from attack
  by cytotoxic T cells

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• d) It is found that NEF is important for HIV
  replication in vivo but there seems to be much
  less effect of NEF in an in vitro cell culture
  situation. Why this is so has long been obscure.
  Recently, this question seems to have been
  solved. The answer is found in the macrophages
  which are changed in two ways when they are
  infected with a NEF-expressing HIV (remember that
  macrophages are the cells that bring HIV into the
  body and the initial strains of HIV in an
  infected patient are macrophage-tropic).

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• HIV-infected macrophages secrete MIP-1alpha and
  MIP-1beta. These are two chemokines that  bind to
  the co-receptors for HIV infection of macrophages
  but here these chemokines have another  function.

• They cause resting CD4 T cells to migrate
  (undergo chemotaxis) towards the infected
  macrophages. This is important in vivo since,
  initially, HIV-infected cells are not very
  numerous and uninfected T cells may not be in the
  vicinity of the infected cells. Moreover, HIV
  does not have a very long half life in the
  circulation before becoming non-infectious.

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• Migration of uninfected cells towards infected
  cells increases the probability that the T cells
  will encounter infected macrophages before they
  leave the reticuloendothelial system.  This
  explains why NEF seems not to be of much
  consequence in cell culture where the cells are
  already close together. The infected macrophages
  do something else. They make a factor that has
  not yet been identified that activates the
  resting T cells that have been attracted towards
  them allowing the T cells to be productively
  infected and to shed new virus (Remember,
  lentiviruses, unlike most other retroviruses can
  infect non-dividing cells.

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• Normally, retroviruses can only enter the nucleus
  and integrate during mitosis when the nuclear
  membrane is broken down however, proteins of
  lentiviruses such as HIV have nuclear targeting
  signals that allow the nucleocapsid to find the
  nuclear pore. Thus, the virus can integrate into
  the host cell chromosome where it can now code
  for more viral RNA and protein. HIV, unlike some
  other lentiviruses, does not transcribe its
  genome to RNA in resting T cells since the
  activation of the promoter in the LTR requires
  transcription factors that are only made when a
  resting T cell becomes activated).  These finding
  explain why macrophages are vital for the spread
  of HIV.

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• Note In vivo, HIV can infect a resting T cell
  but cannot replicate in that cell. Although NEF
  can activate the cell, it cannot be made in the
  resting T cell. The above observations solve this
  conundrum. Macrophages are infected by HIV and
  make NEF without any activation process. As  a
  result they make factors that activate resting T
  cells that can now support a productive
  infection!

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VPU

• After activation of the T cell, the virus faces
  another problem CD4 antigen and Gp120 are being
  made in the endoplasmic reticulum of the same
  cell. They are likely to bind to one another
  before reaching the plasma membrane and such
  complexes are usually targeted by the cell for
  degradation. To stop this unfortunate state of
  affairs, another of the small HIV proteins (VPU)
  promotes the proteolysis of the CD4 antigen of
  the host cell as it is made!

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