History
In 1981, clusters of cases of Kaposi's sarcoma were observed in patients in San Francisco and New York. Kaposi's sarcoma normally occurs in the United States in elderly men of Jewish or Mediterranean ancestry, but these new clusters of patients were all were young male homosexuals. Other diseases associated with immuno-compromisation also occurred in this same population; particularly were in the occurrence of Lymphadenopathy and Pneumocystis pneumonia (an opportunistic infection). Pneumocystis pneumonia, which is caused by an intracellular parasite (Pneumocystis carinii), was also a very rare disease. During the period between 1967 and 1978, only five cases of this disease occurred in Los Angeles, but in 1981 alone, five cases occurred in the same area. Again, all patients were young male homosexuals. The number of sex partners of these patients appeared to beimportant that the disease was particularly prevalent among promiscuous individuals and the partners of those individuals. Later, a similar immunodeficiency was found in intravenous drug users who shared needles, persons who received blood transfusions and hemophiliacs. Moreover, the sex partners of these patients also got the disease. In view of these, it was obvious that an infectious agent was involved; this agent was either passed during sexual intercourse or by receiving blood (or blood products) from another person. The selective loss of T4 helper (CD4+) cells suggested the involvement of a virus. But the causative agent was difficult to identify at first because it does not grow on resting T4 cells. The discovery of HIV depended on the ability to grow the virus in vitro. The disease was originally termed Gay-Related Immune Deficiency (GRID), now it is know has Acquired Immuno-Deficiency Syndrome (AIDS). AIDS is invariably fatal (unless chemotherapeutic intervention occurs). The epidemic has resulted in the deaths of more than half of AIDS patients.The clue to growing the virus came with the realization that, while it did not grow on resting T4 lymphocytes, it would grow on T4 cells that had been activated with interleukin-1 cytokine. This virus was finally isolated by Luc Montanier of Pasteur Institute and Robert Gallo of NIH. Montanier called the virus lymphadenopathy virus (LAV) and Gallo, who had discovered the first human leukemia virus (HTLV-1), named the virus HTLV-3. Today it is known as human immunodeficiency virus (HIV).
Biological Aspects of HIV
HIV has similar structure to other retroviruses, and HIV can be distinguished genetically and antigenically into two subtypes. HIV-1 is the cause of the current pandemic in western countries while HIV-2 is found in west Africa but rarely elsewhere. The HIV-2 type is closely related to simian immunodeficiency virus (SIV) found in west Africa.There are also 10 different HIV-1 subtypes. The major one in the US and Europe is type B. In some countries, mosaics between different subtypes have been found.
Components of HIV
a) Surface Structures
Viral membrane: This is host-derived asa result of buddingfrom the cell surface. Some host proteins become incorporated into theviral membrane.
Surface glycoprotein: A single polypeptide Gp160 is encoded by the env (envelope) gene, and after translation Gp160 is cleaved to form Gp120 (SU) and Gp41 (TM) by a host enzyme in the Golgi apparatus since it gets to the cell membrane via the exocytic pathway. While Gp41 is embedded in the membrane, Gp120 is not. But, Gp120 is held to Gp41 by non-covalent interactions. It is easily shed from the virus particle. There are a large number of sugar chains on Gp120 (which may pose a problem for a vaccine). Gp120 is the protein that interacts with a receptor on the cell to be infected. Gp41 is the fusogen that is exposed after Gp120 has bound to the cell.
b) Internal Structures
Internal structural proteins: These are all encoded by gag gene (group-specific antigen). P17 lines the inner surface of viral membrane to which it is attached by covalently bound myristic acid. Other proteins associated with the nucleocapsid include p24 and p9. The group-specific antigen is made as a polyprotein and is cleaved after budding of the virus by a virally-encoded protease encoded by the pol gene.Other internal proteins: These are encoded by pol gene (polymerase). They are enzymes that participate in integration and replication: reverse transcriptase, integrase and protease.
Genome: Like other retroviruses, the genome is diploid positivesense RNA, 9749 nucleotides with nine open reading frames for 15 proteins.
c) Genome of HIVHIV genome is about the same size as any other retrovirus, for example, Rous sarcoma virus (RSV). However, the genome of HIV is more complex than RSV, since it has extra open reading frames that clearly code for small proteins. Some of these are protein synthesis-controlling proteins. The gag gene and the gag/pol genes together are translated into 9 large polyproteins.i) gag polyprotein: cleaved into four proteins that are found in the mature virus: MA (matrix), CA (capsid), NC (nucleocapsid) and p6.
ii) pol polyprotein: cleaved to PR (protease), RT (reverse transcriptase) and IN (integrase).
iii) env: translated to a polyprotein (Gp160), which is then cleaved to SU (Gp120) and TM (Gp41) by a host cell protease in the Golgi Body.
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 assists indirectlyin assembly. The genes that encode these proteins are known as follows:tat (Trans-Activator of Transcription), rev (Regulator of Virion protein expression), nef (Negative Regulatory Factor), vif (Virion Infectivity Factor), vpu (Viral Protein U) and vpr(Viral Protein R).
These genes encode small proteins. They overlap with the structuralgenes (especially env), but are in different reading frames, someare encoded in two exons (unlike the structural genes) and therefore theirmRNAs 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 proteinsare both necessary for virus production.
iv) 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.v) rev: rev binds to an element only in the mRNA for structural proteins (gag/pol/env ) and regulates the ratio of GAG/POL/ENV tonon-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 life style. 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.
vi) nef: Nef protein is synthesized early in infection. The name, nef, comes from negative factor, despite its small size, nef has severalfunctions. Virus produced in the presence of nef is little moreinfectious than virus produced in its absence.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.
Reverse Transcription and Integration
Formation of polyproteins and their cleavage of HIV are similarto other retroviruses. HIV uses reverse transcriptase imported during infectionas part of the virus. Assembly of new virus takes place at the membraneof the host cell. Three types of protein make up the virion. These arethe membrane protein complex (Gp120 and Gp41, which are originally derivedfrom Gp160) along with two internal precursor proteins, the gagpolyprotein and thegag/pol polyprotein (the latter is the resultof a frameshift that allows the ribosome to continue translation from thegag gene into the pol gene).The proteins aggregate at the cell membrane and the membrane pinchesoff. The larger internal precursor draws two strands of the positive strandRNA into the nascent virion and the protease cuts itself free. Theprotease completes the cleavage of the larger internal precursor to liberate other enzymes (reverse transcriptase, integrase and protease). The protease cleaves the remainder of the largerinternal precursor and the smaller internal precursor into structural proteins, p24, p7 and p9 form the bullet-shaped core.
This protease is vital as the viral proteins are not functionalunless separated. This specific HIV protease has become a good candidatefor a site for an anti-HIV drug . The gag and the gag/pol are attached to the viral membrane via a fatty acid that is covalently bound. The cleavages result in p17 remaining attached to the membrane.
Index of the Enzyme Report:
Introduction (Abstract):