http://galaxy.einet.net/galaxy/Science/Chemistry/vineet-gupta/secondarticle/article.html (World Wide Web Directory, ~04/1995)
This is the second article in the series by guest editor Vineet Gupta. The first one was titled DNA and The Central Dogma of Life.
December 1st was marked as the World AIDS Day and although just a day cannot show the importance of our fight against HIV, it can very well remind us of what a difficult struggle it has been, as we still don't have a reasonable drug against it. HIV infects vast number of people every year, and there are no signs of it letting up. This article is just to give everyone a general understanding of the Chemistry behind the infection and how it is being used to gain better insight into the problem of synthesizing more effective drugs. This article, by no means, is comprehensive and there are plethora of resources on the INTERNET (mentioned at the end of the article) as well as in the text for anyone intersted in going into further details.
Retroviruses are viruses which have RNA as their genetic material enveloped in proteins. The RNA is of positive sense (i.e.; its of same sense as mRNA) and is usually in the form of a ribonucleo- protein complex containing an enzyme called Reverse Transciptase among other proteins. This enzyme, after the viral entry into the infected host cell, reverse transcribes RNA into viral DNA to be inserted into cellular DNA which takes over the cellular synthesis machinery to synthesize more viruses.
Spumaviruses or foamy viruses (Latin spuma = foamy) particles are spherical (100-140 nm diameter) and consist of a ring-shaped nucleoprotein core surrounded by a bilayered membrane envelope. These viruses induce syncytia formation (i.e.; cell accumulation) in the infected cells. In the cell cultures they were observed to induce extensive intracellular vacuoles which under a microscope have a foamy appearance and hence the name spumaviruses. One example of these viruses is HSRV (Human Spuma RetroVirus).
Lentiviruses are non-oncogenic which fuse into and kill their host cells. The virus particles are spherical (100-120 nm diameter) having a conical nucleoprotein covered by a bilayered phospholipid envelope with surface glycoprotein embedded. They are exogenously acquired viruses and are associated with slow, persistent and debilitating life long infections, inducing pathological changes in the immune system of the animals which result in severe immuno - deficiency syndromes. Some examples include HIV- I and II, CAEV (Caprine Arthritis Encephalitis Virus), SIV and BIV.
Viral Structure : Human Immunodeficiency Virus - I is the more prevalent of the two kinds of viruses round the globe. The viroid particle is spherical, about 110 nm in diameter and has its surface spiked with a glycoprotein - gp120. The viral core is made up of a protein p24 which envelopes a nucleoprotein having two copies of single stranded viral (+) RNA, each about 9600 nucleotides long, and proteins including Reverse Transcriptase (p55/p61), endonuclease (p32) and RNA-binding Gag protein (p9). This has an outer shell comprising of myristilated Gag protein (p17) which has its amino terminus inserted into the lipid membrane envelope, which is derived from the infected cell membrane. This envelope has exrtacellular surface glycoprotein gp120 and transmembrane glycoprotein gp41 in the form of noncovalent complexes (fig 1).
Viral Binding And Entry : One of the first lines of defense against any alien molecule in the human body comprise of lymphocytes, which are of two kinds ; B - Cells (which comprise humoral immune response) and T - Cells (which comprise cell-mediated immune response) and which again have further subdivisions based upon their function. Infection in the circulation or extracellular fluids (e.g.; influenza, polio, diphtheria, tetanus toxins) is neutralized by the humoral (which means fluid) arm of the immune system through antibodies (B-Cells) that bind to pathogens or toxins and lead them to their destruction. Infection by intracellular pathogens (e.g.; tuberculosis causing bacteria, syphillis) is protected against by cell-mediated immune response (T-Cells, macrophages). After the viral entry into the blood stream, an immune response is launched to counter these virus particles and helper T- cells lead this attack. These cells have a protein on their surface called CD4. This CD4 protein on CD4+ T - Cells acts as the major receptor for HIV, binding strongly to the gp120 on the viral surface. The transmembrane protein gp41 then mediates the fusion of viral membrane with the cellular membrane and unloads the viral load into the cell. This is the first step in HIV infection.
Viral Hijacking : Once the viral load, in the form of nucleoprotein complex, is in the cellular cytoplasm of the susceptible cell the viral proteins become functional. Reverse Trancriptase synthesizes double stranded viral DNA, reverse transcribing the viral RNA present in the nucleoprotein complex. This enzyme is a DNA polymerase and an RNaseH since it synthesizes DNA and chews up the parent RNA during the process. The DNA then migrates to the nucleus in the form of another nucleo- protein complex. Once inside the nucleus it is integrated into the residual DNA by viral integrase, present in the transported complex, and thereafter it behaves exactly like the cellular DNA and uses cellular enzymes to its benefit.
In-Cell Viral Synthesis : The transcription of integrated viral DNA inside the cell nucleus is controlled not only by cellular enzymes but also by viral proteins that migrated with the complex or which it encodes. This viral DNA encodes a number of genes which can be divided into three categories - structural genes which include gag, pol and env; regulatory genes including tat, rev and nef; and accessory genes which include vif, vpr, vpu, vpt and tev/tnv. The viral genome, in 5' to 3' direction, is organized as 5'-LTR-gag-pol-env- LTR-3', where LTR stands for Long Term Repeats which contain the enhancer and promoter regions for the DNA transcription. The tat and rev genes are present as two exons before and after env; other genes are present inbetween too.
The gag gene encodes for structural proteins used in final virion assembly and release, pol gene encodes for enzymes reverse transcriptase, integrase and protease and env gene encodes for envelope glycoproteins gp120 and gp41.
The tat gene encodes for Tat protein which is absolutely essential for the viral replication. It is a 14kiloDalton(kDa) protein and is made up of 72 amino acid(AA) residues. It promotes transcription of viral DNA by binding to a region on LTR called TAR (trans-activation response element). It is slightly downstream of binding region used by cellular transcription factors Sp1 and TFIID, on LTR. The rev gene encodes for Rev protein which too is necessary for viral transcription. It is a 19kDa protein and consists of 116 AA residues. It binds to Rev Response Element (RRE) on the genome and helps in RNA splicing and transport. The Nef protein is encoded by nef gene. It is a 25-27 kDa protein and helps in the signal transduction.
The accessory genes encode for the proteins with the same name with diverse
functions, but they probably are not absolutely critical.
The viral replication cycle can be shown in the following figure.
The mature viroids are then released into the blood stream which go on to
infect other cells and the cycle continues.
a. Binding induced Programmed Cell Death (apoptosis)
Programmed Cell Death or apoptosis is a natural phenomenon for the cells. It is a natural suicide mechanism by which body takes care of extensively damaged cells, especially when their chromosomal DNA is damaged or cleaved. It is also essential for developing self-tolerance by evolving T-cells and thus maturation of immune system. Apoptosis in cells is usually triggered by stimulation by a number of signals. Mature T-cells, normally, are resistant to apoptotic stimuli. However, under certain circumstances, cell signaling machinery goes haywire and abnormally induces apoptosis in the mature T-cells. This probably is a major cause of the massive depletion of these cells in the blood stream.
Apoptosis in mature CD4+ T-Cells has been proposed to be induced in at least two prominent ways. The first involves signals generated upon binding of HIV surface proteins to CD4 of activated (by other antigens etc.) T-Cells. This can not only occur upon binding of HIV itself to the T-Cells, but also upon binding of infected T-Cells, having viral protein gp120 on their surface, to uninfected cells. Thesecond mechanism involves modification of accessory-cell functions by HIV, changing the balance of activation signals required to prevent apoptosis in the mature T-Cells. This can be accomplished either by enhancing the amount of apoptotic signals or reducing the amount of balancing preventive signals.
b. Syncytia Formation
Syncytia formation has also been proposed to explain the severe loss of CD4+ T-Cells in HIV infected patients' blood. The infected T-Cells having viral protein gp120 on their surfaces can bind to uninfected, perfectly normal T-Cells, owing to the great affinity of binding of gp120 with CD4. This results in formation of big clumps (syncytia). Since this is harmful to the body, an immune response is generated against it, and the whole thing is removed either by the action of killer T-Cells (cell-mediated immune response) or by antibody generation (humoral immune response). Although the syncytia is usually taken care of fairly quickly, a consequence of this phenomenon, also known as bystander effect, is that a great number of uninfected, neighboring cells are lost because of a handful of infected cells, which form the nucleus of dying cells.
Although currently there are only these three drugs in use against HIV, a number of new and more specific drugs are either under development or trial phase. As we start to know more details about the mechanism of action of the virus as well as the human immune system, better and more effective drugs, with enhanced specificities and diminished side effects could be developed. These rationally designed drugs hold better promise than the empirically obtained earlier ones. Already some of the newer drugs look like good candidates for therapy against AIDS, although due to HIVs hyper variable nature, multiple drug therapy seems like the best approach.
These newer drugs target specific components in the viral life-cycle, trying to disable it in every possible way. A number of inhibitors for enzymes like RT, Integrase, Protease, Tat and Rev are being pursued. A number of studies are being conducted in the field of antisense, where researchers are trying to develop ribozymes and other nucleic acid molecules to inhibit HIV synthesis in the nucleus of infected cells. The outer surface as well as the glycoprotein of the virus has also been a subject of target for the new drugs. The following figure gives a picture of the targets for new drugs in making.
Write to Vineet at gupta@chem.chem.rochester.edu or vtga@uhura.cc.rochester.eduA search engine is also provided for Searches on the Web :