HIVbio: HIV Bioinformatics

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Viral Vital Enzymes

1. Reverse Transcriptase
In molecular biology and biochemistry, a reverse transcriptase, also known as RNA-dependent DNA polymerase, is a DNA polymerase enzyme that transcribes single-stranded RNA into double-stranded DNA. It also helps in the formation of a double helix DNA once the RNA has been reverse transcribed into a single strand cDNA. Normal transcription involves the synthesis of RNA from DNA; hence, reverse transcription is the reverse of this.
Reverse transcriptase was discovered by Howard Temin at the University of Well studied reverse transcriptases include:
* HIV-1 reverse transcriptase from human immunodeficiency virus type 1 (PDB 1HMV)Wisconsin.
Ribonuclease H
The enzyme RNase H (EC 3.1.26.4) is a ribonuclease that cleaves the 3'-O-P-bond of RNA in a DNA/RNA duplex to produce 3'-hydroxyl and 5'-phosphate terminated products. RNase H is a non-specific endonuclease and catalyzes the cleavage of RNA via an hydrolytic mechanism, aided by an enzyme-bound divalent metal ion.
Members of the RNase H family can be found in nearly all organisms, from archaea and prokaryota to eukaryota. In DNA replication, RNase H is responsible for cutting out the RNA primer, allowing completion of the newly synthesized DNA.
Retroviral RNase H, a part of the viral reverse transcriptase enzyme, is an important pharmaceutical target, as it is absolutely necessary for the proliferation of retroviruses, such as HIV. Inhibitors of this enzyme could therefore provide new drugs against diseases like AIDS. As of 2004, there are no RNase H inhibitors in clinical trials, though some approaches employing DNA aptamers are in the preclinical stage.
In a molecular biology laboratory, as RNase H specifically degrades the RNA in RNA:DNA hybrids and will not degrade DNA or unhybridized RNA, it is commonly used to destroy the RNA template after first-strand complementary DNA (cDNA) synthesis by reverse transcription, as well as procedures such as nuclease protection assays. RNase H can also be used to degrade specific RNA strands when the cDNA oligo is hybridized, such as the removal of the poly(A) tail from mRNA hybridized to oligo(dT), or the destruction of a chosen non-coding RNA inside or outside the living cell. To terminate the reaction, a chelator, such as EDTA, is often added to sequester the required metal ions in the reaction mixture.
Integrase
ntegrase is an enzyme produced by a retrovirus (such as HIV) that enables its genetic material to be integrated into the DNA of the infected cell. It is also produced by viruses containing double-stranded DNAs for the same purpose.
It is a key component in the pre-integration complex (PIC).
Structure
The integrase protein contains three domains:
* an N-terminal HH-CC zinc finger domain believed to be partially responsible for multimerization
* a central catalytic domain
* a C-terminal.
Both the Central catalytic domain and C-terminal domains have been shown to bind both viral and cellular DNA. At the present time, no crystal structure data with Integrase bound to its DNA substrates exists.
Biochemical data and structural data suggest that integrase functions as a dimer or a tetramer.
In addition, several host cellular proteins have been shown to interact with integrase and may facilitate the integration process.
Function
Integration occurs following production of the double-stranded viral DNA by the viral DNA polymerase, reverse transcriptase.
Integrase acts to insert the proviral DNA into the host chromosomal DNA, a step that is essential for HIV replication.
Integrase catalyzes two reactions:
* 3'-end processing, in which two deoxynucleotides are removed from the 3' ends of the viral DNA. * the strand transfer reaction, in which the processed 3' ends of the viral DNA are covalently ligated to the host chromosomal DNA.
Integration of the proviral DNA is essential for the subsequent transcription of the viral genome, which leads to production of new viral genomic RNA and viral proteins needed for the production of the next round of infectious virus.
In essence, the process involving integrase is a key step in allowing viral DNA to become a permanent member of the host genome. This integrated proviral DNA is then translated using host cell machinery (see translation) into viral proteins.
2. HIV integrase
HIV integrase is a 32 kDa protein produced from the C-terminal portion of the Pol gene product, and is an attractive target for new anti-HIV drugs. In November 2005, data from a phase 2 study of an investigational HIV integrase inhibitor, MK-0518, demonstrated that the compound had potent antiviral activity.On October 12, 2007, the Food and Drug Administration (U.S.) approved the integrase inhibitor Raltegravir (MK-0518, brand name Isentress TM). As of April 2008, this is the only integrase inhibitor approved for treating HIV Infection.
3. HIV Protease
HIV-1 protease (HIV PR) is an aspartic protease that is essential for the life-cycle of HIV, the retrovirus that causes AIDS.HIV PR cleaves newly synthesized polyproteins at the appropriate places to create the mature protein components of an infectious HIV virion. Without effective HIV PR, HIV virions remain uninfectious.Thus, mutation of HIV s active site or inhibition of its activity disrupts s ability to replicate and infect additional cells, making HIV PR inhibition the subject of much pharmaceutical research.
With its integral role in HIV replication, HIV PR has been a prime target for drug therapy. HIV PR inhibitors work by specifically binding to the active site by mimicking the tetrahedral intermediate of its substrate and essentially disabling the enzyme. However, due to the high mutation rates of retroviruses, and considering that a single amino acid change within HIV PR can render it invisible to an inhibitor, the active site of this enzyme can change rapidly when under the selective pressure of replication-inhibiting drugs.One approach to minimizing the development of drug-resistance in HIV is to administer a drug cocktail composed of drugs which inhibit several key aspects of s replication cycle simultaneously, rather than one drug at a time. Other drug therapy targets include reverse transcriptase, virus attachment, membrane fusion, cDNA integration and virion assembly.