AZT-resistant HIV-1 reverse transcriptase

Human Immunodeficiency Virus (HIV)Human Immunodeficiency Virus (HIV)

HIV is a virus that damages the immune system of those affected by it. When a person is HIV positive, the virus infects the host's Helper T cells by attaching to the CD4 receptor on the host cell and fusing the viral envelope with the host cell membrane. The viral particles are then released into the host cell. Once inside the T cell cytoplasm, the viral enzyme, reverse transcriptase, coverts the virus' single stranded RNA into double stranded DNA. This capability classifies HIV as a retrovirus. The viral DNA is then incorporated into the host genome using the viral enzyme, integrase. The host genome now contains viral information and each time the host replicates, the viral genetic information is passed on to daughter cells. This makes HIV particularly difficult to treat, as it protects itself by incorporating its genetic information into that of the host. In addition, HIV is able to remain dormant in a host's body for a period of time before it hijacks the host cell and uses the host to create new viral particles.

Eventually, HIV infection leads to a weakened immune system, making a host more susceptible to opportunistic infections. The host's damaged immune system makes it more difficult to fight off infections and thus those who are HIV positive are more likely to have serious complications from common infections. This is especially concerning in those who have Acquired Immune Deficiency Syndrome (AIDS). Patients are considered to have AIDS when their CD4 cell count drops below 200 per microliter of blood. This means that their immune systems have been significantly damaged and puts them at great risk for opportunistic infections.

There is currently no cure for HIV/AIDS, but treatments that inhibit the replication of the virus do exist. These treatments, particularly AZT, will be discussed in the "Inhibition of Reverse Transcriptase Activity" section. Researchers are also attempting to create an HIV vaccine, but this is proving difficult because HIV is a retrovirus. The ability to convert single stranded RNA into double stranded DNA also creates for more opportunity for mutations of the virus to occur. The high frequency of viral mutations creates many challenges for scientists in the quest to create a vaccine.

Role of Reverse Transcriptase in HIV ReplicationRole of Reverse Transcriptase in HIV Replication

Reverse transcriptase has two enzymatic activities: DNA polymerase and RNase H. DNA polymerase is capable of copying either a DNA or an RNA template, while RNase H cleaves RNA that is part of the RNA/DNA duplex. These functions work together to create double-stranded linear DNA from RNA, which can then be incorporated into the host genome.

The viral RNA serves as the template for DNA polymerase, although a host tRNA primer initiates the synthesis. The primer allows reverse transcriptase to form the minus strand by copying the 5' end of the RNA. An RNA/DNA duplex is then formed from the synthesis of the minus strand and this duplex is the substrate for RNase H. RNase H degrades the RNA strand, leaving the newly formed minus strand of DNA. As DNA polymerase continues to create DNA, RNase H degrades the RNA. This process continues and the plus strand of DNA is created, forming a double-stranded linear DNA molecule.

Without reverse transcriptase, HIV would be incapable of incorporating itself into the host genome. Without this ability, it would be much easier for the host's immune system to destroy the virus, thus making it less harmful. Reverse transcriptase not only plays an important role in HIV replication, but also in the overall effectiveness of the virus.

Structure of Reverse TranscriptaseStructure of Reverse Transcriptase

Reverse transcriptase is a heterodimer of two related : p66 and p51. The p66 subunit has 560 amino acid residues, while the p51 subunit has 440 amino acids. The larger subunit, p66, contains the active sites for both enzymatic activities (DNA polymerase and RNase H), while the smaller subunit, p51, has a structural role.

Two distinct domains, polymerase and RNase H form the p66 subunit. The polymerase domain consists of four subdomains: fingers (residues 1-85 and 118-155), palm (residues 86-117 and 156-236), thumb (residues 237-318), and connection (residues 319-426). The p51 subunit folds into the same subdomains, but the positions of the subdomains relative to each other are different.

Fingers SubdomainFingers Subdomain

The nucleic-acid binding cleft is primarily formed by the p66 polymerase and RNase H subdomains. The p51 thumb and connection subdomains form the floor of the binding cleft. In this way, the binding cleft is positioned so that both the polymerase and the RNAse H active sites come in contact with the nucleic acid.

When polymerase activity begins, reverse transcriptase binds to the nucleic acid substrate.

Inhibition of Reverse Transcriptase ActivityInhibition of Reverse Transcriptase Activity

Approximately half of all anti-HIV drugs target the polymerase activity of reverse transcriptase. Azidothymidine (AZT) is one such medication that affects reverse transcriptase. AZT is classified as a Nucleoside analog Reverse Transcriptase Inhibitor (NRTI), meaning that it is a nucleoside analog which impacts the polymerase activity of the enzyme. AZT is specifically a thymidine analog and AZT-sensitive HIV binds azidothymidine triphosphate (AZTTP) almost as efficiently as DNA thymidine triphosphate (dTTP). When reverse transcriptase binds AZTTP, the polymerase activity is halted. Without completion of polymerase activity, the virus is unable to use its RNA as a template for DNA and incorporate its genetic information into the host genome. Preventing reverse transcriptase from creating viral DNA can greatly impact the progression of the virus by slowing viral replication. This is complicated by the fact that many strains of HIV are able to develop resistance to AZT and other NRTIs.

ReferencesReferences

Sarafianos et al. "Structure and Function of HIV-1 Reverse Transcriptase: Molecular Mechanisms of Polymerization and Inhibition." Journal of Molecular Biology Volume 385, Issue 3. 23 January 2009. 693-713

"Science." AIDS & HIV Information from the AIDS Charity AVERT. Web. 15 Nov. 2010. <http://www.avert.org/>.