Vanderhoff 1
Aaron Vanderhoff
Genetics 303 Section 510
Dr. Bert Ely
November 5, 2009
Prevention Techniques and Attempts at Controlling HIV
HIV is a retrovirus that undergoesgenetic transformations to survive. The mechanisms for reproduction and HIV’s ability to manipulate the human cell have made it a formidable disease. Vaccines are tough to create for HIV because it mutates so fast and generates various strains. The research into defending against HIV recently has been about promoting the human body’s immune system to naturally combat HIV. This research concerns protein families, regulatory factors involved in transcription, and natural small molecules in the body to combat the infection rate and reproduction speed of HIV. Janis Hutchins wrote a history of the HIV virus that describes the evolution of HIV research and why attempts at stopping HIV have been unsuccessful.
Janis Hutchinson describes the current knowledge on HIV in, “The Biology and Evolution of HIV” and talks about the future of fighting HIV/AIDS (Hutchinson). HIV has a fast mutation rate, recombinogenic effect, and uses human defenses to replicate, vaccines and cures for HIV can only be strain or subtype specific (Hutchinson 99-100). HIV adapts fast and passes on the genetic material for drug resistance meaning that for every vaccine made the virus begins fighting back immediately. Since clinical trials for vaccines are based on specific subtypes the hope is that research into immune system and drug related selection may broaden the clinical studies.
In the hopes of boosting the immune system aresearch grouptested the role of tetherin in binding HIV-1 to the outside of the cell to prevent its release. Tetherin is an anchor protein in the body that binds to the plasma membrane of cells with one anchor and then catches HIV virus cells with another anchor. The first anchor is a transmembrane anchor and the one that attaches to the viruses is a putative glycophosphatidylinositol lipid anchor. They employed biochemical and EM assaysto determine whether tetherin is incorporated into virion envelopes (Perez-Caballero et al.). Immunogold labeling of cellsurfaces with anti-HA antibodies revealed that tetherin-HA wasfrequently associated with virions. The research team then tested if tetherin anchored to the plasma membrane of cells on its own and found it did. Following was the hope that tetherin would bind to the HIV virion. They used proviral plasmids to deliver HIV-1 (Wild Type) and HIV-1 (delVpu) into cells. Infectious particle and western blot assays let the researchers track virion release. This showed only a small amount of tetherin binding to HIV-1 (WT) but the release of HIV-1 (delVpu) was strongly inhibited (Perez-Caballero et al.).
The next step was developing an artificial tetherin-like protein to replicate the tether ability and make it effective on HIV-1. The researchers used immunofluorescence to measure the expression of tetherin. This let them see the location of tethering and the distribution. The mutant forms of tetherin that lacked an anchor were devoid of antiviral activity. The artificial tetherin was successful in binding to HIV-1 particles. The research team was excited about being able to use an artificial protein.
The assays that were used to measure tetherin activity were performed with 293T cells in 24 well plates that were transfected usingpolyethylenimine and then were harvested40 hr after transfection. They used centrifugation to separate virion-containing supernatants and then western blot assays to determine particle yield (Perez-Caballero et al 510). EM analyses showed that thetherin tethered budding HIV-1 particles. Tetherin mutants bearing one of the two membrane anchors were still able to efficiently be inserted into the envelope of released HIV-1 virions but unable to tether virion particles (Perez-Caballero et al 510).
Since the introduction of tetherin molecules into the body would require further research other means of arresting HIV infection are being explored. Previous research showed that HIV-1 encodes four auxillary proteins and two regulatory proteins, Tat and Rev, along with the canonical structural proteins and enzymes found in all retroviruses (Cullen 2009). In the transcription process, “Tat first interacts with a cellular factor called cyclin T1, which, along with the kinase cdk9, constitutes positive transcription elongation factor b (P-TEFb)” (Cullen 593). Thanks to P-TEFb phosphorylation of negative regulators of transcription elongation and RNAP II molecules the entire HIV-1 genome can be transcribed. After transcribing the HIV genome they saw that many of the transcription factors overlapped with cellular factors. The reason HIV overlaps with cellular factors is because it relies on the host cell to perform the splicing of its mRNA. Understanding the replication and regulatory factors involved with the virus are a step. The study of these factors allows for research into stopping virion reproduction at the replication stage.
Regulatory factors are important in RNA transcription. HIV relies on the transcription of its RNA in order to replicate and further infect surrounding cells. A study on the inhibition of regulatory factors of HIV showed that RNA transcription, if interfered with, can reduce virus replication (Baird et al.). Polyamides, small molecules that are a couple base pairs in length, can be used to inhibit transcription. They performthe function of inhibition by targeting sequences that are adjacent to transcription factor binding sites. The experiment by Baird and others used four different polyamides in culture to occupy DNA-binding sites. Polyamide 1 and 3 were useful in inhibiting HIV-1 replication through DNA binding-sites. The experiment opens the door for further research into specific sequences that could be used as a general HIV inhibitor.
Viral mini-genesused to be how researchers tested viral gene expression (Jablonski 991). These mini-genes could show what proteins and substrates are created by some viral genes. The downfall of this method was that it didn’t demonstrate what would happen on a macro-scale, or in relation to the other processes and regulatory elements present in viral genomes. Experiments that included overlapping viral genes produced different results from the experiments of the mini-genes separately. This evolution of research led to the study of viral gene families and proteins. Selected hnRNPs and SR family proteins are the target of future research into viral mRNA metabolism and its parallels to cellular metabolism. The reason these protein families are being used is because they have multiple effects based on competition within the viral genome and redundant regulatory factors. Through over-expression of the protein families,they showed that when certain mRNA species were down-regulated, the virus increased the production of other mRNA species (Jablonski 990).
The numerous subtypes of HIV are preventing a uniform vaccine from being developed to fight at least 80% of HIV cases (Hutchinson 2001). The studies on regulation factors mentioned above focused on finding molecules that would inhibit replication. The hope of this research is to develop a molecule that can be used on a broad base to target gene sequences involved in all cases of infection and prevent the virus from spreading. The other method described was promoting the use of the body’s immune system. Tetherin is a natural forming molecule in the body. HIV deactivates it during infection. The development of an artificial version could be used as gene replacement or enhancement to keep HIV virions from spreading to other cells. HIV is a tough retrovirus to combat but experiments are being done to find a way to remove it.
References
1. Baird, Eldon E., Dervan, Peter B., Dickinson, Liliane A., Gottesfeld, Joel M., Gulizia, Richard J., Mosier, Donald E., and Trauger, John W. “Inhibition of RNA polymerase II transcription in human cells by synthetic DNA-binding ligands.” Proceedings of the National Academy of Sciences of the USA95 (1998): 12890-12895.
2. Cullen, Bryan R. "Viral RNAs: Lessons from the Enemy." Cell 136 (2009): 592-97.
3. Hutchinson, Janis Faye. "The Biology and Evolution of HIV." Annual Review of Anthropology 30 (2001): 85-108.
4. Jablonski, Joseph A., and Massimo Caputi. "Role of Cellular RNA Processing Factors in Human Immunodeficiency Virus Type 1 mRNA Metabolism, Replication, and Infectivity." Journal of Virology 83.2 (2009): 981-92.
5. Perez-Caballero, David, Trinity Zang, Alaleh Ebrahimi, Matthew W. McNatt, Devon A. Gregory, Marc C. Johnson, and Paul D. Bieniasz. "Tetherin Inhibits HIV-1 Release by Directly Tethering Virions to Cells." Cell 139 (2009): 499-511.