Nicholas Schmidt Schmidt-1

Nicholas Schmidt Schmidt-1

Biology 303 Term Paper

Dr. Ely

8 November, 2008

Human Immunodeficiency Virus (HIV) is a deadly infection which affects approximately 33.2 million people worldwide (Youth AIDS). HIV is a virus which targets T-cell lymphocytes in order to hijack their cellular machinery for the purpose of viral reproduction (Merck). This targeted attack of helper T-cells compromises the ability of the human immune system to properly defend the body against infection. Because of this, people with HIV/AIDS are susceptible to an array of opportunistic infections, or infections which a person with a healthy, uncompromised immune system would not contract. HIV is also problematic in that it has an exceptionally long incubation period of approximately ten years (Osmond 1998). This long incubation period facilitates the spread of the virus due to the fact that people can spread the virus while unaware of its presence in their body. HIV belongs to a class of viruses known as retroviruses, so named because of their use of the enzyme reverse transcriptase to convert viral RNA to DNA for implantation inside of the host cell. Currently, no cure for HIV exists, although treatment through the use of various anti-retrovirals has proven effective in prolonging the life of many AIDS sufferers. Many studies have been conducted in order to investigate the possibility of an alternate, more effective treatment for HIV, and to investigate the effect that the different mutations of HIV have on its treatment.

Schmidt-2

Nazari and Joshi (2008) investigated the effect that splicing introns into the DNA of HIV-1 proviruses has on viral replication. These introns were inserted into the DNA of the HIV-1 provirus at specific locations inside the integrase-coding region. Because of the specific locations into which these introns were spliced, they were given the names I4021 and I4069. In this experiment, plasmids pACD-4021s and pACD-4069s also needed to be modified in order to allow for the insertion of the introns into the DNA of the virus. The splicing of the introns into the virus, and the subsequent cloning of the virus were performed inside E. coli due to the fact that this procedure had not been successfully performed in mammalian DNA. The cloned HIV-1 proviruses containing the inserted introns were used to transfect human embryonic kidney 293T cells (Nazari and Joshi 2008). Compared to the HIV-1 proviruses which did not receive group II introns, the introns-inserted HIV-1 proviruses produced similar amounts of progeny viruses. Although similar amounts of progeny viruses were produced, those produced by the introns-spliced proviruses proved to be non-infectious due to the failure of the integration of dsDNA in the progeny. In addition, the 91 and 107 aa integrases were also non-functional. Due to these results, Nazari and Joshi (2008) concluded “that group II introns can confer ‘complete’ inhibition of HIV-1 replication at the intended step and should be further exploited for HIV-1 gene therapy and other targeted genetic repairs”. In order for this method to be used as a gene therapy against the virus, Nazari and Joshi (2008) claim that the group II introns would have to be further modified to provide more of a therapeutic protection against viral replication.

Schmidt-3

To gather information about which amino acid sites were being positively selected and involved in the immune control of HIV, Oliveira et al (2004) used codon based substitution models and maximum likelihood methods. An analysis of a total of 71 full-length sequences from subtype B, subtype C, and group M, a group consisting of subtypes A-K, was performed. To exclude the occurrence of recombination between subtypes, only nonrecombinant sequences were included in this study. The number of positively selected amino acid sites in the M group was significantly higher than in subtypes B and C. An unexpected discovery was the high frequency of positively selected sites in the early regulatory proteins Tat and Rev. Before this experiment, Env and Vpu were considered to be the most variable of all of the HIV-1 proteins. However in this experiment, Tat and Rev had proportionately more selected sites than Env and Vpu (Oliveira et al 2004). According to Oliveira et al (2004), “Tat plays a major role in the upregulation of HIV-1 gene expression, Rev controls the switch from chronic abortive infection to full-length mRNA expression and productive infection”. 30 codons in the Tat amino acid and 17 codons in the Rev proved to be influenced by positive selection in the subtype C group, while 19

codons in Tat and 32 in Rev were observed to be under positive selection in the subtype B viruses. Of these positively selected codons, 21 (12 Tat, 9 Rev) were common to both subtypes. Due to these findings, Oliveira et al (2004) concluded that these amino acids might be sites that frequently respond to host selective pressures.

Champenois et al. (2008) conducted an investigation into the impact of natural protease resistance mutations on the effectiveness of an anti-retroviral treatment with Lopinavir/ritonavir (LPV/r.) In this experiment, data were collected from 175 HIV-positive patients in France. All patients enrolled in the experiment were confirmed to have HIV-1 infection by Western-Blot, and had never before been treated with LPV/r containing antiretroviral treatment (ART). For each of the patients enrolled, viral load and CD4 lymphocyte cell counts, genotype resistance, subtype analysis, and ART effectiveness tests were performed. Two criteria were used to determine ART effectiveness, the slope of the viral load decrease from the initiation of ART to 1 month and the time necessary to achieve viral load (VL) undetectablility after the initiation of anti-retroviral treatment (Champenois et al. 2008). However, no significant association between ART effectiveness and natural protease mutations was observed (See Exhibit A from Champenois et al 2008). In addition, Champenois et al (2008) found no correlation between any particular mutation and viral load decrease or undetectablility.

All three of these studies have contributed important knowledge about the HIV-1 virus to those who are attempting to develop a vaccine, or even a cure, for the virus. Nazari and Joshi (2008) gave researchers the method of group II intron splicing which could be modified to be helpful in gene therapy. Champenois et al (2008) investigated the polymorphisms of HIV-1 and discovered that there is no significant difference in the effectiveness of anti-retroviral treatment between these polymorphisms. Were this study never performed, scientists may never have known if a correlation between different polymorphisms and ART effectiveness existed. Oliveira et al (2004) discovered that two early stage proteins, Tat and Rev were most often the locations of positive selection and host-selective pressure, indicating that these proteins may have a role in the high mutation rate of HIV. Because these two proteins have been discovered to have a heavy impact on the behavior of the HIV virus, researchers may use this information by targeting these proteins with treatments in an attempt to stop the virus. In summary, the findings of these three key studies may facilitate the development of a vaccine against HIV-1, a vaccine which would prevent the deaths of millions of people worldwide.

References

Champenois, Karen, Sylvie Deuffic-Burban, Laurent Cotte, Patrice Andre, Philippe Choisy, Faiza Ajana, Laurence Bocket, and Yazdan Yazdanpanah. "Natural Polymorphisms in HIV-1 Protease: Impact on Effectiveness of a First-line Lopinavir-Containing Antiretroviral Therapy Regimen." Journal of Medical Virology 80 (2008): 1871-879.

De Oliveira, Tulio, Marco Salemi, Michelle Gordon, Anne-Mieke Vandamme, Estrelita J. Van Rensburg, Susan Engelbrecht, Hoosen M. Coovadia, and Sharon Cassol. "Mapping Sites of Positive Selection and Amino Acid Diversification in the HIV Genome." Genetics 167 (2004): 1047-058.

"Human Immunodeficiency Virus (HIV) Infection." Feb. 2003. Merck. 2 Nov. 2008 <

Nazari, Reza, and Sadhna Joshi. "Exploring the potential of group II introns to inactivate human immunodeficiency virus type 1." Journal of General Virology 89 (2008): 2605-610.

Osmond, Dennis H. "Epidemiology of Disease Progression in HIV." May 1998. University of California, San Fransisco. 3 Nov. 2008 <