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INVESTIGATING THE ROLE OF T CELL ACTIVATION ON THE ESTABLISHMENT OF HIV LATENCY IN PRIMARY CD4+ T CELLS USING A DUAL-REPORTER VIRUS

  • Author(s): Chavez, Leonard
  • Advisor(s): Verdin, Eric
  • Greene, Warner
  • et al.
Abstract

Acquired Immunodeficiency virus (AIDS) has killed 36 million people since the first cases were reported in 1981, and is considered a worldwide pandemic. AIDS is caused by the human immunodeficiency virus (HIV), which first originated in Africa in the early 1900s, and by the 1980s had spread around the globe. HIV is a lentivirus that slowly destroys the immune system by infecting and killing CD4+ T cells. There is currently no cure or vaccine for HIV. However, drug therapy exists that is capable of controlling HIV infection, and prevents the progression to AIDS. Drug therapy cannot eradicate HIV because the virus can become transcriptionally silent after integrating its genetic material into the host cell's genome, resulting in latent infection. HIV latency is a reversible state, and allows the virus to remain within an infected individual for the entirety of their life-span. The study of HIV latency has been ongoing since 1986, but has been hindered by two major problems: 1) latently infected cells are extremely rare, in vivo; and 2) latently infected cells are indistinguishable from uninfected cells. The work of this dissertation focused on the development and utilization of an HIV dual-reporter virus that could identify latently infected cells, and distinguish them from productively infected and uninfected cells. The HIV dual-reporter viruses utilize two different fluorescent proteins. One fluorescent protein is under the control of the HIV LTR promoter, and identifies cells that are productively infected, while the other fluorescent protein is under the control of a constitutive promoter, independent of the viral promoter, and identifies cells that have integrated provirus. Infection of Jurkat cells and primary CD4+ T cells with these dual-reporter viruses allows for the identification and purification of latently infected cells. This isolated latent population which only expresses the fluorescent protein that is under the control of the constitutive promoter, is devoid of viral transcripts and viral proteins. One of these dual-reporter viruses, HIV Duo-Fluo I, was used to study the role of T cell activation on the establishment of HIV latency in primary CD4+ T cells. Resting primary CD4+ T cells are capable of supporting productive HIV infection, but show a propensity towards latent infection, while activated primary CD4+ T cells are capable of supporting latent infection, but show a propensity towards productive infection. Further, activated primary CD4+ T cells are capable of supporting latent infection without the need to return to a resting state. Our findings suggest that HIV latency is not completely dependent on T cell activation and that infection of both resting and activated primary CD4+ T cells can contribute to the HIV latent reservoir.

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