GSBS Dissertations and Theses

Title

Envelope Determinants of HIV-1 Cytopathicity

Approval Date

February 1998

Document Type

Doctoral Dissertation

Department

Graduate School of Biomedical Sciences, Program in Immunology and Virology

Subjects

HIV-1; Cytopathogenic Effect, Viral; Academic Dissertations

Abstract

In vivo infection with HIV-1 is typically characterized by progressive clinical and immunological deterioration associated with a decrease in CD4 T-cells. The mechanism of CD4 T-cell depletion in vivo and its role in HIV-1 pathogenesis and development of AIDS is not well understood.

My research has focused on understanding the mechanism or mechanisms by which HIV-1 induces cell death. To address this aim, a panel of primary HIV-1 isolates were characterized in vitro for replication kinetics, syncytium formation, and cytopathic effects. From this panel two viral isolates, one from patient A and the other patient D, were selected for further evaluation. Patient A was asymptomatic with absolute CD4 cell count of 2302 at the time of virus isolation and remains so nine years later. Patient D was symptomatic with absolute CD4 cell count of 64 and has subsequently died from AIDS. Both of these patients maintained high viral load in vivo. When the viruses were examined in vitro, they also replicated to high titers. However, there were dramatic differences regarding the induction of cell death by HIV-1 isolates. Viruses obtained from patient A did not induce cell death although they replicated to high titers. In contrast, viruses obtained from patient D were extremely cytopathic in PBMCs with comparable viral replication. Therefore, viral replication alone does not predict the single-cell killing capacity of primary HIV-1 isolates. HIV-1 viruses isolated from an individual with normal CD4 T-cell numbers and an individual with CD4 T-cell depletion replicated to equivalent levels in primary CD4 T-cells. However, the virus isolated from the symptomatic individual induced cell death and the virus isolated from the asymptomatic individual was non-cytopathic in vitro.

It is known that HIV-1 exists in the host as a group of related viruses known as quasispecies. This diversity allows the virus a broad spectrum of genotypes which result in multiple phenotypic properties. It follows then that a single viral isolate may contain a number of variants which differ in their ability to form syncytia, cell tropism, replication kinetics, as well as cytopathic potential. To address this, biological clones were obtained from each of the patients viral quasispecies and characterized for replication and cytopathicity. These clones, GC6 8-4 (isolated from patient A) and HC4 (isolated from patient D) maintained the same viral phenotype as the parental virus. In other words, HIV-1 biological clone GC6 8-4 was non-syncytium inducing (NSI) and non-cytopathic in vitro. In contrast, HIV-1 biological clone HC4 was syncytium inducing (SI) and cytopathic in vitro.

It has been reported that the envelope gene of HIV-1 contains the major determinants of HIV-1 induced CD4 T-cell depletion (17). To understand what may be responsible for the differences in cytopathic behavior between the biological clones, GC6 8-4 and HC4 (42), I analyzed their envelope genes. Chimeric viruses were constructed by switching env regions from V2 through V3 of the biological clones with the corresponding region from the molecular clone NL4-3. These HIV-1 chimeric viruses exhibited similar replication kinetics as well as syncytium inducing abilities. The chimeric virus containing the env region of biological clone, GC6 8-4, was NCP in the single cell killing assay, while the chimeric virus containing the env region of biological clone, HC4, was CP in the single cell killing assay. These studies suggest the presence of a cytopathicity determinant which maps to the envelope region downstream from V2 and through V3 (Stu I at position 6822 to Nhe I at position 7250 based on NL4-3 sequence (101)).

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