GSBS Dissertations and Theses

Approval Date

January 1993

Document Type

Doctoral Dissertation

Department

Graduate School of Biomedical Sciences

Subjects

Chronic Disease; Gene Expression Regulation; HIV-1; Academic Dissertations; Dissertations, UMMS

Abstract

Human immunodeficiency virus type 1 (HIV-1) infections have different patterns of expression in different T-cell lines. HIV-1 encodes regulatory as well as structural genes. The role of HIV-1 regulatory gene expression in determining different patterns of infection was explored in four T-cell lines: C8166, H9, A3.01, and Jurkat. The hypothesis being tested was that differences in the expression of regulatory genes would determine differences in the kinetics of infection.

To study patterns of regulatory and structural gene expression, RNA was isolated from cultures infected with HIV-1-NL4-3 (NL4-3). During the early and acute phases of infection, the absolute amounts of viral RNA differed in the four T-cell lines. However, the relative proportions of messages for regulatory and structural genes were similar. Thus, differences in the kinetics of infection in C8166, H9, A3.01 and Jurkat cells were not determined by differences in the relative levels of expression of regulatory and structural genes.

Analyses of RNA samples from the chronic phase of infection revealed the consistent appearance of novel RNase sensitive sites in H9 and Jurkat cultures. These marked the emergence of viral variants with high ability to establish chronic virus producers. These variants were specifically selected in the chronic phase since they did not undergo selection during serial passage of the virus through the lytic phase of infection. Sequence analysis of the region with the novel RNase sensitive sites revealed the co-mapping of nucleotide changes with each of the novel sites. Most of these differences represented a sense mutation in tat and the abrogation of the initiator methionine of vpu. However, the selected mutations in tat and vpu were not sufficient, by themselves, to affect the ability of NL4-3 to establish chronic virus producers (Chapters I and II).

Further studies on the roles of viral sequences in the chronic phase of infection were undertaken using constructed viruses. Two molecularly cloned viruses, NL4-3 and HIV-1-HXB-2 (HXB-2), were used as parents. NL4-3 has a low ability to establish chronic virus producers. In contrast, HXB-2 has a high ability to establish chronic virus producers. NL4-3 encodes all known HIV-1 genes, whereas HXB-2 is defective for three auxiliary genes: vpr, vpu, and nef. In addition, both viruses differ at other positions throughout the genome. The first series of constructed viruses tested whether differences in auxiliary gene expression determined differences in the ability of NL4-3 and HXB-2 to establish chronic virus producers. NL4-3 mutants containing all possible combinations of the three defective genes in HXB-2 were constructed. Analysis of the ability of these mutants to establish chronic virus producers revealed that vpr and nef limit the ability of NL4-3 to establish chronic virus producers. This was shown by viruses with defects in both of these genes having high ability to establish chronic virus producers (Chapter III).

The second series of constructs tested for the roles of non-auxiliary as well as auxiliary gene sequences on chronic virus production by creating recombinants between NL4-3 and HXB-2. Tests of these recombinants revealed that a gag, pol, vif, and vpr fragment could affect the ability of fragments containing defective auxiliary genes to establish chronic virus producers. Taken together, these results indicate that vpr, nef, and 5' internal sequences play important roles in determining the ability to establish chronic virus producers (Chapter IV).

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