Thesis Abstract and Dissertation Abstracts
HIV-1 Envelope Gene and Protein: Differential Tropism Among Viral Strains
Abstract Category: Science
Course / Degree: Anatomy and Cell Biology; Immunology; Pathology
Institution / University: SUNY Health Sciences Center at Brooklyn, New York, United States
Published in: 1998
This thesis addressed specific aspects of HIV-1 tropism and extended the conventional understanding by looking beyond virus/receptor interactions as an explanation for tropism.
The consequence of known differences between genomic sequences within the V3 region of T-cell (T-) tropic HIV-1 and those in macrophage/monocyte (M-) tropic viruses was investigated at an intracellular level. Strains of HIV-1 can be classified as either T-tropic or M-tropic based on characteristic nucleotide changes within V3, in the HIV-1 envelope (env) gene. These changes are minimal yet result in a reduced capacity of T-tropic isolates to productively infect macrophages. A function for env in addition to virus binding and entry was hypothesized, and the block to productive infection of T-tropic HIV-1 in macrophages investigated. Particular attention was given to the role of M-tropic env and its protein product.
HIV-1 replication in T cells was studied to evaluate the kinetics of viral DNA synthesis. Infection of macrophages was studied using human peripheral blood monocytes (induced to differentiate to mature macrophages), and also the human cell line HL60, which could be induced to differentiate into macrophage-like cells. An alteration in the tropism of a T-tropic HIV-1 was achieved when previously permissive HL60 cells were induced to differentiate into macrophage-like cells. Differentiated HL60 cells did not support productive infection by the same T-tropic virus. Manipulation of the HIV-1 genome was developed and utilized in order to more closely examine the inability of T-tropic HIV-1 to infect macrophages.
Pseudotypes of HIV-1 composed of a T-tropic, but env-deleted, genome and either M-tropic or T-tropic envelope protein, both entered human monocyte-derived macrophages (MDM). Reverse transcription was evaluated by polymerase chain reaction (PCR) assay for strong-stop, full-length, or nuclear forms of viral cDNA. Binding and entry of T-tropic HIV-1 occurred in MDM followed by reverse transcription but complete progression through the viral replication cycle did not.
Additional experiments utilized pseudotype viruses constructed from a parent HIV-1 clone (pGP) which was made incapable of expressing M-tropic env (pGPmsk). This was accomplished by PCR-directed mutagenesis of pGP env using two sets of primers designed for this purpose. Infectivity of pGPmsk was restored by either T- or M-tropic envelope protein supplied in trans from protein expression vectors during co-transfection of a producer cell line (RD cells). Resultant pseudotypes were identical except for their T-tropic or M-tropic envelope. They were competent only for single-cycle infection due to termination of env expression, rather than by env deletion. PCR analysis of viral DNA revealed that each pseudotype reverse transcribed its RNA in MDM. This confirmed that T-tropic HIV-1 entered macrophages but further suggested that intracellular M-tropic (ADA V3) genomic sequences influenced subsequent reverse transcription. Differences in reverse transcription were observed and no productive infection was detected in either system. The block to T-tropic HIV-1 infection in MDM was delimited as occurring after the initiation of reverse transcription but before virus expression can take place. The intracellular presence of M-tropic (ADA V3) genomic sequences led to enhanced T-tropic HIV-1 replication in MDM. In other experiments, exogenous M-tropic envelope protein influenced the reverse transcription of two different strains of T-tropic HIV-1.
These data confirm earlier reports that T-tropic HIV-1 can indeed enter and begin reverse transcription in an alternate host cell-type. They also support the notion that although initiated, infection by T-tropic HIV-1 in macrophages is blocked at an intracellular level. Taken together, the results presented negate the notion that the HIV-1 env-mediated binding to cellular surface receptors alone determines the capacity of a given HIV-1 to infect a target cell. Additional virus/host-cell interactions may be important in determining the overall replicative capacity of a given HIV-1.
Thesis Keywords/Search Tags:
HIV-1; tropism; macrophages; AIDS; reverse transcription
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Submission Details: Thesis Abstract submitted by Michael G. Pellegrino from United States on 11-May-2006 20:37.
Abstract has been viewed 2808 times (since 7 Mar 2010).
Michael G. Pellegrino Contact Details: Email: consultmgp@aol.com
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