Important note: Information in this article was accurate in 1991. The state of the art may have changed since the publication date.
Biological characterization of SIV molecular clones from tissue of an immunodeficient macaque.
Symp Nonhum Primate Models AIDS. 1990 Nov 28-30;8:22 (abstract no. 6). Unique Identifier : AIDSLINE PRIM8/900006 Hirsch VM; Dapolito G; Goldstein S; Johnson PR; Department of Microbiology, Georgetown University and Laboratory; of Infectious Diseases, NIAID, NIH, Rockville, MD
Abstract:
Tissues of immunodeficient macaques experimentally infected with SIVsm contain multiple diverse genotypes, best described as a proviral swarm. We wished to assess the biologic relevance of these multiple genomes. For this purpose, we used PCR to derive molecular clones of the 3' half (4.3 kb) of the proviral genome from total cellular DNA isolated from the spleen of an immunodeficient pigtail macaque (Pt62). Ten full-length chimeras were constructed between these clones and the 5' end of the infectious molecular clone, smH-4i. Four of the clones thus constructed were evaluated by: (i) sequence analysis, (ii) transfection into CEMX174 cells, (iii) western blot analysis, (iv) cell-free infections of various cell lines, PBMC, and macrophages, and, (v) electronmicroscopy (EM). Each clone was genetically unique; for example, approximately 3% amino acid variation in the envelope was observed between individual clones. Each clone had the potential of encoding all the known structural and regulatory proteins. All four clones produced virus particles after transfection of CEMX174 cells, as shown by western blot analysis and EM. We compared the tropism of cell-free virus in various human cell lines, PBMC, and rhesus peritoneal macrophages. Virus produced from the SIVsm clone, smH-4, replicates in a wide range of human cell lines (CEMX174, CEMss, MT4, H9, AA2, and MT2) and in PBMC of three species (human, pigtail, and rhesus macaque). In contrast, the tissue-derived clones exhibited extremely restricted tropism; for example, only one clone infected CEMX174 cells. However, three clones appeared to replicate in rhesus macrophages. This was not surprising since macrophages were the predominant infected cell type in the source tissue (spleen). Three of these clones were selected for experimental inoculation of pigtail macaques; all macaques were infected as evidenced by seroconversion and/or virus isolation. These highly related clones with varying tropism afforded us the opportunity to dissect the viral determinants of tropism. Interestingly, the region of gp120 implicated in CD4 binding of HIV-1 was strictly conserved in all clones. Assuming that tropic properties reside within gp120, we constructed a chimeric virus consisting of the gp120 coding region of clone SIVsm62a in an smH-4i background. As expected, the gp120 sequences of SIVsm62a conferred its restricted tropism to this chimera. Further constructs subdividing gp120 are ongoing. Our findings suggest that a second cell surface receptor (in addition to CD4) may be important in determining fine specificities of tropism. In addition, considering the restricted tropism of tissue derived clones, isolation of viruses in human cell lines probably causes significant loss of viral genotypes. The individual importance of the diverse genotypes in terms of pathogenesis is unknown.
Keywords: Animal Cell Line Chimera Clone Cells/ULTRASTRUCTURE DNA, Viral/BIOSYNTHESIS Genotype Human HIV Envelope Protein gp120/GENETICS HIV-1/METABOLISM Macaca Polymerase Chain Reaction Proviruses/GENETICS Receptors, Virus/METABOLISM Simian Acquired Immunodeficiency Syndrome/*GENETICS Spleen/CHEMISTRY SIV/GROWTH & DEVELOPMENT/*GENETICS/ULTRASTRUCTURE Virus Replication ABSTRACT
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Important note: Information in this article was accurate in 1991. The state of the art may have changed since the publication date.
