Replication of a macrophage-tropic strain of human immunodeficiency virus type 1 (HIV-1) in a hybrid cell line, CEMx174, suggests that cellular accessory molecules are required for HIV-1 entry

Abstract

To investigate the mechanism underlying one aspect of the cellular tropism of human immunodeficiency virus type 1 (HIV-1), we used a macrophage-tropic isolate, 89.6, and screened its ability to infect a number of continuous cell lines. HIV-1(89.6) was able to replicate robustly in a T-cell/B-cell hybrid line, CEMx174, while it replicated modestly or not at all in either of its parents, one of which is the CD4-positive line CEM.3. Analysis by transfection of a molecular clone, a virus uptake assay, and polymerase chain reaction all provided strong evidence that the block to HIV-1(89.6) replication in the CEM.3 line lies at the level of cellular entry. These results were complemented by preparing a CD4-expressing derivative of the B-cell parent, 721.174, and demonstrating that it is permissive for productive HIV-1(89.6) replication. Given these experimental findings, we speculate that there exist cellular accessory factors which facilitate virus entry and infection in CD4-positive cells. Furthermore, these cellular accessory factors may be quite virus strain specific, since not all macrophage-tropic strains of HIV-1 were able to replicate in the CEMx174 hybrid cell line. This experimental model provides a system for the identification of one or more of these putative cellular accessory factors.

Human immunodeficiency virus type 1 infection of SK-N-MC cells: domains of gp120 involved in entry into a CD4-negative, galactosyl ceramide/3' sulfo-galactosyl ceramide-positive cell line

“…Using chimeras obtained between viruses that infect or do not infect SK-N-MC, we have identified a 193-amino-acid region encompassing V3, V4, and V5 of the HIV-1 HxB2 gp120 that confers the ability to infect SK-N-MC to a phenotypically negative isolate, HIV-1 89.6 . Although HIV-1 89.6 is a macrophage-tropic virus (17,19,45), experiments with a small panel of HIV-1 isolates showed that macrophage tropism bore no clear relationship to SK-N-MC infectability (Table 1). Interestingly, when this 193-amino-acid region was subdivided into two pieces, one of which contained the V3 loop, the other more distal regions, either half allowed the 89.6 virus to infect SK-N-MC, although quantitative competitive PCR identified quantitative differences among the recombinants.…”

“…High-titer cell-free viral supernatants were then filtered and stored in 1.0-ml aliquots at Ϫ80ЊC until use. The 50% tissue culture infectious dose (TCID 50 ) was determined by inoculation of CEMx174 cells with 10-fold dilutions of virus stocks and observing the cells for cytopathological changes (45); the endpoint was calculated by the method of Reed and Muench (43). HIV-1 primary isolates contributed by the WHO Network for HIV Isolation and Characterization were obtained from the AIDS Reagent Program and amplified in interleukin-2-stimulated peripheral blood mononuclear cells (54a).…”

“…The aqueous phase was removed, and the DNA was precipitated by the addition of 2 volumes 100% ethanol at Ϫ80ЊC and pelleting in a microcentrifuge for 10 min. (45). Reaction mixtures that contained the GAG04 (CATICTATTTGTTCITGAAGGGTACTAG, nt 1081 to 1054 of HxB2)-GAG06 (GCITTIAGCCCIGAAGTIATACCCATG, nt 822 to 848 of HxB2) primer pair were subjected to three cycles of 97ЊC for 1 min, 55ЊC for 2 min, and 72ЊC for 1 min followed by 37 cycles of 94ЊC for 1 min, 55ЊC for 2 min, and 72ЊC for 1 min and finally 5ЊC for 5 min (39,40).…”

“…SK-N-MC/CD4 cells express the CD4 molecule. SK-N-MC/CD4 cells were prepared as described previously(45) and analyzed by flow cytometry. Fluorescence with a combination of monoclonal antibodies OKT4 and OKT4A (CD4), positive control antibody W632 (HLA class I), or a negative control (NEG CNTRL) antibody is shown in the flow cytometry tracings.VOL.…”

Human plasma enhances the infectivity of primary human immunodeficiency virus type 1 isolates in peripheral blood mononuclear cells and monocyte-derived macrophages

“…Virus binding/internalization assay. To more directly measure the effect of human plasma on the binding of HIV-1 to primary cells, the amounts of cellbound viral particles were measured by a quantitative p24 enzyme-linked immunosorbent assay (ELISA) as previously described (40,52). In each tube, 6 ϫ 10 5 cells were incubated with 80% (vol/vol) human plasma or medium, with the addition of 20% (vol/vol) virus inoculum at a total volume of 600 l. At each time point, infected cells were centrifuged, washed two times with PBS, resuspended in 200 l of 1% Triton X-100, and incubated for 1 h at 37ЊC.…”