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We investigated whether cells derived from the fetal central nervous system can support productive infection by a human immunodeficiency virus type 1 (HIV-1) isolate termed UHC-1, produced by a cellular clone derived from HIV-1 strain HIV-IIIB chronically infected U-937 promonocytic cells, and what the effect of nucleoside analogs might be on viral replication in this system. Fractionation of human fetal brain tissue into two different populations, enriched for either astrocytes or macrophages, showed that only the latter were able to support productive UHC-1 replication and generation of detectable progeny virus. Pretreatment of fetal brain macrophages with either of two nucleoside analogs, 3'-azido-3'-deoxythymidine (AZT) or the (-) enantiomer of 2'-deoxy-3'-thiacytidine, efficiently blocked production of progeny virus. Generation of unintegrated proviral DNA and HIV-1 transcripts were inhibited by pretreatment of fetal brain macrophages with 1 microM AZT. Administration of AZT at 24 h postinfection led to a slight reduction in viral transcript levels and viral progeny production by day 15 postinfection; however, brain macrophages under these conditions did not contain detectable amounts of unintegrated viral DNA. These results suggest that AZT may interfere with the accumulation of unintegrated HIV-1 DNA in brain macrophages. This is the first demonstration that nucleoside analogs are able to block HIV-1 replication in primary cultures of brain cells.

During retroviral assembly, tRNAs are incorporated into the virion, one of which serves as a primer for the reverse transcription reaction. Using two dimensional polyacrylamide gel electrophoresis, we have studied the patterns of tRNAs incorporated into HIV-1 (3B) produced either in the lymphoid cell line H-9 or in the monocytic cell line U937. We have also examined viral tRNA patterns incorporated in a non-infectious, mutant virion which lacks pol gene products and processed gag protein. Our results lead to the following conclusions: 1) tRNA incorporated into HIV-1 is a select subpopulation of the host-cell's tRNA. 2) The type of tRNA incorporated into the virion is dependent upon cell type. 3) There can be multiple species of tRNA of similar mobilities tightly associated to the viral genome. 4) The packaging of putative primer tRNA into virions requires either the synthesis of pol gene products, the processing of gag proteins, or both, while the incorporation of non-primer tRNAs does not.

Limiting-dilution techniques were employed to derive single-cell clones from U-937 cells that had been chronically infected with human immunodeficiency virus type 1. All clones thus obtained were positive for the presence of viral antigens; however, not all of the clones produced infectious progeny virus, as detected by the presence of reverse transcriptase (RT) activity in culture fluids. Six of these clones were monitored over time to determine whether their phenotype of human immunodeficiency virus type 1 expression was stable. Three clones maintained production of RT activity at a high level and showed a very high percentage of cells positive for viral p24 antigen, as determined by indirect immunofluorescence. The other three clones showed variations in either their levels of RT activity or the number of cells positive for p24, after which they stabilized. Infectious virus could be recovered from only three clones, as assessed by coculture experiments with different cell types. Two other clones were shown to produce noninfectious viruses. Molecular analyses at the DNA, RNA, and protein levels showed extensive variations between the viral isolates recovered from each clone.

The effects of human immunodeficiency virus 1 (HIV-1) infection on cellular differentiation and NF-kappa B DNA binding activity have been investigated in a new model of myeloid differentiation. PLB-985 cells represent a bipotential myelomonoblastic cell population capable of either granulocytic or monocytic differentiation after induction with appropriate inducers. By virtue of the presence of CD4 on the cell surface, PLB-985 cells were chronically infected with HIV-1 strain IIIB. PLB-IIIB cells clearly possessed a more monocytic phenotype than the parental myeloblasts, as determined by differential staining, increased expression of the myeloid-specific surface markers, and transcription of the c-fms proto-oncogene. NF-kappa B binding activity was inducible by tumor necrosis factor and phorbol myristate acetate in PLB-985. However, in PLB-IIIB cells, constitutive expression of a novel NF-kappa B complex was detected, composed of proteins ranging between 70 and 110 kD. These proteins interacted specifically with the symmetric NF-kappa B site from the interferon beta (IFN-beta) promoter. Mutations affecting the 5' guanine residues of the kappa B site were unable to compete for these NF-kappa B-related proteins. Inducibility of endogenous IFN-beta and IFN-alpha RNA was also increased in PLB-IIIB cells. These studies indicate that HIV-1 infection of myelomonoblastic cells may select for a more mature monocytic phenotype and that unique subunit associations of NF-kappa B DNA binding proteins may contribute to differential NF-kappa B-mediated gene expression.

Single-cell clones derived from the U-937 monocytic cell line were studied for susceptibility to infection by human immunodeficiency virus type 1 (HIV-1). Of four such clones, we found that three (UC12, UC14, and UC18) supported replication of HIV-1 more efficiently than parental U-937 cells, as measured by reverse transcriptase activity and p24 core antigen production. In contrast, another clone (UC11) showed only baseline infection throughout an 8-week culture period, before finally becoming positive for expression of viral antigen. This differential susceptibility to infection directly correlated with accumulation of intracellular viral DNA. Furthermore, the UC11 clone expressed lower levels of Sendai virus-inducible tumor necrosis factor alpha mRNA than did the UC12 or UC18 clones. Susceptibility to infection did not correlate with expression of cell surface CD4, since all clones expressed similar levels of CD4 mRNA and surface membrane CD4 protein. Prior exposure of both susceptible UC18 and resistant UC11 clones to Leu3a antibody completely blocked infection by HIV-1, suggesting that no other independent receptors were recognized by the virus.

Our results demonstrate that the formation of intracellular complexes between the envelope glycoprotein precursor gp160 of human immunodeficiency virus type 1 and CD4 is a major event, leading to the disappearance of CD4 at the cell surface of infected U937 cells. Using both productively and defectively infected clones of U937 cells, we assessed the effect of CD4-gp160 intracellular association on the maturation of both proteins. Pulse-chase labeling followed by sequential immunoprecipitation was used to analyze the processing of both free and associated CD4 and gp160, and the results showed that the trimming, proteolytic cleavage, and degradation of gp160 were completely abrogated after intracellular binding to CD4. Similarly, the maturation process which normally transforms 80% of CD4 to a partially endoglycosidase H-resistant species was also impaired subsequent to the formation of these complexes. A comparison of gp160 maturation either in free form or as a CD4 complex revealed that neither inefficient transport nor degradation of gp160 can account for the observed blockage of CD4 maturation. Moreover, this impairment was independent of gp120 and gp41, since a defective clone of human immunodeficiency virus type 1-infected cells, unable to cleave gp160, showed binding of CD4 and inhibition of CD4 transport and maturation with the same efficiency as occurred in productively infected cells. Expression of gp160 is thus necessary and sufficient to cause CD4 receptor down-modulation for both productively and defectively infected cells.

Polyethylene glycol was used to induce polykaryon formation among U-937 cell subclones carrying defective human immunodeficiency virus (HIV) type 1 proviral DNA. Fusion of cells which produced gp120-defective virions (UHC15.7) with cells unable to generate reverse transcriptase (RT) activity (UHC8 and UHC18) yielded polykaryons which made infectious viral progeny that showed normal protein profiles. Southern blot analysis of proviral DNA of cells infected with such fusion-derived virus revealed a restriction map identical to that of cells harboring infectious parental-type HIV type 1 (U-937/UHC1). These results suggest that repair mechanisms involving genetic recombination(s) play a role in the generation of infectious virus after fusion of cells which harbor defective HIV.

The effect of cyclosporine A (CyA) on the ability of the human immunodeficiency virus type 1 (HIV-1) to infect the H-9 T-cell leukemic line, as well as interleukin-2 (IL-2)-grown human peripheral blood- derived lymphocytes, has been studied. Pretreatment of H-9 cells and human lymphocytes with CyA over 24 hours completely prevented viral infection over a 21-day period, whereas the addition of drug at two hours postinfection with HIV-1 had a significant inhibitory effect on viral replication and expression of the virus-specific antigens p17 and p24. However, if CyA was added at later times to these lymphocytic cells, this inhibitory effect was lost. Indeed, the removal of CyA from cultures that had been treated from two hours after infection led to the rapid production of progeny virus. HIV-1 was able to infect peripheral blood lymphocytes obtained from each of four kidney allograft recipients on long-term CyA antirejection therapy, as long as drug was not included in the culture medium. In addition, we asked what effect pretreatment with CyA of cells of the U-937 monocytic line and primary cultures of human monocytes/macrophages might have on infection by HIV-1. CyA had no demonstrable effect on the ability of HIV-1 to infect cells of either type.