Retention of HIV-1 inside infected MOLT-4 cells in association with adhesion-induced cytoskeleton reorganization.

OBJECTIVE: To study the mechanism of the suppression of HIV release during cell-to-cell adhesion. DESIGN AND METHODS: To investigate the effects of cell-to-cell adhesion on HIV release in association with cytoskeletal elements, chronically HIV-infected T cells were cocultured with different adherent cell lines, cultured on a fibronectin-coated surface, or treated with cytochalasin D. The amount of viral protein released in the culture supernatant and retained inside the cells was monitored by a p24 enzyme-linked immunosorbent assay and Western blotting. For F-actin staining, cells were stained with FITC-labelled phalloidine and examined by immunofluorescence microscopy. RESULTS: Cocultivation resulted in a reduced amount of virus in the culture supernatant and induced the retention of viral protein inside the infected cells. On adhesion to cells, the F-actin of the infected cells was polarized towards the cell periphery from a diffuse cytoplasmic distribution. Similar data were obtained when the infected cells were treated with cytochalasin D or adhered to fibronectin. CONCLUSION: Cell-to-cell adhesion induced polarization of F-actin, which might facilitate HIV retention inside infected T cells.

2,3 Dimercapto-1-propanol inhibits HIV-1 tat activity, viral production, and infectivity in vitro.

We have examined the effect of 2,3 dimercapto-1-propanol (DMP), which is known as an anti-heavy metal-poisoning drug, against human immunodeficiency virus type 1 (HIV-1). We demonstrate that DMP inhibited transactivation directed by tat protein, which is a metal containing transcriptional transactivating factor and also interfered with viral production. Furthermore, treatment and pretreatment of cells with DMP strongly reduced their sensitivity for HIV-1 infection through unknown mechanisms. These results indicate that DMP reveals pleuripotent effects on HIV-1 infection and production in vitro and thus may provide an exploitable hypothesis for designing new drugs against AIDS.

The mycoplasma-related inhibitor of HIV-1 reverse transcriptase has a DNase activity and is present in the particle-free supernatants of contaminated cultures.

Drastic inhibition of the human immunodeficiency virus (HIV) reverse transcriptase (RT) by mycoplasma has been noted in many laboratories causing confusion in data interpretation. The mycoplasma-related inhibitor of HIV-1 RT was identified as a soluble protein in the particle-free supernatant of a contaminated culture. Gel filtration studies revealed the molecular mass of this protein to be about 70 kDa. This RT-inhibitor contained a DNase with strong activity on both linear and circular DNAs. Addition of this inhibitor after completion of reverse transcription still reduced the final outcome of the RT assay significantly, implying that the inhibitory mechanism occurred mainly by its DNase activity. Treatment of the culture with an antimycoplasma drug cured the mycoplasma contamination, removed the RT-inhibitor and abolished the DNase activity.

Syncytium-inducing capacity of human immunodeficiency virus (HIV): analysis by the use of cloned viruses.

The marked cytopathic effects of human immunodeficiency virus HIV for susceptible cells are caused mainly by fusion between cells expressing viral envelope glycoproteins and cells expressing CD4 molecule. In this study, we tested the ability of different clones of HIV to induce syncytia in CD4-positive cells. We have reported marked difference in syncytium-inducing capacity of 2 clones of human T lymphotropic virus type III (HTLV-IIIB) isolate despite no detectable difference in expression of viral glycoprotein (gp120). This difference in syncytium induction could be explained by the difference detected in their infectivity and binding activities to CD4-positive cells. Meanwhile we reported difference in syncytium-inducing capacity of 2 clones of lymphadenopathy associated virus (LAV1) isolate parallel to the different amounts of gp120 and other viral proteins expressed by these 2 clones. These results suggest that viral factors like infectivity and binding affinity of the virus to the susceptible cells and the amount of viral gp120 expressed by the infected cells may interact in a complex manner affecting fusion activity and syncytium induction in CD4-positive cells.

Synergistic infectivity of highly and minimally infectious clones of human immunodeficiency virus in vitro.

To explore the biologic significance of the presence of a heterogeneous human immunodeficiency virus (HIV) population within infected individuals with regard to ultimate disease progression, the effect of coinfection of more than one variant of HIV on infectivity and cytopathogenicity in CD4-positive cells was examined. Using the lowest and highest infectious clones of HIV obtained by the plaque-cloning method, a clear consistent synergism of infectivity and cytopathic effects was detected when different cell lines were coinfected with a mixture of the two clones. These data suggest that the emergence of a highly infectious variant of HIV due to mutation may modify the infectivity of a minimally infectious latent variant with the final progression of HIV infection to overt AIDS.

Impaired infectivity of HIV-1 after a single point mutation in the POL gene to escape the effect of a protease inhibitor in vitro.

To examine whether the mutation of protease in an HIV-1 resistant to a protease inhibitor affects the virus phenotype in vitro, the infectivity of the protease inhibitor-escape-virus was compared to that of the parent virus. In different T-cell lines, the infectivity of the escape virus was impaired by 10-fold compared to the parent virus. MT-4 cell killing by the escape virus, measured using the MTT assay, was much weaker than that by the parent virus. The escape virus contained more unprocessed Pr55gag than the parent virus. A delayed appearance of mature p24 in cells chronically infected with the escape virus was also noticed by the pulse-chase method. The same findings were obtained using pNL432 (HIV-1 DNA molecular clone) with the same mutation in the protease gene. Despite the lack of a significant difference in virus binding, less unintegrated and integrated DNA was detected in MT-4 cells infected with the escape virus compared to the parent virus. The impaired infectivity of the escape virus may be explained by the inefficient maturation of Gag proteins, due to the mutated protease, which may affect an early step in the virus life cycle.

Exposure of p19 matrix protein of human T-cell leukemia virus type I (HTLV-I) on the surface of MOLT-4#8 cells after virus adsorption.

The p19 matrix (MA) protein of human T-cell leukemia virus type I (HTLV-I) was exposed on the surface of MOLT-4#8 cells in the very early step of the virus infection. Transfer of the virus-binding MOLT-4#8 cells from 4 degrees C to 37 degrees C resulted in increased detection of the viral gp46 and p19 MA protein on the cells, which was, however, inhibited by 4 degrees C or cytochalasin B treatment. These data showed that increased temperature and fluidity of the cell membrane were required for the increased detection of gp46 and p19 after viral adsorption. On the other hand, exposure of the p19 MA protein was not observed on the virus-treated U937 cells although gp46 was detected. This was not due to inefficient binding of the HTLV-I to the U937 cells, since the methanol-fixed cells were p19 MA protein-positive. MOLT-4#8 cells induced marked cell fusion when co-cultured with MT-2 cells, but U937 cells induced no fusion. All of these results indicated that these two cell lines differed in the property of plasma membrane in terms of degradation of HTLV-I envelope after viral adsorption. Uncoating of the HTLV-I might occur on the plasma membrane, especially on MOLT-4#8 cells.

In vitro modification of human immunodeficiency virus type 1 (HIV-1) infectivity by the U937 cells.

The effect of host cell factors on infectivity of human immunodeficiency virus type 1 (HIV-1) was studied by infecting a monoblastoid cell line (U937) or a T-cell line (MOLT-4) with a highly infective single clone of HIV-1 and comparing the infectivity of the produced viruses to different cell lines. Chronically infected U937 cells consistently produced viruses with minimal infectivity. This phenotypic change was host-dependent as the back-passage of the U937-produced low infective viruses into MOLT-4 cells resulted in regaining their original high infectivity. Southern and Northern blot analyses of the HIV-1 grown in U937 cells did not reveal any genomic difference between it and the virus grown it MOLT-4 cells. The radioimmunoprecipitation analysis of viral proteins showed that the HIV-1-infected U937 cells had a different pattern of envelope glycoproteins and core proteins, which well correlated with the low infectivity of the produced viruses. This experimental system using MOLT-4 and U937 cell lines would be useful to further explore host cell factor(s) which play an important role in the regulation of HIV-1 infectivity.

Analysis of 3' terminals of human immunodeficiency virus type 1 transcripts in persistently infected cells.

To examine the 3' terminal processing of human immunodeficiency virus type 1 (HIV-1) transcripts and the effects of phorbol ester (TPA) on this processing, cellular RNAs from persistently infected T cells (MOLT-4) or promonocytes (U937), with or without TPA treatment, were analyzed. To map the 3' terminals of viral transcripts, the RNA samples were examined by RNase-protection assay with an HIV-1 long terminal repeat (LTR) antisense riboprobe. Without TPA treatment, the viral transcripts initiated at the cap site in 5' LTR and polyadenylated at poly(A) site in 3' LTR were dominantly detected in both types of cells. This analysis demonstrated that some occlusion mechanism inactivating the poly(A) site in 5' LTR might exist in these infected cells. After TPA treatment, we found a dramatic shift in the protected patterns of viral transcripts in MOLT-4 cells, while the shift in U937 cells was less dramatic. These results suggested that the primary factor(s) involved in the observed effect of TPA might be cellular. We also demonstrated that the shift in the protected patterns of viral transcripts was associated with increased steady-state levels of viral transcripts. These results indicated that the factors involved in the TPA-induced shift might have some relation to the trans-activation of HIV-1 by similar substances.

Isolation of human T-cell leukemia virus type I from a transformed T-cell line derived spontaneously from lymphocytes of a seronegative Egyptian patient with mycosis fungoides.

Mycosis fungoides (MF) is a rare form of cutaneous T-cell lymphoma that may be associated with human T-cell leukemia virus type I (HTLV-I) infection. Using the polymerase chain reaction, the HTLV-I pX region was constantly detected in the genomic DNA extracted from peripheral blood mononuclear cells (PBMCs) of an HTLV-I antibody-seronegative Egyptian MF patient enrolled in a study to isolate HTLV-I from North Africa. A CD4+ and interleukin-2 (IL-2) receptor-positive T-cell line was established when the phytohemagglutinin-stimulated PBMCs of that patient were maintained in IL-2-containing culture medium. The cell line (EMF) was initially IL-2 dependent and then became IL-2 independent after gradual withdrawal of the IL-2. The cells reacted positively with monoclonal antibodies specific for the HTLV-I Env or HTLV-I Gag proteins. Using the Southern blot analysis, HTLV-I provirus could be detected in the genomic DNA extracted from the EMF cells. Limited nucleotide sequence of the env region showed more than 95% homology between the EMF provirus and other known HTLV-I isolates. Western blot analysis of the cell lysates showed the expression of the HTLV-I structural proteins. These data imply that a transforming HTLV-I provirus may be present, at least in certain cases of MF, regardless of the presence or absence of the specific antibodies.

Generation and characterization of a human immunodeficiency virus type 1 (HIV-1) mutant resistant to an HIV-1 protease inhibitor.

A synthetic peptide, RPI 312, that specifically inhibits the protease of the human immunodeficiency virus type 1 (HIV-1) showed a potent inhibition on virus production, maturation, and infectivity. Treatment with this agent prevented the cleavage of Gag protein at the site between p17 and p24 in HIV-1 chronically infected MOLT-4 cells as well as in the released virus. Passage of HIV-1 in the presence of gradually increasing concentrations of this protease inhibitor resulted in emergence of a variant that could evade the drug effects. In the resistant variant the maturation of Gag proteins appeared normal, but its infectivity was reduced compared with that of the parent virus. The nucleotides coding the amino acids at and around the cleavage site between Gag proteins p17 and p24 were not changed. One point mutation (A-->G) at site 2082 of the pol gene that resulted in one amino acid change at site 84 of the protease from isoleucine to valine (I-84-->V) could be detected in the resistant variant. An HIV-1 infectious DNA clone with the I-84-->V mutation also showed reduced sensitivity to this protease inhibitor. The findings that the resistant variant had lower infectivity and was still affected by higher doses of the drug support the speculation that resistance to protease inhibitors may not be as problematic as other drug resistance.

Inhibition of human immunodeficiency virus replication in a human T cell line by antisense RNA expressed in the cell.

The effect of the expression of antisense RNA against the human immunodeficiency virus (HIV) genome in a human T-cell line CEM on HIV replication was investigated. A 2.7 kilobase (kb) fragment of the HIV genome, including tat and a part of rev and env, was cloned into the retroviral vector WB in the antisense orientation under the SV40 or H-2K promoter. CEM cells transduced with this antisense gene via recombinant retrovirus expressed the RNAs of three different molecular sizes containing the antisense construct. CEM cells and these transduced cells were infected with HIV. HIV replication was evaluated 4-10 days later by an immunofluorescence assay and by determining the reverse transcriptase activity in the culture supernatant. The results indicate that although the recombinant retrovirus WB strongly enhanced the HIV replication in CEM cells, the expression of antisense RNA in the cells was highly effective in impeding the replication of HIV. The inhibitory effect was especially high in CEM cells transduced with the antisense gene under the control of SV40 promoter. In this case, HIV antigen-positive cells and reverse transcriptase activity in the culture supernatant of transduced cells were reduced to 30-50% and 5-10% of those in CEM cells and in the CEM cells transduced with WB, respectively.

Sporadic carriers of human T-lymphotropic virus type I in northern Egypt.

Sera from 3,158 individuals living in northern Egypt were tested for the presence of antibodies against human T-lymphotropic virus type I (HTLV-I) by the newly developed particle agglutination (PA) test. Ten sera gave a positive reaction in the PA test. Eight of these sera were examined further by Western blotting and all of them gave several bands corresponding to HTLV-I structural proteins. Two of the 8 sera gave positive results in the indirect immunofluorescence test. The results indicate the presence of HTLV-I carriers in this area, although at very low incidence (0.063%).