Characterization and use of a recombinant retroviral system for the analysis of drug resistant HIV.

A recombinant retroviral system was used for the analysis of early HIV breakthrough infection in the presence of antiviral drugs. The use of replication-defective HIV allowed a quantitative analysis of a single cycle of infection. This report characterizes this recombinant HIV system and demonstrates it's validity in comparison to standard assays. It is demonstrated that the protease inhibitor XM323 inhibits both early and late events in the HIV life-cycle, while dextran sulphate inhibits only early events. In addition, it is shown that this system can be used for detecting and quantitating drug resistant HIV. Thus, the use of this system may provide both novel information about the stage of the viral life-cycle inhibited and a preliminary assessment of the mechanism(s) responsible for breakthrough infection in the presence of antiretroviral drugs.

Improved blood sample processing for PCR.

To simplify our procedure for blood sample processing for PCR, we introduced a simpler, shorter, and more cost-effective method for the separation of peripheral blood lymphocytes (PBL) and DNA extraction for amplification with Taq polymerase. By this method, blood samples are processed in two simple 15-min steps: (1) separation of PBLs from whole blood by red blood cell lysis with the Roche Specimen Washing Solution, and (2) DNA extraction by heat-detergent treatment of separated PBLs. This new method is simpler than the standard Ficoll-Hypaque method for PBLs separation and Proteinase K digestion for DNA extraction. It is not inhibitory to DNA amplification and it allows effective processing of blood samples even after prolonged storage (as long as 8 days) at room temperature.

Cytomegalovirus enhances lysis of HIV-infected T lymphoblasts.

A T4+ lymphoblastoid cell line (CR-10) persistently infected with the human immunodeficiency virus (HIV) and designated CR-10/NIT was superinfected with cytomegalovirus (CMV) isolated from peripheral blood mononuclear cells of a patient with AIDS. A productive CMV cycle in the CR-10/NIT lymphoblasts was demonstrated by fluorescent antibody staining (IF) using a monoclonal antibody (MAb) to the 150-kDa major capsid protein, by infectivity assays and by electron microscopy (EM). Two-color IF analysis showed that a small percentage of the CR-10/NIT cells were producing both CMV and HIV at any one time. EM studies revealed that all doubly infected cells were lysed whereas most cells infected only with HIV appeared intact. Cell lysis appeared 24 hr after superinfection of the CR-10/NIT cells with CMV and progressed to complete destruction of the cell culture between days 9 and 10. Our results suggest that CMV may convert a mildly cytopathic HIV infection of T lymphoblasts into a highly lytic process.

Responses of neurologic complications of AIDS to 3'-azido-3'-deoxythymidine and 9-(1,3-dihydroxy-2-propoxymethyl) guanine. I. Clinical features.

Fourteen patients with AIDS were treated for 23 neurologic complications: four episodes of acute meningoencephalitis; eight episodes of subacute encephalopathy; two cases of progressive multifocal leukoencephalopathy; and nine cases of polyneuropathy. Nine patients were treated with 9-(1,3-dihydroxy-2-propoxymethyl)guanine (DHPG), one with 3'-azido-3'-deoxythymidine (AZT), and four initially with DHPG directed against cytomegalovirus (CMV) retinitis or encephalitis and subsequently with AZT against human immunodeficiency virus (HIV) encephalopathy. CMV retinitis was a helpful clinical observation indicating neurologic involvement. DHPG produced improvement in two of three cases of acute meningoencephalitis but was ineffective in cases of subacute encephalopathy or neuropathy. AZT therapy resulted in resolution in both of the two treated cases of acute confusional state and in two of the four treated cases of polyradiculoneuropathy with paraparesis but was ineffective in the late stage of subacute encephalopathy. These results suggest that CMV is important in some cases of acute meningoencephalitis, whereas HIV is a dominant pathogen in subacute dementia and polyneuropathy in patients with AIDS. DHPG may be beneficial in the former, whereas AZT appears to be effective in the latter complications.

A human T-cell line resistant to cytopathic effects of the human immunodeficiency virus (HIV).

Infection of human helper T lymphocytes with the human immunodeficiency virus (HIV) results in a rapid induction of cytopathic effects and cell lysis. We isolated a variant of the human T-lymphoblastoid cell line, CEM, that is fully susceptible to HIV infection but resistant to virally induced cytopathic effects. Exposure of the cells, designated CR-10, to HIV resulted in the expression of viral antigens in 100% of cells within 6-9 days. Virus-infected cells remained fully viable and could be cultivated under standard culture conditions for a desired period of time. Parental CEM cells died within 9-12 days after HIV infection. Proviral DNA could be detected in the HIV-infected CR-10 cells by Southern blot and molecular hybridization 4-5 days after infection; the relative amount of proviral DNA reached maximum at Days 6-10 and remained stable during an 8-month follow-up period. Virus production by HIV-infected CR-10 cells was documented by electron microscopy and detection of reverse transcriptase activity in cell culture supernatants. HIV-infected CR-10 cells exhibited a down modulation of the OKT-3, OKT-4, OKT-4A, OKT-8, and OKT-11 T-cell surface markers, but not of the OKT-9 (transferrin receptor). One of the HIV persistently infected CR-10 cell clones has been kept in continuous culture for over 8 months. During this period, the cells remained fully viable, 100% positive for HIV antigens, and negative for most of the T-cell surface markers tested and continued to produce biologically active HIV. The CR-10 and HIV-infected CR-10 cell lines will be useful in studies on the biology of HIV and in the isolation and large-scale propagation of this virus.

High prevalence and high titers of LAV/HTLV-III antibodies in healthy hemophiliacs in the midwestern United States.

Twenty-eight patients from the Nebraska Regional Hemophilia Center were studied for the prevalence and titers of antibodies to lymphadenopathy-associated virus/human T cell lymphotropic virus type III (LAV/HTLV-III) and for clinical symptoms of possible progression to the acquired immune deficiency syndrome (AIDS). Ten of 18 (56 percent) patients with hemophilia A who were frequently treated with commercial factor VIII concentrate were seropositive for LAV/HTLV-III antibodies as determined by immunofluorescent study and Western blot testing. Of the four factor VIII-deficient patients who were seronegative, one had received only heat-treated factor VIII concentrates, two had received only cryoprecipitate, and one had received no transfusions since 1983. None of the patients treated only with factor IX concentrate, volunteer donor plasma, or cryoprecipitate had LAV/HTLV-III antibodies. In nine of 10 seropositive hemophiliacs, titers of serum antibodies to LAV/HTLV-III ranged from 1:1,280 to 1:10,240, indicating a strong immune response against LAV/HTLV-III antigens and/or persistent infection with the virus. Serum from seropositive hemophiliacs interacted on Western blot testing with all the major LAV/HTLV-III polypeptides, including envelope proteins gp 42 and gp 120. Despite the possible exposure to LAV/HTLV-III during the past four years, none of the patients in this group had symptoms suggestive of progression towards AIDS. Whether or not immunity to the AIDS retrovirus developed in this group of patients remains to be determined.

Role of Epstein-Barr virus in acquired immune deficiency syndrome.

We have reviewed the biologic characteristics, immune responses, and diverse array of diseases occurring from Epstein-Barr virus infections in immune deficient patients. We have summarized possible roles of the virus in the risk groups for AIDS. Data is convincing that EBV is responsible for some of the cases of lymphadenomegaly and Burkitt-like, non-Hodgkin's lymphomas in patients with pre-AIDS and AIDS. A hypothesis has been proposed wherein EBV and other stimulants of B and T cells allow productive infection by the retrovirus and spread of HTLV-III throughout the helper T cell populations.

Prevalence of AIDS-associated retrovirus and antibodies among male homosexuals at risk for AIDS in Greenwich Village.

LAV/HTLV-III has been closely linked to the acquired immunodeficiency syndrome (AIDS). We have studied and correlated the prevalence of AIDS-associated retrovirus and retroviral antibodies in several groups of male homosexuals from Greenwich Village. Retrovirus was detected in cultured peripheral blood lymphocytes by testing for reverse transcriptase (RT) and confirmed by establishment of virus-producer cell lines, and electron microscopy. Seventy-six percent of patients with AIDS, 93% with AIDS-related complex (ARC), 69% with generalized lymphadenopathy (LAS), and 35% of asymptomatic homosexuals were positive for virus in the RT assay. Transmission of the virus from RT-positive lymphocytes into the CEM cell line was successful in 10 of 11 randomly chosen cases. No virus isolates were obtained from lymphocytes of 8 heterosexual individuals. Serum antibodies against AIDS-associated virus were detected by indirect immunofluorescence assay and confirmed by Western blotting, using an LAV/HTLV-III-producer cell line, LAV-N1, which we established. LAV/N1 virus was purified by ultracentrifugation through sucrose gradient and the pattern of its proteins was determined by SDS-gel electrophoresis and Western blotting using sera from an AIDS patient. The major polypeptides of LAV/HTLV-III (19, 25-27, 32, 42 and 54 kilodalton) were present. These proteins did not react in Western blots with sera positive for Adult T cell leukemia virus (ATLV). thus, LAV-N1 and ATLV were not antigenically related. In our assay for LAV/HTLV-III antibodies, 18 (100%) of patients with AIDS, 13 (100%) of patients with ARC, 24 (69%) of 35 patients with LAS and 9 (39%) of 23 asymptomatic homosexuals were sero-positive. Heterosexual controls were negative. All IF-positive sera tested by Western blot contained antibodies against specific viral proteins. High titers (greater than or equal to 1:1280) of serum antibodies against LAV/HTLV-III virus were detected in 71% of AIDS patients, 62% with ARC, 38% LAS and 13% among asymptomatic homosexuals. Our data show that the presence of LAV/HTLV-III antibodies correlates with the presence of infectious virus. Antibody titers may also correlate with progression toward AIDS.

Isolation and characterization of an EBV superinfection-enhanced, host-cell-coded, 53,000-dalton protein.

Microsomal membrane fraction proteins with enhanced synthesis after P3HR-1 Epstein-Barr virus (EBV) superinfection of Raji cells were identified with [35S]-methionine labeling and SDS-PAGE. One 53,000-dalton protein, which was found in both the microsomal membrane and cytosol fractions, was purified by ion-exchange chromatography, and specific rabbit antisera were prepared to it. This protein was found to be present in Raji cells, but its expression was enhanced after P3HR-1 EBV superinfection. It was more abundant in the cytosol than in the microsomal membrane fraction of the cell, and its synthesis was not affected by treatment of the cells with phosphonoacetic acid. It was present in several EBV-genome-negative cell lines and in activated B lymphocytes and consequently represents a host-cell-coded protein which is enhanced by EBV superinfection or by lymphocyte activation.

Establishment of retrovirus-, Epstein-Barr virus-positive B-lymphoblastoid cell lines derived from individuals at risk for acquired immune deficiency syndrome (AIDS).

Peripheral blood lymphocytes from male individuals at risk for AIDS were cultured in the presence of interleukin-2. Approximately 90% of cultures originating from pre-AIDS and AIDS patients were retrovirus-positive as detected by the reverse transcriptase (RT) assay and confirmed by electron microscopy. Prolonged incubation of the retrovirus-positive cells resulted in the establishment of several interleukin-2-independent B-lymphoblastoid cell lines. These cells were positive for Epstein-Barr virus (EBV)-specific antigens and contained EBV particles. When irradiated cells from the new lines were cocultivated with an RT-negative T-cell line CEM, an efficient transmission of retrovirus was detected. The supernatants from cocultivated cells had 5-10 fold higher levels of RT activity as compared with the supernatant from the cell line alone. Type-C retroviral particles and budding structures similar to those of human T cell leukemia virus type III (HTLV-III) and lymphadenopathy-associated virus (LAV) were found by electron microscopy. HTLV-III/LAV-specific polypeptides were detected by immunoprecipitation with sera from lymphadenopathy and AIDS patients, but not with sera from healthy individuals. Our data suggest that EBV-infected B lymphocytes from individuals at risk for AIDS may serve as a biological reservoir for the AIDS-associated retrovirus.

p105, an Epstein-Barr virus-induced, phosphonoacetic acid-insensitive glycoprotein target of the anti-Epstein-Barr virus immune response.

To describe structures and biological functions of targets for antibody-mediated immune responses to Epstein-Barr virus (EBV)-infected lymphoid cells, we have characterized a membrane-associated protein of 105,000 daltons, p105, which was prominently recognized in immunoprecipitates with some EBV antigen-reactive patients' sera. A rabbit antiserum to immunopurified p105 was developed. [35S]methionine-labeled p105 was specific to EBV-superinfected Raji cells, and its synthesis was not blocked with phosphonoacetic acid, indicating that it is an "early" viral antigen. Phosphonoacetic acid treatment of EBV-superinfected Raji cells was associated with the accumulation, mainly in the cytosol, of an 88,000-dalton protein, p88, which was also recognized with anti-p105 serum, but was not detected in superinfected cells which had not been treated with phosphonoacetic acid. Although anti-p105 serum immunoprecipitated a membrane fraction protein, it did not neutralize P3HR-1 virus and was not considered to be an exposed virion component. We conclude that p105 is an early, EBV-induced, membrane fraction antigen to which EBV-infected patients generate a substantial antibody response.