Genomic structure of an attenuated quasi species of HIV-1 from a blood transfusion donor and recipients.

A blood donor infected with human immunodeficiency virus-type 1 (HIV-1) and a cohort of six blood or blood product recipients infected from this donor remain free of HIV-1-related disease with stable and normal CD4 lymphocyte counts 10 to 14 years after infection. HIV-1 sequences from either virus isolates or patient peripheral blood mononuclear cells had similar deletions in the nef gene and in the region of overlap of nef and the U3 region of the long terminal repeat (LTR). Full-length sequencing of one isolate genome and amplification of selected HIV-1 genome regions from other cohort members revealed no other abnormalities of obvious functional significance. These data show that survival after HIV infection can be determined by the HIV genome and support the importance of nef or the U3 region of the LTR in determining the pathogenicity of HIV-1.

HIV-1 can be recovered from a variety of cells including peripheral blood monocytes of patients receiving highly active antiretroviral therapy: a further obstacle to eradication.

During highly active antiretroviral therapy (HAART), HIV-1 can still persist in circulating, resting CD4+ T lymphocytes, lymph node mononuclear cells, and seminal cells of patients despite sustained suppression of plasma viremia to undetectable levels. Sanctuary sites where antiretroviral drug penetration is not optimal may allow local HIV-1 infection of cells within and passing through these tissues. Factors such as imperfect drug adherence due to complicated drug regimens may also result in tissue compartments with suboptimal drug concentrations allowing viral replication. We have examined blood monocytes from HIV-1-infected subjects being effectively treated with HAART to determine virus carriage in these cells. Monocytes were purified from peripheral blood of patients with plasma HIV-1 RNA below 50 copies/mL and who had maintained levels of plasma RNA below detection for 3 months or more. Replication-competent virus could be recovered from the majority of monocyte populations by co-culture with CD8-depleted, PHA-activated, peripheral blood mononuclear cells. Sequencing of the reverse transcriptase and protease genes of the recovered viruses did not reveal resistance to both reverse transcriptase and protease inhibitors. Continued new infection of this transitory, circulating population of cells even during prolonged, effective HAART most likely reflects ongoing, low-level HIV-1 replication within cellular reservoirs and sanctuary sites in the body.

Accumulation of unintegrated circular viral DNA in monocytes and growth-arrested T cells following infection with HIV-1.

Cytocidal retrovirus infection is characterized by rapid accumulation of unintegrated viral DNA forms. These are thought to be generated by multiple rounds of reinfection and have been suggested to play a central role in cytopathogenesis. Here we have reviewed the work done in this area with HIV-1, mostly using acutely and chronically infected T cell and monocytic cell lines and in some cases T cells blocked at S phase of the cell cycle by aphidicolin treatment. To these studies, we have compared our findings with HIV-1 infected primary peripheral blood monocyte-derived macrophages and untreated and growth-arrested MT-2 cells, two biologically disparate cell populations. Using 1- and 2-long terminal repeat (LTR) circular forms as indicators of unintegrated viral DNA, we found similar rapid accumulation in both untreated and growth-arrested MT-2 cells. In contrast, we found much lower levels in monocyte/macrophages. Our findings suggest that accumulation of unintegrated viral DNA does not require virus production and reinfection in growth-arrested T cells. The significantly lower levels found in monocyte/macrophages may reflect superinfection resistance, allowing the maintenance of a persistent infection.

Direct quantification of HIV-1 infectivity for monocyte--macrophages using an infectious focus assay.

Monocyte--macrophages are important target cells and reservoirs for HIV. The existing methods for the quantification of infectious virus in HIV stocks are not totally satisfactory for use with macrophage cultures. We have developed an infectious focus assay for the direct quantification of virions infectious for human peripheral blood monocyte-derived macrophages adhering to plastic microtitre plates. The combination of an HIV-1 p24-antigen-specific monoclonal antibody and a beta-galactosidase-linked second antibody resulted in a sensitive and very specific assay. With 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside as substrate, the assay proved to be as sensitive as p24 antigen quantification in culture supernatants.

HIV-1 DNA and mRNA concentrations are similar in peripheral blood monocytes and alveolar macrophages in HIV-1-infected individuals.

OBJECTIVE: To determine the relative contribution of alveolar macrophages, peripheral blood monocytes (PBM) and peripheral blood lymphocytes (PBL) from HIV-infected individuals to HIV-1 viral load. METHODS: Alveolar macrophages were obtained by flexible bronchoscopy, and PBM and PBL by venipuncture from HIV-1-infected individuals. Alveolar macrophages and PBM were purified using immunomagnetic bead selection to deplete CD3+ and CD19+ cells from bronchoalveolar lavage and peripheral blood mononuclear cells, respectively. DNA and mRNA were extracted and gag copy number quantified using polymerase chain reaction (PCR) and reverse transcriptase PCR. The titres of infectious cell-associated HIV-1 in cells were determined by the endpoint dilution coculture technique for alveolar macrophages and PBM. RESULTS: Alveolar macrophages and PBM from HIV-1-infected subjects (n=11) contained equivalent concentrations of HIV-1 DNA and HIV-1 mRNA as determined by PCR and reverse transcriptase PCR, respectively. Antiretroviral therapy was associated with reduced viral DNA concentrations in alveolar macrophages but not in PBM. PBL had a significantly higher level of proviral DNA and mRNA than alveolar macrophages or PBM. CONCLUSIONS: Although alveolar macrophages infected in vitro are more permissive for HIV-1 replication than PBM, this difference could not be demonstrated in vivo.

Monocytes harbour replication-competent, non-latent HIV-1 in patients on highly active antiretroviral therapy.

OBJECTIVE: To determine whether HIV-1 can be recovered from blood monocytes as well as resting, memory CD4 T lymphocytes of patients on highly active antiretroviral therapy (HAART) with undetectable plasma viraemia and whether infection is active or latent. DESIGN: Five patients with plasma HIV-1-RNA levels of less than 500 copies/ml for at least 3 months and less than 50 copies/ml at the time of sampling were initially selected, followed by an additional five patients with viral loads of less than 50 copies/ml for 3 months or more. METHODS: Monocytes were isolated from blood by plastic adherence, then further purified by a second adherence step or CD3 depletion before co-culture with CD8-depleted donor peripheral blood mononuclear cells. Virus isolates were examined for mutations conferring resistance to reverse transcriptase or protease inhibitors and for genotype. The highly purified monocytes were also analysed for the presence of proviral and unintegrated viral DNA and multiply spliced (MS) viral mRNA by polymerase chain reaction. RESULTS: Virus was recovered from monocytes of five patients. Sequencing of the recovered viruses did not reveal multiple drug resistance, and was consistent with a non-syncytium-inducing/CCR5 phenotype. Proviral DNA was detectable in monocytes from all subjects, and unintegrated HIV-1 DNA and MS RNA was found in four out of five populations examined. CONCLUSION: Recovery of replication-competent virus from some HAART patients indicates that monocytes can also harbour HIV-1. Detection of circular, viral DNA and spliced RNA, albeit at very low levels, in these cells suggests that their infection is recent and transcriptionally active rather than latent.

Granulocyte-macrophage colony-stimulating factor inhibits HIV-1 replication in monocyte-derived macrophages.

BACKGROUND: Previous studies of the effect of granulocyte-macrophage colony-stimulating factor (GM-CSF) on HIV-1 replication in macrophages have had inconsistent results, variously reporting no effect, augmentation or inhibition of viral replication. OBJECTIVE: To investigate the regulation of HIV-1 in monocyte-derived macrophages (MDM) by GM-CSF in vitro. METHODS: The role of GM-CSF on HIV-1 replication was assessed as supernatant and intracellular p24 antigen concentrations and by HIV-1 DNA and mRNA production under different culture conditions. Expression of CD4 and CCR5 receptors was examined. The effect of GM-CSF with an E21R mutation, which binds only to the alpha-chain of GM-CSF receptor, was used as an additional control. RESULTS: GM-CSF consistently suppressed HIV-1 replication in human MDM in vitro, as assessed by supernatant and intracellular p24 antigen concentrations and HIV-1 gag mRNA expression. The inhibitory effect of GM-CSF on HIV-1 replication was observed regardless of HIV-1 strain, source of GM-CSF, stage of MDM maturation or timing of GM-CSF exposure in relation to HIV-1 infection. The effect was dose dependent and reversed by addition of a neutralizing monoclonal antibody (4D4). Flow cytometric analysis of surface expression of CD4 and CCR5 indicates that GM-CSF does not affect HIV-1 entry into MDM. Analysis of intracellular HIV-1 DNA and mRNA suggests that HIV-1 replication is inhibited at or before transcription. E21R GM-CSF had no effect on HIV-1 replication in MDM. CONCLUSIONS: GM-CSF regulates HIV-1 replication in MDM, inhibiting HIV-1 replication through binding to the beta-chain of the GM-CSF receptor.

Surface CD4 is critical to in vitro HIV infection of human alveolar macrophages.

The CD4 glycoprotein is the major cellular receptor for HIV. CD4 surface expression of monocytes decreases with time in culture while their susceptibility to HIV-1 increases. Our aim was to investigate whether this phenomenon occurs in macrophages that have differentiated in vivo by investigating CD4 expression and HIV-1 infection of human alveolar macrophages (AMs). Using flow cytometry to detect CD4 expression by Leu-3a labeled indirectly with fluorescein isothiocyanate or allophycocyanin, we found that CD4 was expressed at low but detectable levels, despite the high background autofluorescence well described in AMs. This finding was supported by the detection of CD4 mRNA in AMs using RT-PCR. T cell contamination of mRNA extracts of AMs was excluded by amplifying in parallel with primers to the constant region of the T cell receptor. Despite this low level of surface CD4, recombinant soluble CD4 and anti-CD4 antibody completely inhibited HIV-1 infection of AMs. We conclude that CD4, although expressed at low levels on the surface of AMs, appears to be critical to HIV-1 infection of these cells.

Susceptibility of human monocytes to HIV type 1 infection in vitro is not dependent on their level of CD4 expression.

Monocytes from HIV-seronegative persons were analyzed for CD4 expression and susceptibility to infection with HIV-1 on the day of isolation and following 1, 2, and 7 days in culture. Although surface CD4 was readily detected on freshly isolated monocytes, these cells were relatively resistant to infection. After 1 to 2 days in culture, when surface expression of CD4 had decreased over 90% to near background levels, cells became susceptible to infection with HIV-1. CD4 expression on monocytes cultured for 7 days was more than four times higher than that on freshly isolated cells, and the cultured cells were fully permissive to infection. These observations suggest that the differing susceptibility of monocytes and monocyte-derived macrophages to infection with HIV-1 is not simply proportional to the level of surface CD4 expression.

Coproantibody response to rotavirus infection.

During three months after a family outbreak of diarrhoeal disease, rotavirus-specific immunoglobulins of the IgA, IgG and IgM classes were measured by enzyme-linked immunosorbent assay in faecal extracts from the four people involved. Shortly afterwards, sequential extracts were obtained from another infant after a proven rotavirus infection. Rotavirus infection was diagnosed by electron microscopy in three of the patients from whom acute-phase faecal samples were obtained, and all five patients developed a transient specific-antibody response. Antirotaviral IgA, IgM and IgG all reached peak titres between two and four weeks after infection, then dropped back to undetectable levels after two months. If these findings are confirmed in larger numbers of cases, they will provide the basis for simple diagnosis of recent rotavirus infections, without the need of even a single sample of serum.

Measurement of immunoglobulin A, G, and M class rotavirus antibodies in serum and mucosal secretions.

A solid-phase, enzyme-linked immunospecific assay for measurement of different immunoglobulin classes of human rotavirus antibodies is described. The antigen, which was adsorbed directly to polyvinyl microtiter plates, consisted of a clarified cell culture stock of the simian rotavirus SA 11. The assay was sensitive and reproducible and could readily be calibrated to determine concentrations of each class of antibody. The assay was applied to measurements of rotavirus antibodies in serum, colostrum, milk, and fecal samples. It particularly facilitates investigations of the role of immunoglobulin A antibodies in immunity to rotavirus infections.

Immunogenic glycoproteins of infectious laryngotracheitis herpesvirus.

The viral glycoproteins produced in cells infected with either vaccine strain or virulent isolates of infectious laryngotracheitis virus, an avian herpesvirus, were identified by in vitro labeling using [14C]glucosamine and [14C]mannose. Chicken antisera to the vaccine strain and to a virulent isolate, and rabbit antisera to the vaccine strain, immunoprecipitated four major viral glycoproteins of 205, 115, 90, and 60K mol wt. Additional glycoprotein bands were recognized by immune chicken and rabbit sera in Western blotting using a glycoprotein fraction purified from extracts of virus-infected cells. Monoclonal antibodies to the immunogenic glycoproteins were produced and characterized by immunoprecipitation and Western blotting. One group of monoclonal antibodies reacted only with the 60K glycoprotein, by both techniques, while a second group reacted with the 205, 115, and 90K glycoproteins in immunoprecipitation and with additional bands of 85 and 160K in Western blotting.

Derivation of neutralizing monoclonal antibodies against rotavirus.

Monoclonal antibodies were derived against the SA11 simian, NIC bovine, and Wa human rotavirus strains and characterized by enzyme-linked immunosorbent assay, plaque neutralization, and hemagglutination inhibition. Several strain SA11-specific antibodies were found to have neutralizing and hemagglutination-inhibiting capacity.

The major surface glycoprotein of simian rotavirus (SA11) contains distinct epitopes.

The polypeptide specificities on monoclonal antibodies previously derived against the SA11 simian, NIC bovine, and Wa human strains of rotavirus were determined by radioimmunoprecipitation of infected cell lysates. All the monoclonal antibodies derived using NIC and Wa were found to be directed against the major component of the inner capsid, while most of the SA11 monoclones were directed against the major outer capsid glycoprotein. When several SA11 glycoprotein-specific monoclonal antibodies were used in competitive binding studies, four distinct epitopes, which correlated with the functional activities of the antibodies, were defined. One epitope appeared most critical for virus neutralization, another was involved to a lesser extent, and the remaining two epitopes seemed to have no role. A possible topographical arrangement of these epitopes is suggested.

Antigens of infectious laryngotracheitis herpesvirus defined by monoclonal antibodies.

Monoclonal antibodies to glycoprotein and protein antigens of infectious laryngotracheitis virus (ILTV) were divided into five groups on the basis of their reactivity in immunofluorescence and Western blotting. Group I antibodies recognised a single band of 60 K and Group II antibodies recognised bands of 205, 160, 115, 90 and 85 k in Western blotting. In immunofluorescence both these groups of antibodies reacted with antigens located in the cytoplasm of fixed virus-infected cells and they also reacted with unfixed cells, suggesting that these antigens are on the surface of virus-infected cells. While Group I monoclonal antibodies did not react with extracts of tunicamycin-treated cells, some Group II antibodies recognised bands of decreased molecular weight compared to those present in untreated cells. The reactivity of the Group II antibodies with extracts of tunicamycin-treated cells suggested that they recognised at least three different epitopes which was confirmed by ELISA additivity assays. Monoclonal antibodies of Group III, Group IV and Group V recognised several low molecular weight proteins from 45 to 24 k. Immunofluorescence studies showed that these were nuclear and cytoplasmic antigens that were not present on the surface of virus-infected cells.

Intracellular localization of rotaviral proteins.

The differential distribution of two SA 11 rotaviral capsid antigens in thin sections of infected cells was examined using antibody-coated colloidal gold electron-dense particles as specific post-embedding immunocyto-chemical labels. The treatment of thin sections of conventionally fixed and embedded tissue specimens with sodium metaperiodate allowed specific localization of the antigens in tunicamycin-treated, infected CV-1 cells. Both protein antigens were investigated with specific anti-rotavirus hyper-immune sera and with specific monoclonal antibodies. These studies showed that the major outer capsid glycoprotein, gp34, of SA11 rotavirus particles was mainly located within the cisternae and along the membranes of the rough endoplasmic reticulum. The antigen of the major inner capsid protein, p42, was identified attached to enveloped virus particles, and even more obviously, on laminar crystalline structures in the nucleus and cytoplasm of the infected cells.

HIV-1 infection of monocyte-derived macrophages reduces Fc and complement receptor expression.

Fc receptor (FcR) and complement receptor (CR) expression on HIV-infected monocyte-derived macrophages may be an important determinant of immune function. We studied the effects of HIV-1 infection of macrophages in vitro on FcR and CR expression. Macrophages were infected with HIV-1DV 7 days following isolation, and the expression of Fc gamma RI-III and CR3 were measured at intervals thereafter by flow cytometry. We found a reduction in receptor expression with the percentage of cells expressing FcRI 14 days post infection declining from 77% to 13%, FcRII fell from 96% to 85%, FcRIII from 45% to 9%, and CR3 from 91% to 67% 14 days following infection. As these receptors are important for macrophage function, their down-modulation may contribute to the pathogenesis of HIV-related disease.

Mutational analysis of the HIV-1 LTR as a promoter of negative sense transcription.

The HIV-1 gene promoter is a bi-directional promoter of transcription. We report the characterization of the negative sense promoter (NSP) by analysis of the effect on negative sense transcription of a series of LTR U3 region substitution mutants. Mutations in the region nt -58 to -183 (positive sense transcription initiation nt +1) reduced transcription to <15% of wild type NSP activity. This region, essential for NSP activity, was designated the core basal promoter. Over expression of NF-kappaB RelA(p65) and LEF-1 increased negative sense expression, as did over expression of H-ras oncogene, consistent with the presence of cognate sequence motifs for NF-kappaB, LEF-1 and RBF. We were also able to confirm that the NSP is a TATA-less promoter inhibited by HIV-1 Tat. Based on our findings, we propose a model for the interaction between the NSP and PSP, and the role of Tat in regulating the interaction.