Simian immunodeficiency virus (SIV(mac)251) membrane lipid mixing with human CD4\u3csup\u3e+\u3c/sup\u3e and CD4\u3csup\u3e-\u3c/sup\u3e cell lines in vitro does not necessarily result in internalization of the viral core proteins and productive infection

The cell binding site of simian immunodeficiency virus (SIV) is believed to be the CD4 molecule. Several CD4+ cell lines are, however, resistant to infection by SIV(mac)251 in vitro and additional cell membrane molecules have been implicated in SIV(mac)251 entry. We investigated the binding, envelope fusion and entry of the viral core proteins (p27) of SIV(mac)251 into two human CD4+ cell lines (H9 and Sup-T1) which are infectible, and one CD4+ (A3.01) and two CD4- cell lines (K562 and Raji) that are resistant to infection. The fusion of the viral and cellular membranes was monitored by a fluorescence assay for lipid mixing. Cell entry of the viral core was evaluated following virus-cell incubation and cell surface trypsinization. We found that SIV(mac)251 can bind to and fuse (membrane lipid mixing) in a temperature dependent but pH-independent fashion with CD4+ and CD4- human-derived cell lines. In contrast. lipid mixing with CD4 expressing EL-4 mouse T cells or Mv-1-lu mink lung fibroblasts was absent or limited, suggesting that certain components of human cell membranes in addition to CD4 are involved in SIV(mac) envelope-cell fusion. Lipid mixing with the human cells was inhibited partially by soluble CD4. Anti-CD4 antibodies inhibited the lipid inter-mixing with H9, but not with Raji cells, whereas neutralizing anti-SIV(mac) sera inhibited fusion with both CD4+ and CD4- cells. Out of the five human cell lines tested, efficient entry of p27 and productive infection took place only with H9 and Sup-T1 cells. In these two cases, the amounts of p27 internalized during virus-cell fusion correlated with the extent of infection

Enhancement of human immunodeficiency virus type 1 infection by cationic liposomes: The role of CD4, serum and liposome-cell interactions

We have reported previously the enhancement of the infectivity of human immunodeficiency virus type 1 (HIV- 1) by liposomes composed of the cationic lipid N[2,3-(dioleyloxy) propyl]-N,N,N-trimethylammonium chloride (DOTMA). To determine the mechanism by which this process occurs, we have investigated the role of CD4, serum concentration and liposome-cell interactions in the DOTMA-mediated stimulation of HIV-1 infection of A3.01 cells. Serum alone significantly inhibited the binding and infectivity of HIV-1, but DOTMA-mediated enhancement of infectivity was more pronounced in the presence of serum than in its absence. HIV-1 binding to cells was increased in the presence of DOTMA liposomes, DEAE-dextran and polybrene, all of which also enhanced infectivity to a similar extent at comparable concentrations. Fluorescence dequenching measurements indicated that DOTMA liposomes fused with HIV-1, but not with cell membranes, in the presence of serum. The enhancing effect of DOTMA liposomes on HIV-1 infectivity was CD4-dependent, and appeared to involve virus-liposome fusion and liposome binding to the cell surface. DOTMA liposomes did not mediate infection of the CD4- K562 and Raji cell lines

P04-15. Prevalence of Broadly Neutralizing Antibody Responses During Acute/Early HIV Infection

Background: Determining how cross-reactive neutralizing antibody (NAb) responses develop during natural HIV-1 infection may provide key information to understand the role they play in controlling the infection and in disease progression. Here we investigated the frequency and breadth of broadly NAb responses during acute and early infection in a well controlled cohort, and attempted to characterize the factors associated with the development of such responses. Methods: The plasma of 38 clade B acutely-infected, antiretroviral-naive subjects from two cohorts was screened for breadth of neutralization against a panel of heterologous isolates in an Env pseudovirus neutralization assay. Clade A, B and C variants were chosen from reference panels of viruses, created to evaluate the NAb responses elicited during infection or immunization. Results: Our preliminary screening strategy demonstrated that broadly neutralizing antibodies can be detected in a third of the infected subjects. Breadth of neutralization can develop as early as one year during natural HIV infection, however the majority of breadth was observed at over two years post infection. Conclusion: Cross-reactive NAbs are developed more frequently during early infection than previously thought. The consequences of this 'early' development of cross-reactive NAbs on plasma viremia and disease progression are under investigation. The work described was supported by National Institute of Health grant number R01 AI047708-11

Fusion activity and inactivation of influenza virus: Kinetics of low pH-induced fusion with cultured cells

The kinetics of fusion of influenza virus (A/PR/8/34) with human promyelocytic leukaemia (HL-60), human T lymphocytic leukaemia (CEM) and murine lymphoma (S49) cells were investigated. Fusion was demonstrated by electron microscopy, and monitored by fluorescence dequenching of octadecylrhodamine incorporated in the virus membrane. Rapid fusion was induced upon mild acidification of the medium. At pH 5, all virus particles were capable of fusing with the cells. The initial rate and the extent of fusion were maximal between pH 4.9 and 5.2 and declined sharply below and above this range. The rate constants of adhesion of influenza virus to cells or erythrocyte ghosts were large, indicating a diffusion-controlled process. The rate constants of fusion of the virus with cells were smaller than those found previously for fusion with various liposomes. Although preincubation of the virus at acidic pH in the absence of target membranes almost completely inactivated the virus in its ability to fuse with erythrocyte ghosts, it reduced the extent of fusion with cultured cells by only 20 to 40%. Kinetic analysis of fusion revealed a mode of inactivation of the virus bound to erythrocyte ghosts or suspension cells, below pH 5.4, different from that of the virus preincubated at low pH without target membranes