Analysis of nonhuman primate peripheral blood mononuclear cells for susceptibility to HIV-1 infection and HIV coreceptor expression.

HIV-1 infection of nonhuman primates does not lead to the acquired immunodeficiency syndrome seen in humans. The basis for this lack of disease progression in these animals is still unknown. In this study, primary nonhuman primate peripheral blood mononuclear cells (PBMC) were tested for their susceptibility to in vitro infection by several different primary HIV-1 isolates representing distinct subtypes or clades. None of the five HIV-1 subtypes tested were able to readily establish an infection in chimpanzee or baboon PBMC, as determined by p24 antigen capture assays. To address the mechanism of in vitro resistance to HIV-1 infection, PBMC were analyzed for HIV coreceptor mRNA expression and cell surface expression. Flow cytometry analysis of the nonhuman primate PBMC demonstrated that they do express CD4, CCR3, CCR5, and CXCR4 on their cell surface. Therefore, the level of restriction in the virus replication cycle does not appear to lie at the point of entry in these cells.

Sequence comparisons of non-human primate HIV-1 coreceptor homologues.

Infection of non-human primate peripheral blood mononuclear cells (PBMCs) in vitro with primary human immunodeficiency virus type 1 (HIV-1) isolates is extremely inefficient and often unattainable. The mechanism of resistance to infection by primary HIV-1 isolates in chimpanzee and baboon PBMCs is unknown. In this study, two HIV-1 coreceptors, CCR5 and CXCR4, were sequenced from chimpanzee and baboon PBMCs to determine if any sequence variations or mutations in these genes could be responsible for resistance to HIV infection. Primers were designed from the human coreceptor sequences and were able to amplify the CCR5 and CXCR4 genes from these non-human primate cells. No 32 base pair deletion (delta32) mutations were found in any of the non-human primate samples tested. CXCR4 sequence analysis showed chimpanzee and baboon share 99.7 and 98% nucleotide sequence homology and 100 and 98.9% amino acid sequence homology, respectively, compared to the human sequence. CCR5 sequence analysis demonstrated that chimpanzee and baboon share 99.6 and 98% nucleotide homology and 100 and 98% amino acid homology, respectively, with the human sequence. These data indicate that no variations in these coreceptor gene sequences exist that can explain the lack of susceptibility to infection with primary HIV-1 isolates in non-human primate PBMCs.

Cross-clade inhibition of human immunodeficiency virus type 1 primary isolates by monoclonal anti-CD4.

A murine monoclonal antibody (MAb) with human CD4 specificity was tested for the ability to inhibit primary human immunodeficiency virus type 1 (HIV-1) isolates clades A through E. Human peripheral blood mononuclear cells (PBMC) were used as target cells for infectivity. The HIV-1 primary isolates were examined for the capacity to infect PBMC targets in the presence or absence of the anti-CD4 MAb, designated P1. P1 broadly inhibited clade A, C, D, and E isolates, based on a reduction of HIV-1 p24 antigen concentrations compared with untreated controls. Little to no virus-inhibiting activity was observed with a primary HIV-1 clade B isolate, designated BZ167. Additionally, a second primary clade B isolate was efficiently inhibited from infecting PBMC targets by P1. The data indicate that P1 exhibits group-specific inhibiting activity against non-clade B primary HIV-1 isolates in vitro.

The outcome of poliovirus infections in K562 cells is cytolytic rather than persistent after hemin-induced differentiation.

K562-Mu erythroleukemia cells readily establish a long-term persistent poliovirus infection characterized by continuous virus production in the absence of complete p220 cleavage and host translation shutoff (R. E. Lloyd and M. Bovee, Virology 194:200-209, 1993). The mechanism of resistance appears to be modulated at the intracellular level and to be related to decreased virus-mediated cytopathic effects (P. A. Benton, J. W. Murphy, and R. E. Lloyd Virology 213:7-18, 1995). It is well documented that hemin induces the differentiation of K562 cells and alters the expression of several host proteins. We report here that growth of K562 cells in hemin prior to poliovirus infection results in a dose-dependent increase in virus-induced cell lysis and thereby alters the normally persistent outcome of infection to a more lytic phenotype. K562 cells infected after hemin treatment displayed increased host translation shutoff, p220 cleavage, viral protein synthesis, and viral RNA accumulation compared with nontreated cells. Since hemin treatment of K562 cells also induced the increased expression of several heat shock proteins (Hsp70, Hsc70, Hsp90, and cohort p60), we tested the hypothesis that their increased expression may play a role in altering poliovirus infection in hemin-treated K562 cells. However, neither heat stress nor oxidative stress, inducers of heat shock protein synthesis, altered the outcome (of virus infections. In addition, we report the novel finding that subunits of two translation initiation factors, p220 (eIF-4G) and eIF-2alpha, are cleaved as a result of hemin treatment of K562 cells. It is proposed that hemin alters the expression of specific host proteins in K562 cells, probably other than heat shock proteins, which changes the initial response to poliovirus infections from persistent to lytic.

K562 cell strains differ in their response to poliovirus infection.

Poliovirus readily establishes a persistent infection in the K562-Mu erythroleukemia cell strain. In this study, three additional K562 cell strains were analyzed for their responses to poliovirus infection and found to be quite variable. K562 cells obtained from the ATCC established a persistent infection, similar to the K562-Mu cell strain, while the majority of cells from two other strains, K562-KI and K562-We, were killed by 4 or 11 days postinfection (p.i.), respectively. Several characteristics of the uninfected and infected cell strains were examined to determine if differences existed which could explain the dramatically different responses to infection. Since K562 cell strains can differentiate toward several cell lineages, the four strains were analyzed for physical and functional likeness to the original K562 cell line using well-established functional criteria to determine whether gross changes in differentiation state had occurred. Based on the lack of MHC class I antigen expression and a dose-dependent increase in globin synthesis in response to hemin, all three laboratory K562 cell strains were indistinguishable from the ATCC reference strain. Surface poliovirus receptor levels were also similar in all K562 cell strains, although four- to fivefold lower than those in HeLa cells. Most biochemical events in virus replication either were very similar among K562 cells or were slightly variable and did not correlate with the degree of cell killing. These included levels of virus production, levels of viral protein produced, and processing and turnover of viral polypeptides. The key difference between the cell strains which consistently correlated with cell killing was the degree of virus-induced host translation shutoff, which was always greatest in the most virus-sensitive K562-KI cells. In addition, levels of 2Apro produced in K562 cell strains did not appear to correlate with the levels of host protein shutoff. A related and novel finding in these studies which also strongly correlated with the outcome of infection was the ability of levels of intact p220 to recover by 24 hr p.i. in virus-resistant K562-Mu and -ATCC cells. These data suggest that the key determinants of outcome of infection in this cell model are cytoplasmic host factors related to cytopathology and not factors which may modulate levels of viral protein synthesis or RNA synthesis.