CD4-independent infection by human immunodeficiency virus type 1 after phenotypic mixing with human T-cell leukemia viruses.

Although human immunodeficiency virus (HIV) is the causative agent of the acquired immunodeficiency syndrome and related disorders, it has been suggested that viral cofactors may accelerate the progression of the disease. We present evidence that human T lymphoid cells productively coinfected by HIV type 1 (HIV-1) and human T-cell leukemia virus type I (HTLV-I) or HTLV-II generate a progeny of phenotypically mixed viral particles that allow the penetration of HIV-1 into previously nonsusceptible CD4- human cells, including mature CD8+ T lymphocytes, B lymphoid cells, epithelial cells, and skeletal muscle cells. The infection is independent of the major HIV-1 receptor, (i.e., the CD4 glycoprotein) since OKT4a, a neutralizing anti-CD4 monoclonal antibody, fails to block the penetration of HIV-1. Similarly, infection is not inhibited by monoclonal antibody M77, directed toward the neutralizing loop of the gp120 envelope glycoprotein of HIV-1. In contrast, pretreatment of the virus stock with HTLV-I-neutralizing human serum completely abolishes the penetration of phenotypically mixed HIV-1 into CD4- cells. These results suggest that HTLV-I or HTLV-II may increase the pathogenicity of HIV-1 by broadening the spectrum of its cellular tropism and, thus, favoring its spread within the organism of coinfected hosts.     

Reduced glycosylation of human cell lines increases susceptibility to CD4-independent infection by human immunodeficiency virus type 2 (LAV-2/B).

The human immunodeficiency virus type 2 (HIV-2) strain LAV-2/B is able to infect a variety of human cell lines via a CD4-independent pathway. We have used the glycosylation inhibitors tunicamycin, swainsonine, and deoxymannojirimycin to further characterize this putative alternative receptor for HIV-2 (LAV-2/B). These antibiotics resulted in an increase (5- to 30-fold) in the susceptibility of a variety of CD4- human cell lines to infection by LAV-2/B (RD, HeLa, HT29, Rsb, Heb7a, Hos, and Daudi). Several nonprimate cell lines (mink Mv-1-lu, rabbit SIRC, hamster a23, mouse NIH 3T3, cat CCC, and rat HSN) remained resistant to infection by LAV-2/B after treatment with glycosylation inhibitors, suggesting that they do not express the HIV-2 CD4-independent receptor. Two of these nonprimate cell lines are readily infected by HIV-2 when they express CD4 (Mv-1-lu and CCC). Treatment of human cells with neuraminidase had no effect on subsequent infection by LAV-2/B, suggesting that the increase in susceptibility to infection of deglycosylated cells is not due to a change in the electrostatic charge of the cell surface. Treatment of RD CD4- cells and HeLa CD4+ cells with a variety of proteases resulted in a 75 to 90% decrease in infection by LAV-2/B when compared with untreated cells. Taken together, all these data suggest that HIV-2 can utilize a membrane glycoprotein other than CD4 to attach and fuse with a variety of human cells.     

A trans-receptor mechanism for infection of CD4-negative cells by human immunodeficiency virus type 1.

Chemokine receptors, particularly CCR5 and CXCR4, act as essential coreceptors in concert with CD4 for cellular entry by human immunodeficiency virus type 1 (HIV-1; reviewed in [1]). But infection of CD4(-) cells has also been encountered in various tissues in vivo, including astrocytes, neurons and microvascular endothelial cells of the brain [2] [3] [4] [5] [6], epithelial cells [5] [7], CD4(-) lymphocytes and thymocytes [8] [9], and cardiomyocytes [10]. Here, we present evidence for the infection of CD4(-) cell lines bearing coreceptors by well-known HIV-1 strains when co-cultured with CD4(+) cells. This process requires contact between the coreceptor-bearing and CD4(+) cells and supports the full viral replication cycle within the coreceptor-bearing target cell. Furthermore, CD4 provided in trans facilitates infection of primary human cells, such as brain-derived astrocytes. Although the pathobiological significance of infection of CD4(-) cells in vivo remains to be elucidated, this trans-receptor mechanism may facilitate generation of hidden reservoirs of latent virus that confound antiviral therapies and that contribute to specific AIDS-associated clinical syndromes.     

Cofactor requirement for human immunodeficiency virus type 1 entry into a CD4-expressing human cell line.

Expression of the human immunodeficiency virus type 1 (HIV-1) receptor CD4 on many nonhuman and some human cell lines is not sufficient to permit HIV-1 infection. We describe a human glioblastoma cell line (U373-MG) which remains resistant to HIV-1 despite the added expression of an authentic CD4 molecule. The block to HIV-1 infection of these cells is strain independent and appears to be at viral entry. Heterokaryons of CD4-expressing U373-MG (U373-CD4) cells fused to HeLa cells allow HIV-1 entry. A U373-CD4/HeLa hybrid clone allows efficient HIV-1 replication. These results suggest that HeLa cells express a factor(s) that can complement the viral entry defect of U373-CD4 cells and is necessary for efficient CD4-mediated HIV-1 infection.     

A seven-transmembrane domain receptor involved in fusion and entry of T-cell-tropic human immunodeficiency virus type 1 strains.

Entry of human immunodeficiency virus type 1 (HIV-1) into cells requires binding to CD4 and fusion with a cellular membrane. Fusion does not occur in most nonhuman cells even when they express human CD4, indicating that one or more human accessory factors are required for virus infection. Recently, a seven-transmembrane domain protein has been shown to serve as an accessory factor for T-cell-tropic (T-tropic) HIV-1 isolates (Y. Feng, C. C. Broder, P. E. Kennedy, and E. A. Berger, Science 272:872-877, 1996). Here we show that expression of this glycoprotein, termed fusin, in murine, feline, simian, and quail cell lines, in conjunction with human CD4, rendered these cells fully permissive for HIV-1 envelope glycoprotein (Env)-mediated membrane fusion. Expression of CD4 or fusin alone did not permit fusion. In addition, introduction of fusin and CD4 into a human cell line, U87MG, that is resistant to HIV-1 induced syncytium formation and to infection by HIV-1 when expressing CD4 alone made this cell line permissive for Env-mediated cell-cell fusion. Fusion was observed only with T-tropic Env proteins. Macrophage-tropic (M-tropic) Env proteins from the SF162, ADA, and Ba-L HIV-1 strains did not fuse with cells expressing fusin and CD4, suggesting that M-tropic viruses utilize an accessory molecule other than fusin. Finally, coexpression of fusin and CD4 made both a murine and feline cell line susceptible to virus infection by T-tropic, but not M-tropic, HIV-1 strains.     

Human Immunodeficiency Virus Type 1 (HIV-1) Infection and Transcytosis Activity of a HIV-1 Susceptible Clone from HeLa Cell

In order to clarify the transmission process of human immunodeficiency virus type 1 (HIV-1) through the epithelial cell barrier, HeLa cells susceptible and non-susceptible to HIV-1 were cloned and designated as P6 HeLa and N7 HeLa cells, respectively. P6 HeLa cells could be infected with the LAI strain of HIV-1 and mediated HIV-1 transcytosis. In contrast, N7 HeLa cells exhibited neither HIV-1 infection nor transcytosis. CD4 and galactosylceramide as the receptors for HIV-1 were not detected on P6 HeLa cells, although an anti-CD4 monoclonal antibody (mAb) blocked HIV-1 infection. Since HIV-1-infected P6 HeLa cells exhibited no fusion and survived, we speculated that the P6 HeLa cells expressed molecules other than CD4 which facilitated HIV-1 infection. Two mAbs (A-14 ITK and C57 a9-9) which inhibited the HIV-1 infection of P6 HeLa cells were generated. Each mAb recognized distinct molecule(s) as shown by Western blotting. Transcytosis by the P6 HeLa cells was inhibited by C57 a9-9 but not by A-14 ITK or anti-CD4 mAb. Both infection and transcytosis may be responsible for HIV-1 transmission through epithelial cells in a complex manner. Although infection and transcytosis occurred via different mechanisms, the molecule(s) recognized by C57 a9-9 mAb may be associated with both processes.     

Opsonization of HIV-1 by semen complement enhances infection of human epithelial cells

In the present study we demonstrate that both X4- and R5-tropic HIV-1 strains are able to infect the human epithelial cell line HT-29. Infection was enhanced 2-fold when HIV was added to semen before contact with the cell cultures. The enhancing effect of semen was complement dependent, as evidenced by blockage of generation of C3a/C3a(desArg) in semen by heat or EDTA treatment of semen and suppression of semen-dependent enhancement with mAbs directed to complement receptor type 3 (CD11b/CD18) and soluble CD16. Infection of HT-29 cells was assessed by the release of p24 Ag in cultures and semiquantitative PCR of the HIV-1 pol gene. Inhibition of infection of HT-29 by stromal cell-derived factor 1 was decreased in the case of semen-opsonized X4- and R5-tropic virus compared with unopsonized virus. In contrast, inhibition of infection by RANTES was increased for opsonized X4-tropic HIV-1 compared with unopsonized virus. Taken together these observations indicate that activation of complement in semen may play an enhancing role in mucosal transmission of HIV-1 by facilitating infection of epithelial cells and/or enhancing infection of complement receptor-expressing target cells in the mucosa.     

Modulation of surface transferrin receptors in lymphoid cells de novo infected with human immunodeficiency virus type-1

To investigate whether transferrin receptor (CD71) expression is affected by acute HIV-1 infection, three different lymphoid cell lines (MT-4, SUPT-1, H9) were infected with HIV-1 and tested for surface CD71 expression after different incubation periods depending on cell survival after infection. We found that expression of surface CD71 was lower in cells infected with HIV-1 than in uninfected controls: the timing and extent of this down-modulation depended apparently on the different susceptibility of the cell lines to HIV-1 infection and cytopathogenicity. Citrate, a molecule capable of chelating iron, dose-dependently prevented down-modulation of surface CD71 in HIV-1 infected cells as well as viral cytopathic effects. We conclude that (i) expression of surface transferrin receptors is down-modulated by acute HIV-1 infection in T lymphoid cells, that (ii) this cell phenotypic modulation is associated with the cytopathic effects of the virus, and that (iii) these phenomena are modulated by iron chelation. These results support the view that iron metabolism may be an important area for interaction between HIV-1 and human cells.     

Human immunodeficiency virus type 1 infection of cells and tissues from the upper and lower human female reproductive tract.

Viable tissue sections and isolated cell cultures from the human fallopian tube, uterus, cervix, and vaginal mucosa were examined for susceptibility to infection with human immunodeficiency virus type 1 (HIV-1). We examined infectivity by using the monocytotropic strain HIV-1(JR-FL) and several primary isolates of HIV-1 obtained from infected neonates. HIV-1 infection was measured by p24 production in short-term culture and by immunofluorescence detection of HIV-1 Nef and p24 proteins by laser scanning confocal microscopy. Three-color immunofluorescence was used to phenotype HIV-infected cells within tissue sections from each site. Our findings indicate that epithelial, stromal, and dendritic cells and cells with CD14+ CD4+, CD14-CD4-, and CD4+ CD14- phenotypes from the female reproductive tract are infectable with HIV-1. Of importance is the finding that tissues from the upper reproductive tract are susceptible to infection with HIV-1. Moreover, tissue samples from women in all stages of the menstrual cycle, including postmenopausal women (inactive), could be infected with HIV-1. Female reproductive tract cells required a minimum of 60 min of exposure to HIV-1 in order for infection to occur, in contrast to peripheral blood lymphocytes, which became infected after being exposed to HIV-1 for only 1 min. These findings demonstrate that HIV-1 can infect cells and tissues from different sites within the female reproductive tract and suggest that multiple cell types, including epithelial cells, may be targets for the initial infection by HIV-1.     

The effects of cytomegalovirus on human immunodeficiency virus replication in brain-derived cells correlate with permissiveness of the cells for each virus.

Human cytomegalovirus (HCMV) is commonly found in the brains of patients with AIDS and in some cases can be detected in the same cells as can human immunodeficiency virus type 1 (HIV-1). In this study, we analyzed the patterns of replication of HIV-1 and HCMV in singly infected cells and the effects of dual infection in human brain-derived cell lines of three different origins: neuroblastoma cell lines SK-N-MC and SY5Y; astrocytoma/glioblastoma cell lines U373-MG and Hs 683; and undifferentiated glioblastoma cell lines A172 and T98G. To bypass the restriction at the adsorption/penetration step in these CD4-negative cells, we used HIV-1 (amphotropic retrovirus) pseudotypes. These HIV-1 pseudotypes infected the majority of the cells in the cultures and expressed high levels of HIV-1 gene products in all except the SY5Y cells. The cell lines differed in the ability to support HCMV infection, but coinfection with HIV-1 had no effect on HCMV replication. The A172 cells were completely nonpermissive for HCMV gene expression, while HCMV replication in the singly infected T98G and SK-N-MC cell lines was restricted at the level of some early gene products. This resulted in complete and partial inhibition, respectively, of viral DNA synthesis. Dual infection of the A172, T98G, and SK-N-MC cells had no effect on HIV-1 replication. The other three cell lines, U373-MG, Hs 683, and SY5Y, were fully permissive for HCMV replication. In the U373-MG and Hs 683 cells, HCMV markedly inhibited the synthesis of HIV-1 gene products. In contrast, a transient stimulation of HIV-1 production followed by a repression was observed in the dually infected SY5Y cells. We conclude from these results that under conditions in which both HIV-1 and HCMV can undergo fully permissive infection, HCMV can repress HIV-1 gene expression. In cells in which HCMV replication is limited but HIV-1 replicates well, there is no effect on HIV-1 gene expression. However, activation of HIV-1, at least transiently, may occur in cells in which HIV-1 gene expression is limited. These studies suggest that a threshold level of some HIV-1 gene product(s) may obscure activation or promote repression of HIV replication by HCMV.     

CD4-independent, productive human immunodeficiency virus type 1 infection of hepatoma cell lines in vitro.

Five hepatoma cell lines, including CZHC/8571, PLC/PRF/5, Hep3B, HepG2, and HUH7, were inoculated with three diverse isolates of human immunodeficiency virus type 1 (HIV-1). Productive infection was noted in all hepatoma cell lines, and expression of viral p24 antigen lasted for over 3 months, but its level decreased in proportion to the number of viable cells. HIV-1 antigens were also found in the cells by immunohistochemical staining and radioimmunoprecipitation assay, as were viral RNA by in situ hybridization and HIV-1-like particles by electron microscopy. Virus yield assays were also positive on supernatant fluids collected from hepatoma cultures inoculated with HIV-1. Despite their susceptibility to infection, all five hepatoma cell lines were negative for CD4 by immunofluorescence and for CD4 mRNA by slot-blot hybridization. In addition, HIV-1 infection of hepatoma cell lines was not blocked by anti-CD4 monoclonal antibody or soluble CD4. Together, these findings clearly demonstrate that all five hepatoma cell lines were susceptible to productive infection by HIV-1 in vitro via a CD4-independent mechanism.     

Establishment of a new system for determination of coreceptor usages of HIV based on the human glioma NP-2 cell line.

CD4 and one of the G-protein-coupled receptors (GPCRs) on the cell surface function as a receptor and a coreceptor, respectively, in infection of cells with human and simian immunodeficiency viruses (HIV/SIV). To determine which GPCRs can be coreceptors for HIV (HIV-1 and HIV-2) or SIV infection, several cell lines, including human osteosarcoma HOS-T4 cells and human glioma U87/CD4 cells, have been used. However, these cells often show susceptibilities to some HIV or SIV strains before transduction of GPCRs. The results of this study showed that a CD4-transduced human glioma cell line, NP-2/CD4, a human erythroleukemia cell line, K562/CD4, and a human ovarian cancer cell line, TYK/CD4, were completely resistant to the HIV-1 and HIV-2 strains tested. After transduction of several GPCRs into NP-2/CD4, K562/CD4, or TYK/CD4 cells, NP-2/CD4 cells but not K562/CD4 or TYK/CD4 cells mostly showed expected susceptibilities to several HIV strains. Therefore, an NP-2 cell system would be useful to determine the coreceptor usage of HIV isolates, to find a new coreceptor for HIV/SIV, and to analyze the early stages of HIV/SIV infection.     

Susceptibility of mink (Mustera vision)-derived cells to replication by human immunodeficiency virus type 1

In vivo studies for understanding viral transmission and replication, host immune responses, and pathogenesis of human immunodeficiency virus type 1 (HIV-1) infection would greatly benefit from the establishment of a small-animal model. In this study, we explored the potential of American mink (Mustera vison) as a susceptible host. We found that primary cells and cell lines derived from this species efficiently supported trans-activation of the HIV-1 long terminal repeat by Tat. Accordingly, the cysteine residue at position 261, which has been shown to be important for interaction of the human cyclin T1 with the HIV-1 regulatory protein Tat, is conserved in the mink homologue. No species-specific defect in Rev function could be detected in mink cells. In addition, primary splenocytes, fibroblasts, and the Mv.1.Lu cell line from American mink supported early as well as late HIV-1 gene expression following infection with vesicular stomatitis G protein-pseudotyped HIV-1 viruses, at levels comparable to those seen with permissive human cells. Furthermore, the mink Mv.1.Lu cell line stably expressing human CD4 and CCR5 receptors supported a spreading HIV-1 infection with few, if any, deficiencies compared to findings in human cell lines. This indicates the potential of HIV-1 to replicate in these cells once the blockade at the stage of virus entry has been removed. These results clearly show that cells from American mink generally pose no functional intracellular block to HIV-1 replication, and collectively they raise the possibility that this animal species could be engineered to support HIV-1 infection, providing a useful small-animal model for evaluating de novo infection by HIV-1.     

Neutralizing antibodies against the V3 loop of human immunodeficiency virus type 1 gp120 block the CD4-dependent and -independent binding of virus to cells.

The CD4 molecule is an essential receptor for human immunodeficiency virus type 1 (HIV-1) through high-affinity interactions with the viral external envelope glycoprotein gp120. Previously, neutralizing monoclonal antibodies (MAbs) specific to the third hypervariable domain of gp120 (the V3 loop) have been thought to block HIV infection without affecting the binding of HIV particles to CD4-expressing human cells. However, here we demonstrate that this conclusion was not correct and was due to the use of soluble gp120 instead of HIV particles. Indeed, neutralizing anti-V3 loop MAbs inhibited completely the binding and entry of HIV particles into CD4+ human cells. In contrast, the binding of virus was only partially inhibited by neutralizing anti-CD4 MAbs against the gp120 binding site in CD4, which, like the anti-V3 loop MAbs, completely inhibited HIV entry and infection. Nonneutralizing control MAbs against either the V3 loop or the N or C terminus of gp120 had no significant effect on HIV binding and entry. HIV-1 particles were also found to bind human and murine cells expressing or not expressing the human CD4 molecule. Interestingly, the binding of HIV to CD4+ murine cells was inhibited by both anti-V3 and anti-CD4 MAbs, whereas the binding to human and murine CD4- cells was affected only by anti-V3 loop MAbs. The effect of anti-V3 loop neutralizing MAbs on the HIV binding to cells appears not to be the direct consequence of gp120 shedding from HIV particles or of a decreased affinity of CD4 or gp120 for binding to its surface counterpart. Taken together, our results suggest the existence of CD4-dependent and -independent binding events involved in the attachment of HIV particles to cells; in both of these events, the V3 loop plays a critical role. As murine cells lack the specific cofactor CXCR4 for HIV-1 entry, other cell surface molecules besides CD4 might be implicated in stable binding of HIV particles to cells.     

CD4-independent, CCR5-dependent simian immunodeficiency virus infection and chemotaxis of human cells

Most simian immunodeficiency virus (SIV), human immunodeficiency virus type 2 (HIV-2), and HIV-1 infection of host peripheral blood mononuclear cells (PBMCs) is CD4 dependent. In some cases, X4 HIV-1 chemotaxis is CD4 independent, and cross-species transmission might be facilitated by CD4-independent entry, which has been demonstrated for some SIV strains in CD4(-) non-T cells. As expected for CCR5-dependent virus, SIV required CD4 on rhesus and pigtail macaque PBMCs for infection and chemotaxis. However, SIV induced the chemotaxis of human PBMCs in a CD4-independent manner. Furthermore, in contrast to the results of studies using transfected human cell lines, SIV did not require CD4 binding to productively infect primary human PBMCs. CD4-independent lymphocyte and macrophage infection may facilitate cross-species transmission, while reacquisition of CD4 dependence may confer a selective advantage for the virus within new host species.     

Effects of the human CD38 glycoprotein on the early stages of the HIV-1 replication cycle.

CD38 displays lateral association with the HIV-1 receptor CD4. This association is potentiated by the HIV-1 envelope glycoprotein gp120. The aim of this work was to evaluate the CD38 role in T cell susceptibility to HIV-1 infection. Using laboratory X4 HIV-1 strains and X4 and X4/R5 primary isolates, we found that CD38 expression was negatively correlated to cell susceptibility to infection, evaluated as percentage of infected cells, release of HIV p24 in the supernatants, and cytopathogenicity. This correlation was at first suggested by results obtained in a panel of human CD4(+) T cell lines expressing different CD38 levels (MT-4, MT-2, C8166, CEMx174, Supt-1, and H9) and then demonstrated using CD38 transfectants of MT-4 cells (the line with the lowest CD38 expression). To address whether CD38 affected viral binding, we used mouse T cells that are non-permissive for productive infection. Gene transfection in mouse SR.D10.CD4(-).F1 T cells produced four lines expressing human CD4 and/or CD38. Ability of CD4(+)CD38(+)cells to bind HIV-1 or purified recombinant gp120 was significantly lower than that of CD4(+)CD38(-) cells. These data suggest that CD38 expression inhibits lymphocyte susceptibility to HIV infection, probably by inhibiting gp120/CD4-dependent viral binding to target cells.-Savarino, A., Bottarel, F., Calosso, L., Feito, M. J., Bensi, T., Bragardo, M., Rojo, J. M., Pugliese, A., Abbate, I., Capobianchi, M. R., Dianzani, F., Malavasi, F., and Dianzani, U. Effects of the human CD38 glycoprotein on the early stages of theHIV-1 replication cycle.     

Intracellular expression of human immunodeficiency virus type 1 (HIV-1) protease variants inhibits replication of wild-type and protease inhibitor-resistant HIV-1 strains in human T-cell lines.

The enzymatic activity of the human immunodeficiency type 1 (HIV-1) protease (PR) is crucial to render HIV-1 virions mature and infectious. Hence, genetic intervention strategies based on trans-dominant (td) variants of the HIV-1 PR might be an alternative to current pharmacological and gene therapy regimens for AIDS. CD4-positive human CEM-SS T-cell lines were generated which constitutively expressed HIV-1 td PR variants. HIV-1 infection experiments demonstrated severely reduced HIV-1 replication in these td PR CEM-SS cell lines compared with control T cells expressing wild-type PR. Furthermore, replication of an HIV-1 isolate bearing a PR inhibitor-resistant PR was blocked, showing that genetic intervention strategies based on td PRs can be effective against HIV-1 isolates containing PR inhibitor-resistant mutants.     

Inhibition of human immunodeficiency virus fusion by a monoclonal antibody to a coreceptor (CXCR4) is both cell type and virus strain dependent.

CXCR4 (also termed fusin, LESTR, or HUMSTR) is a member of the G-protein-coupled chemokine receptor family with seven membrane-spanning domains. CXCR4 acts as a coreceptor for syncytium-inducing human immunodeficiency virus type 1 (HIV-1) strains, conferring entry into CD4+ cells. We show here that a novel mouse monoclonal antibody (12G5) that recognizes CXCR4 blocked cell-to-cell fusion and cell free-virus infection of CXCR4+ CD4+ RD rhabdomyosarcoma cells by seven HIV-1 and HIV-2 strains that had various cell tropisms for different CD4+ human cell types. Yet the majority of the members of the same virus panel resisted 12G5 inhibition on T-cell lines. When inhibition was observed on these cell types, it was both cell type and virus strain dependent. In at least one situation, 12G5 failed to block LAI infection of cells expressing CXCR4 as the only available coreceptor. Our observations suggest that CXCR4 could be processed or presented differently depending on the cell type, allowing some strains to evade 12G5 inhibition. Alternatively, since several of the viruses could infect certain CXCR4- CD4+ cell lines, it is conceivable that alternative coreceptors are active, enabling individual HIV strains to choose between compatible coreceptors during entry into cells. Moreover, the strain dependency of 12G5 inhibition implies that the interaction of different HIVs with CXCR4 varies.