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.     

Transgenic Mice Expressing Human Immunodeficiency Virus Type 1 in Immune Cells Develop a Severe AIDS-Like Disease

We have constructed transgenic (Tg) mice expressing the entire human immunodeficiency virus type 1 (HIV-1) coding sequences in cells targeted by HIV-1 infection in humans. These Tg mice developed a severe AIDS-like disease leading to early death (<1 month). They developed muscle wasting, severe atrophy and fibrosis of lymphoid organs, tubulointerstitial nephritis, and lymphoid interstitial pneumonitis. In addition the expression of RANTES was increased in various tissues of these Tg mice relative to that in the normal controls. Disease appearance was correlated with the levels of transgene expression. The numerous pathologies observed in these mice are remarkably similar to those observed in human AIDS and, more specifically, in pediatric AIDS.     

Differential growth kinetics are exhibited by human immunodeficiency virus type 1 TAR mutants.

The human immunodeficiency virus type 1 (HIV-1) TAR element is critical for the activation of gene expression by the transactivator protein, Tat. Mutagenesis has demonstrated that a stable stem-loop RNA structure containing both loop and bulge structures transcribed from TAR is the major target for tat activation. Though transient assays have defined elements critical for TAR function, no studies have yet determined the role of TAR in viral replication because of the inability to generate viral stocks containing mutations in TAR. In the current study, we developed a strategy which enabled us to generate stable 293 cell lines which were capable of producing high titers of different viruses containing TAR mutations. Viruses generated from these cell lines were used to infect both T-lymphocyte cell lines and peripheral blood mononuclear cells. Viruses containing TAR mutations in either the upper stem, the bulge, or the loop exhibited dramatically decreased HIV-1 gene expression and replication in all cell lines tested. However, we were able to isolate lymphoid cell lines which stably expressed gene products from each of these TAR mutant viruses. Though the amounts of virus in these cell lines were roughly equivalent, cells containing TAR mutant viruses were extremely defective for gene expression compared with cell lines containing wild-type virus. The magnitude of this decrease in viral gene expression was much greater than previously seen in transient expression assays using HIV-1 long terminal repeat chloramphenicol acetyltransferase gene constructs. In contrast to the defects in viral growth found in T-lymphocyte cell lines, several of the viruses containing TAR mutations were much less defective for gene expression and replication in activated peripheral blood mononuclear cells. These results indicate that maintenance of the TAR element is critical for viral gene expression and replication in all cell lines tested, though the cell type which is infected is also a major determinant of the replication properties of TAR mutant viruses.     

HIV-1 Infected Lymphoid Organs Upregulate Expression and Release of the Cleaved Form of uPAR That Modulates Chemotaxis and Virus Expression

Cell-associated receptor for urokinase plasminogen activator (uPAR) is released as both full-length soluble uPAR (suPAR) and cleaved (c-suPAR) form that maintain ability to bind to integrins and other receptors, thus triggering and modulating cell signaling responses. Concerning HIV-1 infection, plasma levels of suPAR have been correlated with the severity of disease, levels of immune activation and ineffective immune recovery also in individuals receiving combination anti-retroviral therapy (cART). However, it is unknown whether and which suPAR forms might contribute to HIV-1 induced pathogenesis and to the related state of immune activation. In this regard, lymphoid organs represent an import site of chronic immune activation and virus persistence even in individuals receiving cART. Lymphoid organs of HIV-1⁺ individuals showed an enhanced number of follicular dendritic cells, macrophages and endothelial cells expressing the cell-associated uPAR in comparison to those of uninfected individuals. In order to investigate the potential role of suPAR forms in HIV-1 infection of secondary lymphoid organs, tonsil histocultures were established from HIV-1 seronegative individuals and infected ex vivo with CCR5- and CXCR4-dependent HIV-1 strains. The levels of suPAR and c-suPAR were significantly increased in HIV-infected tonsil histocultures supernatants in comparison to autologous uninfected histocultures. Supernatants from infected and uninfected cultures before and after immunodepletion of suPAR forms were incubated with the chronically infected promonocytic U1 cell line characterized by a state of proviral latency in unstimulated conditions. In the contest of HIV-conditioned supernatants we established that c-suPAR, but not suPAR, inhibited chemotaxis and induced virus expression in U1 cells. In conclusion, lymphoid organs are an important site of production and release of both suPAR and c-suPAR, this latter form being endowed with the capacity of inhibiting chemotaxis and inducing HIV-1 expression.     

CD4-independent infection of human neural cells by human immunodeficiency virus type 1.

A number of studies have indicated that central nervous system-derived cells can be infected with human immunodeficiency virus type 1 (HIV-1). To determine whether CD4, the receptor for HIV-1 in lymphoid cells, was responsible for infection of neural cells, we characterized infectable human central nervous system tumor lines and primary fetal neural cells and did not detect either CD4 protein or mRNA. We then attempted to block infection with anti-CD4 antibodies known to block infection of lymphoid cells; we noted no effect on any of these cultured cells. The results indicate that CD4 is not the receptor for HIV-1 infection of the glioblastoma line U373-MG, medulloblastoma line MED 217, or primary human fetal neural cells.     

Simian virus 40-based replication of catalytically inactive human immunodeficiency virus type 1 integrase mutants in nonpermissive T cells and monocyte-derived macrophages

Integrase function is required for retroviral replication in most instances. Although certain permissive T-cell lines support human immunodeficiency virus type 1 (HIV-1) replication in the absence of functional integrase, most cell lines and primary human cells are nonpermissive for integrase mutant growth. Since unintegrated retroviral DNA is lost from cells following cell division, we investigated whether incorporating a functional origin of DNA replication into integrase mutant HIV-1 might overcome the block to efficient gene expression and replication in nonpermissive T-cell lines and primary cells. Whereas the Epstein-Barr virus (EBV) origin (oriP) did little to augment expression from an integrase mutant reporter virus in EBV nuclear antigen 1-expressing cells, simian virus 40 (SV40) oriT dramatically enhanced integrase mutant infectivity in T-antigen (Tag)-expressing cells. Incorporating oriT into the nef position of a full-length, integrase-defective virus strain yielded efficient replication in Tag-expressing nonpermissive Jurkat T cells without reversion to an integration-competent genotype. Adding Tag to integrase mutant-oriT viruses yielded 11.3-kb SV40-HIV chimeras that replicated in Jurkat cells and primary monocyte-derived macrophages. Real-time quantitative PCR analyses of Jurkat cell infections revealed that amplified copies of unintegrated DNA likely contributed to SV40-HIV integrase mutant replication. SV40-based HIV-1 integrase mutant replication in otherwise nonpermissive cells suggests alternative approaches to standard integrase-mediated retroviral gene transfer strategies.     

Epstein-Barr virus LMP2A-induced B-cell survival in two unique classes of EmuLMP2A transgenic mice

Latent membrane protein 2A (LMP2A) is one of only two viral proteins expressed during latent Epstein-Barr virus (EBV) infections in human peripheral B cells. LMP2A blocks B-cell receptor (BCR) signal transduction in vitro by modulation of the Syk and Lyn protein tyrosine kinases. Five genetically unique LMP2A transgenic mouse lines (EmuLMP2A) with B-cell lineage expression of LMP2A were generated in this study to analyze the importance of LMP2A expression in vivo. These animals can be grouped into EmuLMP2A(BCR+) (TgB, Tg6, and TgC) and EmuLMP2A(BCR-) (Tg7 and TgE) lines based on B-cell phenotype. LMP2A expression in bone marrow cells of EmuLMP2A(BCR-) lines was associated with a bypass of normal B-lymphocyte developmental checkpoints inasmuch as immunoglobulin light-chain gene rearrangement occurred in the absence of complete immunoglobulin heavy-chain gene rearrangement. The resulting BCR-negative B cells were able to exit the bone marrow and colonize peripheral lymphoid organs. LMP2A expression in EmuLMP2A(BCR+) lines was not associated with altered B-cell development in a genetically wild-type background. When crossed into a recombinase activating null (RAG(-/-)) genetic background, LMP2A expression in either RAG(-/-) EmuLMP2A(BCR+) or RAG(-/-) EmuLMP2A(BCR-) animals was able to provide a survival signal to BCR-negative splenic B cells. Additionally, bone marrow cells from all EmuLMP2A animals were able to proliferate in response to interleukin-7-dependent developmental signals in vitro. These studies illustrate that LMP2A can provide a survival signal to BCR-negative B cells in two different groups of EmuLMP2A transgenic mice.     

Susceptibility of rat-derived cells to replication by human immunodeficiency virus type 1

Progress in developing a small animal model of human immunodeficiency virus type 1 (HIV-1) disease would greatly facilitate studies of transmission, pathogenesis, host immune responses, and antiviral strategies. In this study, we have explored the potential of rats as a susceptible host. In a single replication cycle, rat cell lines Rat2 and Nb2 produced infectious virus at levels 10- to 60-fold lower than those produced by human cells. Rat-derived cells supported substantial levels of early HIV-1 gene expression, which was further enhanced by overexpression of human cyclin T1. Rat cells displayed quantitative, qualitative, and cell-type-specific limitations in the late phase of the HIV-1 replication cycle including relative expression levels of HIV-1 Gag proteins, intracellular Gag processing, and viral egress. Nb2 cells were rendered permissive to HIV-1 R5 viruses by coexpression of human CD4 and CCR5, indicating that the major restriction on HIV-1 replication was at the level of cellular entry. We also found that primary rat lymphocytes, macrophages, and microglia expressed considerable levels of early HIV-1 gene products following infection with pseudotyped HIV-1. Importantly, primary rat macrophages and microglia, but not lymphocytes, also expressed substantial levels of HIV-1 p24 CA and produced infectious virions. Collectively, these results identify the rat as a promising candidate for a transgenic small animal model of HIV-1 infection and highlight pertinent cell-type-specific restrictions that are features of this species.     

Plasmacytoid Dendritic Cells Suppress HIV-1 Replication but Contribute to HIV-1 Induced Immunopathogenesis in Humanized Mice

The role of plasmacytoid dendritic cells (pDC) in human immunodeficiency virus type 1 (HIV-1) infection and pathogenesis remains unclear. HIV-1 infection in the humanized mouse model leads to persistent HIV-1 infection and immunopathogenesis, including type I interferons (IFN-I) induction, immune-activation and depletion of human leukocytes, including CD4 T cells. We developed a monoclonal antibody that specifically depletes human pDC in all lymphoid organs in humanized mice. When pDC were depleted prior to HIV-1 infection, the induction of IFN-I and interferon-stimulated genes (ISGs) were abolished during acute HIV-1 infection with either a highly pathogenic CCR5/CXCR4-dual tropic HIV-1 or a standard CCR5-tropic HIV-1 isolate. Consistent with the anti-viral role of IFN-I, HIV-1 replication was significantly up-regulated in pDC-depleted mice. Interestingly, the cell death induced by the highly pathogenic HIV-1 isolate was severely reduced in pDC-depleted mice. During chronic HIV-1 infection, depletion of pDC also severely reduced the induction of IFN-I and ISGs, associated with elevated HIV-1 replication. Surprisingly, HIV-1 induced depletion of human immune cells including T cells in lymphoid organs, but not the blood, was reduced in spite of the increased viral replication. The increased cell number in lymphoid organs was associated with a reduced level of HIV-induced cell death in human leukocytes including CD4 T cells. We conclude that pDC play opposing roles in suppressing HIV-1 replication and in promoting HIV-1 induced immunopathogenesis. These findings suggest that pDC-depletion and IFN-I blockade will provide novel strategies for treating those HIV-1 immune non-responsive patients with persistent immune activation despite effective anti-retrovirus treatment.     

Human immunodeficiency virus type 1 bound to B cells: relationship to virus replicating in CD4+ T cells and circulating in plasma

Human immunodeficiency virus type 1 (HIV-1) virions bind to B cells in the peripheral blood and lymph nodes through interactions between CD21 on B cells and complement-complexed virions. B-cell-bound virions have been shown to be highly infectious, suggesting a unique mode of HIV-1 dissemination by B cells circulating between peripheral blood and lymphoid tissues. In order to investigate the relationship between B-cell-bound HIV-1 and viruses found in CD4+ T cells and in plasma, we examined the genetic relationships of HIV-1 found in the blood and lymph nodes of chronically infected patients with heteroduplex mobility and tracking assays and DNA sequence analysis. In samples from 13 of 15 patients examined, HIV-1 variants in peripheral blood-derived B cells were closely related to virus in CD4+ T cells and more divergent from virus in plasma. In samples from five chronically viremic patients for whom analyses were extended to include lymph node-derived HIV-1 isolates, B-cell-associated HIV-1 and CD4+-T-cell-associated HIV-1 in the lymph nodes were equivalent in their divergence from virus in peripheral blood-derived B cells and generally more distantly related to virus in peripheral blood-derived CD4+ T cells. These results indicates virologic cross talk between B cells and CD4+ T cells within the microenvironment of lymphoid tissues and, to a lesser extent, between cells in lymph nodes and the peripheral blood. These findings also indicate that most of the virus in plasma originates from cells other than CD4(+) T cells in the peripheral blood and lymph nodes.     

Nef enhances human immunodeficiency virus replication and responsiveness to interleukin-2 in human lymphoid tissue ex vivo

The nef gene is important for the pathogenicity associated with simian immunodeficiency virus infection in rhesus monkeys and with human immunodeficiency virus type 1 (HIV-1) infection in humans. The mechanisms by which nef contributes to pathogenesis in vivo remain unclear. We investigated the contribution of nef to HIV-1 replication in human lymphoid tissue ex vivo by studying infection with parental HIV-1 strain NL4-3 and with a nef mutant (DeltanefNL4-3). In human tonsillar histocultures, NL4-3 replicated to higher levels than DeltanefNL4-3 did. Increased virus production with NL4-3 infection was associated with increased numbers of productively infected cells and greater loss of CD4(+) T cells over time. While the numbers of productively infected T cells were increased in the presence of nef, the levels of viral expression and production per infected T cell were similar whether the nef gene was present or not. Exogenous interleukin-2 (IL-2) increased HIV-1 production in NL4-3-infected tissue in a dose-dependent manner. In contrast, DeltanefNL4-3 production was enhanced only marginally by IL-2. Thus, Nef can facilitate HIV-1 replication in human lymphoid tissue ex vivo by increasing the numbers of productively infected cells and by increasing the responsiveness to IL-2 stimulation.     

HIV-1 tracing method of systemic viremia in vivo using an artificially mutated virus pool

The appearance of human immunodeficiency virus type 1 (HIV-1) plasma viremia is associated with progression to symptomatic disease and CD4⁺ T cell depletion. To locate the source of systemic viremia, this study employed a novel method to trace HIV-1 infection in vivo. We created JRCSFξnef, a pool of infectious HIV-1 (strain JR-CSF) with highly mutated nef gene regions by random mutagenesis PCR and infected this mutated virus pool into both Jurkat-CCR5 cells and hematopoietic stem cell-transplanted humanized mice. Infection resulted in systemic plasma viremia in humanized mice and viral RNA sequencing helped us to identify multiple lymphoid organs such as spleen, lymph nodes, and bone marrow but not peripheral blood cells as the source of systemic viremia. Our data suggest that this method could be useful for the tracing of viral trafficking in vivo.     

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.     

Species-specific, postentry barriers to primate immunodeficiency virus infection

By using replication-defective vectors derived from human immunodeficiency virus type 1 (HIV-1), simian immunodeficiency virus (SIV(mac)), and murine leukemia virus (MuLV), all of which were pseudotyped with the vesicular stomatitis virus (VSV) G glycoprotein, the efficiency of postentry, early infection events was examined in target cells of several mammalian species. Titers of HIV-1 vectors were significantly lower than those of SIV(mac) and MuLV vectors in most cell lines and primary cells from Old World monkeys. By contrast, most New World monkey cells exhibited much lower titers for the SIV(mac) vector compared with those of the HIV-1 vector. Prosimian cells were resistant to both HIV-1 and SIV(mac) vectors, although the MuLV vector was able to infect these cells. Cells from other mammalian species were roughly equivalent in susceptibility to the three vectors, with the exception of rabbit cells, which were specifically resistant to the HIV-1 vector. The level of HIV-1 vector expression was very low in transduced cells of rodent, rabbit, cow, and pig origin. Early postentry restriction of primate immunodeficiency virus infection exhibits patterns largely coincident with species borders and applies to diverse cell types within an individual host, suggesting the involvement of species-specific, widely expressed cellular factors.