CXCR4-tropic HIV-1 suppresses replication of CCR5-tropic HIV-1 in human lymphoid tissue by selective induction of CC-chemokines

In infected individuals, human immunodeficiency virus type 1 (HIV-1) exist as a "swarm" of quasi species compartmentalized in tissues where individual viral variants may interact locally. We have used human lymphoid tissue, where the critical events of HIV disease occur, to study local interactions in model HIV-1 binary swarms ex vivo. We infected tissue blocks with binary mixtures consisting either of CCR5-dependent and CXCR4-dependent variants or of 2 dual-tropic HIV-1 variants, of which one is skewed to utilization of CXCR4 and the other of CCR5. HIV-1 variants that use CXCR4 suppress replication of CCR5-dependent HIV-1 variants, whereas CCR5-dependent HIV-1 variants do not affect replication of CXCR4-dependent HIV-1. CC-chemokines that inhibit replication of CCR5-dependent HIV-1 variants were up-regulated by CXCR4-dependent HIV-1, thus possibly contributing to this suppression. Tissue-specific chemokine/cytokine network modulations triggered by individual HIV-1 variants may be an important mechanism of local interactions among HIV-1 quasi species in infected tissue.     

HIV-1 induced activation of CD4+ T cells creates new targets for HIV-1 infection in human lymphoid tissue ex vivo

We demonstrate mechanisms by which HIV-1 appears to facilitate its own infection in ex vivo-infected human lymphoid tissue. In this system, HIV-1 readily infects various CD4+ T cells, but productive viral infection was supported predominantly by activated T cells expressing either CD25 or HLA-DR or both (CD25/HLA-DR) but not other activation markers: There was a strong positive correlation (r=0.64, P=.001) between virus production and the number of CD25+/HLA-DR+ T cells. HIV-1 infection of lymphoid tissue was associated with activation of both HIV-1-infected and uninfected (bystanders) T cells. In these tissues, apoptosis was selectively increased in T cells expressing CD25/HLA-DR and p24gag but not in cells expressing either of these markers alone. In the course of HIV-1 infection, there was a significant increase in the number of activated (CD25+/HLA-DR+) T cells both infected and uninfected (bystander). By inducing T cells to express particular markers of activation that create new targets for infection, HIV-1 generates in ex vivo lymphoid tissues a vicious destructive circle of activation and infection. In vivo, such self-perpetuating cycle could contribute to HIV-1 disease.     

Segregation of R5 and X4 HIV-1 variants to memory T cell subsets differentially expressing CD62L in ex vivo infected human lymphoid tissue

OBJECTIVE: The mechanisms of HIV-triggered immunodeficiency were examined by determining the segregation of R5 and X4 HIV-1 variants into memory T cell subsets expressing differentially a homing receptor, CD62L-selectin, in human lymphoid tissue. METHODS: Subpopulations of CD3 and intracellular p24 gag-positive cells in human lymphoid tissue infected ex vivo with X4 HIV-1 variant NL4-3 and R5 HIV-1 variant AD8 were analysed for expression of the T cell memory markers CD45RO and CD45RA, the T cell homing receptor for lymphoid tissue CD62L, and the HIV-1 coreceptors CCR5 and CXCR4. RESULTS: Memory CD4 T cells were the predominant targets for productive infection of lymphoid tissue ex vivo with both R5 and X4 HIV-1. R5 HIV-1 predominantly infected CD62L-negative memory T cells, which selectively express CCR5. In contrast, X4 HIV-1 variants predominantly infected CD62L+ memory T cells, although CXCR4 coreceptor was equally expressed by memory T cells of both CD62L-positive and CD62L-negative subsets. A high proportion of X4 HIV-1, but not of R5 HIV-1, productively infected T cells, displayed a CD45RA+CD45RO+ phenotype. CONCLUSION: The selective expression of the CCR5 coreceptor by CD62L-negative terminally differentiated memory T cells correlates with the preferential productive infection of these cells with the R5 HIV-1 variant. The predominance of X4 HIV-1 variants in less-differentiated memory T cells may be related to their recent activation state, as suggested by the coexpression of both CD45RA and CD45RO molecules on their surface. Differential homing of CD62L-positive and CD62L-negative cells suggests different routes of dissemination of X4 and R5 viruses.     

HIV-1 pathogenesis differs in rectosigmoid and tonsillar tissues infected ex vivo with CCR5- and CXCR4-tropic HIV-1

Gut-associated lymphoid tissue (GALT) has been identified as the primary target of HIV-1 infection. To investigate why GALT is especially vulnerable to HIV-1, and to determine whether the selective transmission of CCR5-using viral variants (R5) in vivo is the result of a greater susceptibility of GALT to this viral variant, we performed comparative studies of CXCR4-using (X4) and R5 HIV-1 infections of human lymphoid (tonsillar) and rectosigmoid tissues ex vivo under controlled laboratory conditions. We found that the relative level of R5 replication in rectosigmoid tissue is much greater than in tonsillar tissue. This difference is associated with the expression of the CCR5 co-receptor on approximately 70% of CD4 T cells in rectosigmoid tissue, whereas in tonsillar tissue it is expressed on fewer than 15% of CD4 T cells. Furthermore, tonsillar tissue responds to X4 HIV-1 infection by upregulating the secretion of CC-chemokines, providing a potential CCR5 blockade and further resistance to R5 infection, whereas gut tissue failed to increase such innate immune responses. Our results show that rectosigmoid tissue is more prone than tonsillar lymphoid tissue to R5 HIV-1 infection, primarily because of the high prevalence and availability of R5 cell targets and reduced chemokine blockade. The majority of CD4 T cells express CXCR4, however, and X4 HIV-1 readily replicates in both tissues, suggesting that although the differential expression of co-receptors contributes to the GALT vulnerability to R5 HIV-1, it alone cannot account for the selective R5 infection of the rectal mucosa in vivo.     

Pertussis toxin B-oligomer dissociates T cell activation and HIV replication in CD4 T cells released from infected lymphoid tissue

OBJECTIVE: To investigate, in human lymphoid tissue infected with HIV-1 ex vivo, the immunostimulatory and HIV inhibitory properties of pertussis toxin B oligomer (PTX-B) and of the genetically modified non-toxic PT-9K/129G. METHODS: Human tonsils from uninfected donors were infected ex vivo with R5 or X4 HIV-1 in the presence or absence of PTX-B. Virus replication was evaluated in culture supernatants; cells emigrated from tissue blocks were immunostained for lymphocytic and activation markers. HIV DNA and cell proliferation were evaluated with real-time PCR and [H]thymidine incorporation, respectively. RESULTS: Both PTX-B and PT-9K/129G inhibited HIV-1 replication. These compounds activated and stimulated the proliferation of emigrated cells, most of which were CD4 T lymphocytes. Cells emigrated from infected tissues did not produce detectable virus in unstimulated or in PTX-B- or PT-9K/129G-stimulated cultures whereas robust virus production was triggered by phytohemagglutinin (PHA) or interleukin-2 (IL-2). Analysis of HIV DNA content indicated that infected cells were present among emigrated cells and that their number greatly increased following IL-2 stimulation, whereas it remained constant in the presence of PTX-B or PT-9K/129G. CONCLUSIONS: PTX-B and PT-9K/129G inhibit both R5 and X4 HIV-1 replication in human lymphoid tissue ex vivo. In contrast to PHA and IL-2, they promote the proliferation of CD4 T lymphocytes emigrated from tissue, including HIV-infected cells, without triggering virus replication. Therefore, these emigrated CD4 T cells represent a novel model of a latent inducible HIV reservoir. Thus, PTX-B and the clinically approved PT-9K/129G are potential antiretroviral agents endowed with immunostimulatory capacity.     

Effect of R77Q, R77A and R80A changes in Vpr on HIV-1 replication and CD4 T cell depletion in human lymphoid tissue ex vivo

BACKGROUND: It has been suggested that mutations of R77A and R80A in the HIV-1 viral protein R (Vpr) impair its proapoptotic activity and that a naturally occurring R77Q variation is associated with non-progressive HIV-1 infection. RATIONALE: To assess the effect of Vpr R77Q, R77A and R80A mutations on the efficiency of CCR5(R5)- and CXCR4(X4)-tropic HIV-1 replication and cytopathicity in human lymphoid tissue (HLT). METHODS: Vpr mutants of the X4-tropic HIV-1 NL4-3 clone and an R5-tropic derivative were generated by PCR mutagenesis. Virus stocks established by transfection of 293T cells were used to infect macrophages and ex vivo HLT. HIV-1 replication was assessed by measuring p24 core antigen in the culture supernatants and CD4 T-cell depletion and apoptosis were measured by flow cytometric analysis. RESULTS: The R5-tropic HIV-1 Vpr mutants replicated with slightly (R77A, R77Q) to moderately (R80A) reduced efficiency in ex vivo-infected HLT and macrophages. In comparison, the changes in Vpr had negligible effects on replication of the X4-tropic forms in lymphatic tissues. Mutation of R77Q and R80A reduced apoptosis of HIV-1-infected cells in ex vivo-infected HLT independently of the viral coreceptor tropism. However, only the R5-tropic HIV-1 Vpr mutants caused markedly less CD4 T-cell depletion than wild-type HIV-1 at the end of ex vivo HLT culture. CONCLUSIONS: The observation that Vpr R77Q reduces the cytopathicity of R5-tropic HIV-1 in lymphoid tissues supports a role in non-progressive HIV-1 infection but the attenuating effects might be dependent on the viral subtype and coreceptor tropism.     

Hepatocytes are permissive for human cytomegalovirus infection in human liver cell culture and In vivo.

The cytopathic potential of human cytomegalovirus (HCMV) in human liver cells was analyzed in cell culture and in tissue sections from patients with HCMV hepatitis. Liver cell cultures, consisting of hepatocytes, bile duct epithelial cells, and stromal cells were infected by various HCMV strains. Cytopathic effects, viral gene expression, and virus production were detected. Infected cell types were identified by immunocytochemical double labeling. Hepatocytes were the predominant target cells of HCMV infection in liver tissues and in cell culture. Late-stage infected cultured hepatocytes produced infectious progeny virus, and infectious virus was propagated from liver tissue specimens. HCMV infection in cultured liver cells closely resembled in vivo infection of the liver with regard to the target cell spectrum and the permissive course of infection. It is concluded that HCMV can cause direct liver parenchyma damage by efficient cytolytic infection of hepatocytes.     

HIV-infected human Langerhans cells transmit infection to human lymphoid tissue ex vivo

OBJECTIVES: To create a novel ex vivo model for early biologic events involved in sexual transmission of HIV and to demonstrate that Langerhans cells (LC), the purported initial mucosal target cells for HIV, play a critical role in this process. METHODS: Epidermal cells containing LC were isolated from normal-appearing skin of healthy volunteers and exposed to a panel of primary and laboratory-adapted R5- and X4-HIV isolates, washed and applied to the surfaces of allogeneic tonsil tissue blocks. Viral replication was followed by measuring HIV p24 protein in culture supernatants by ELISA. RESULTS: Both R5- and X4-HIV isolates could be transmitted by LC and established high levels of infection in lymphoid tissue (p24 > 10 ng/ml). Depletion of LC within epidermal cell suspensions abrogated the ability of HIV-exposed suspensions to transmit virus to tonsil histocultures. CONCLUSIONS: Using a novel ex vivo model, human LC are shown for the first time to be the major epidermal cell type that is involved in transmission of HIV infection to human lymphoid tissue. Importantly, this system could prove useful in further understanding LC trafficking and other early biological events involved in primary HIV infection.     

Inhibition of HIV-1 replication in human lymphoid tissues ex vivo by measles virus

Human immunodeficiency virus (HIV) type 1 replication and disease progression are enhanced by various pathogens in coinfected individuals. However, acute infection with measles virus (MV) has been found to suppress HIV-1 replication in coinfected children. We investigated the mechanisms of this phenomenon using human lymphoid tissues coinfected ex vivo with HIV-1 and MV. MV inhibited both CXCR4-tropic (X4) and CCR5-tropic (R5) HIV-1, but the inhibitory effect was particularly profound for R5 virus, which transmits infection and dominates the early stages of HIV-1 disease. MV inhibits the replication of R5 HIV-1 in coinfected tissues by up-regulation of the CC chemokine RANTES, a well-known inhibitor of R5 HIV-1 infection, and this up-regulation is augmented in tissues coinfected with R5 HIV-1. Deciphering the molecular mechanisms by which MV and other pathogens alter local cytokine/chemokine networks and cause tissue microenvironments to become detrimental to HIV-1 may significantly contribute to the development of effective anti-HIV therapies.     

In vitro infection of human primary adipose cells with HIV-1: a reassessment

We reassessed the infection ability of human primary preadipocytes. The use of X4, R5 or VSV-G-pseudotyped viral particles indicated that viral entry is the limiting step. However, transfection with HIV-1 receptors restored efficient infection. Analyses of CD4, CXCR4 and CCR5 expression on preadipocytes and adipocytes revealed that receptor co-expression levels did not permit HIV-1 entry into adipose cells from all biopsies tested. We concluded that adipose tissue cannot be infected with HIV-1 in vivo.     

Human immunodeficiency virus type 1 tat gene enhances human cytomegalovirus gene expression and viral replication.

Clonal lines of human rhabdomyosarcoma (RD) cells, constitutively expressing human immunodeficiency virus type 1 (HIV-1) tat gene (RD tat cell lines) showed enhanced expression of human cytomegalovirus (HCMV) immediate-early (IE) and late (L) proteins upon HCMV infection, as compared with control RD cells. One of the RD tat cell lines produced infectious HCMV. The RD-tat cell lines, following transfection with recombinant plasmids containing the full length of the HCMV-IE enhancer/promoter linked to the bacterial chloramphenicol acetyltransferase (CAT) gene, exhibited an increased CAT expression by the tat product. A chronically HIV-1-infected human T-lymphoid cell line, SupT1, superinfected with HCMV, expressed HCMV-IE proteins while the parental SupT1 cells infected with HCMV were negative. Parental SupT1 cells coinfected with HIV-1 and HCMV also expressed HCMV-IE proteins, indicating that HIV-1-encoded proteins exert a positive regulatory effect on HCMV expression.     

Humanized mouse models of HIV-1 latency

The existence of long-lasting cellular reservoirs of HIV-1 is one of the major hurdles in developing effective anti-retroviral therapies. These latently infected cells and tissues efficiently evade immune responses and remain dormant until activated, upon which they can generate a productive HIV-1 infection. This classic scenario of viral latency becomes even more difficult to study and model due to the extreme complexity of translating in vivo virus-cell interactions into a controlled in vitro system. The recent developments and constant improvements upon hematopoietic engraftment of human cells and tissues onto recipient immunocompromised murine scaffolds have made it possible to model complex human innate and adaptive immune responses in a small animal model. Specifically, HIV-1 infection has been successfully modeled in these humanized mice to mimic transmission, pathogenesis, host immune responses, and treatment. Here, we review the complexities surrounding modeling HIV-1 latency in vitro and in vivo and highlight the most recent humanized mouse models that support retroviral infection.     

Primary and recombinant HIV type 1 strains resistant to protease inhibitors are pathogenic in mature human lymphoid tissues

Preserved peripheral CD4+ T cell counts despite virologic failure in patients undergoing protease inhibitor (PI)-containing antiviral regimens are a frequent occurrence in human immunodeficiency virus (HIV) disease. One hypothesis to explain the relative sparing of CD4+ T cells is that HIV strains exhibiting PI resistance concomitantly are attenuated in terms of cytopathicity for mature T cells. To test this hypothesis, we used a three-dimensional human tonsil histoculture microenvironment to assess the pathogenic potential of a panel of primary and recombinant HIV-1 strains derived from patients experiencing PI failure. All the viruses tested replicated efficiently in these cultures and, in some cases, better than comparable wild-type viral isolates. Furthermore, the PI-resistant strains depleted CD4+ T cells potently and comparably with wild-type isolates in these ex vivo lymphoid tissues. These results demonstrate that PI-resistant viruses are not inherently less pathogenic for mature T cells. Therefore, the sustained peripheral lymphocyte counts in patients with selective virologic failure may be due to specific defects in viral replication in other cell compartments or to an undefined host adaptation to viral infection during PI therapy.     

Cytomegalovirus infection and trans-activation of HIV-1 and HIV-2 LTRs in human astrocytoma cells

Susceptibility of a human astrocytoma cell line to human cytomegalovirus (HCMV) infection was investigated. Infection of U-373MG astrocytoma cells with two strains of HCMV resulted in both production of extracellular, infectious virus and expression of immediate early and early antigens within 18 hours and late antigens after 72 hours of infection. The kinetics of infection in U-373MG cells were the same as in human diploid fibroblasts (MRC-5). Since HCMV and human immunodeficiency virus (HIV) have reportedly been found in astrocytic cells in vivo, we studied the possible interaction between HCMV and HIV long terminal repeat (LTR) elements in this cellular environment. HCMV infection transactivated the LTR of HIV-1 and HIV-2 to similar levels. Interestingly, transfection of these cells with infectious HIV-1 provirus did not result in expression of gag, env, or F proteins detectable by immunofluorescence. However, provirus gene expression was not completely silent, since it transactivated HIV-1 LTR. The level of this transactivation was similar to that seen following cotransfection with a tat expression vector. These results suggest that opportunistic infection with HCMV may reactivate latent HIV genomes in glial cells.     

Cytotoxic factors secreted by cells infected by human immunodeficiency virus type I

Conditioned media from human immunodeficiency virus type I (HIV-1) infected cells were tested for cytotoxic cell-derived factors. The assay used a murine fibroblast cell line which is sensitive to the effects of tumor necrosis factors, but nonpermissive for HIV-1 replication. Cytotoxic activity was detected in cultures of peripheral blood mononuclear cells infected with HIV-1. However, no differences in activity were found in conditioned media from infected lymphoid or monocytoid cell lines compared to their uninfected counterparts. These data suggest that cytotoxic activities of this type are not mediators of cell killing resulting from HIV-1 infection. Thus, this cytotoxic activity is a direct or indirect result of virus replication or cytopathicity. One should consider a role for this cytotoxic factor, secreted by HIV-1 infected mononuclear cells, in various aspects of infection in vivo, such as AIDS encephalopathy or the systemic manifestations accompanying ARC.     

Diagnosis and monitoring of human cytomegalovirus diseases in patients with human immunodeficiency virus infection by use of a real-time PCR assay.

We used a real-time PCR assay to measure human cytomegalovirus (HCMV) DNA load in whole blood and plasma of 70 patients who were infected with human immunodeficiency virus type 1. Break points of 3.0 x 10(3) copies/mL in whole blood and 1.0 x 10(3) copies/mL in plasma were well-correlated with the existence of definite HCMV disease (sensitivity, 93% and 86%; specificity, 89% and 85%; positive predictive value, 70% and 63%; and negative predictive value, 98% and 95%, respectively). In patients with < 50 cells/microL of CD4(+) T lymphocytes, positive predictive values increased to 78% and 71%, respectively. The viral loads of all patients who received anti-HCMV therapy declined to < or =2.0 x 10(2) copies/mL in parallel with the improvement of clinical symptoms. These findings show that the HCMV DNA load quantified with our method is a useful tool for diagnosis of HCMV diseases and for monitoring the disease activity in patients infected with HIV-1.