Altered T cell activation and development in transgenic mice expressing the HIV-1 nef gene.

The nef gene, which encodes related cytoplasmic proteins in both human (HIV) and simian (SIV) immunodeficiency viruses is dispensable for viral replication in vitro. In contrast, in vivo experiments have revealed that SIV nef is required for efficient viral replication and development of AIDS in SIV infected rhesus monkeys, thus indicating that nef plays an essential role in the natural infection. We show that expression of the Nef protein from the HIV-1 NL43 isolate in transgenic mice perturbs development of CD4+ T cells in the thymus and elicits depletion of peripheral CD4+ T cells. Thymic T cells expressing NL43 Nef show altered activation responses. In contrast, Nef protein of the HIV-1 HxB3 isolate does not have an overt effect on T cells when expressed in transgenic animals. The differential effects of the two HIV-1 nef alleles in transgenic mice correlate with down-regulation of CD4 antigen expression on thymic T cells. The differential interactions of the NL43 and HxB3 nef alleles with CD4 were reproduced in a transient assay in human CD4+ CEM T cells. Down-regulation of CD4 by nef in both human and transgenic murine T cells indicates that the relevant interactions are conserved in these two systems and suggests that the consequences of Nef expression on the host cell function can be analyzed in vivo in the murine system. Our observations from transgenic mice suggest that nef-elicited perturbations in T cell signalling play an important role in the viral life cycle in vivo, perhaps resulting in elimination of infected CD4+ T cells.     

Interactions between human immunodeficiency virus type 1 and vaccinia virus in human lymphoid tissue ex vivo

Vaccinia virus (VACV) has been attracting attention recently not only as a vector for various vaccines but also as an immunization tool against smallpox because of its potential use as a bioterrorism agent. It has become evident that in spite of a long history of studies of VACV, its tissue pathogenesis remains to be fully understood. Here, we investigated the pathogenesis of VACV and its interactions with human immunodeficiency virus type 1 (HIV-1) in the context of human lymphoid tissues. We found that ex vivo-cultured tonsillar tissue supports productive infection by the New York City Board of Health strain, the VACV strain of the Dryvax vaccine. VACV readily infected both T and non-T (B) lymphocytes and depleted cells of both of these subsets equally over a 12-day period postinfection. Among T lymphocytes, CD8(+) cells are preferentially depleted in accordance with their preferential infection: the probability that a CD8(+) T cell will be productively infected is almost six times higher than for a CD4(+) T cell. T cells expressing CCR5 and the activation markers CD25, CD38, and HLA-DR are other major targets for infection by VACV in lymphoid tissue. As a consequence, VACV predominantly inhibits the replication of the R5(SF162) phenotype of HIV-1 in coinfected tissues, as R5-tropic HIV-1 requires activated CCR5(+) CD4(+) cells for productive infection. Human lymphoid tissue infected ex vivo by VACV can be used to investigate interactions of VACV with other viruses, in particular HIV-1, and to evaluate various VACV vectors for the purpose of recombinant vaccine development.     

The nef gene products of both simian and human immunodeficiency viruses enhance virus infectivity and are functionally interchangeable.

Adult rhesus macaques infected with nef-defective simian immunodeficiency virus (SIV) exhibit extremely low levels of steady-state virus replication, do not succumb to immunodeficiency disease, and are protected from experimental challenge with pathogenic isolates of SIV. Similarly, rare humans found to be infected with nef-defective human immunodeficiency virus type 1 (HIV-1) variants display exceptionally low viral burdens and do not show evidence of disease progression after many years of infection. HIV-1 Nef induces the rapid endocytosis and lysosomal degradation of cell surface CD4 and enhances virus infectivity in primary human T cells and macrophages. Although expression of SIV Nef also leads to down-modulation of cell surface CD4 levels, no evidence for SIV Nef-induced enhancement of virus infectivity was observed in earlier studies. Thus, it remains unclear whether fundamental differences exist between the activities of HIV-1 and SIV Nef. To establish more clearly whether the SIV and HIV-1 nef gene products are functionally analogous, we compared the replication kinetics and infectivity of variants of SIVmac239 that either do (SIVnef+) or do not (SIV delta nef) encode intact nef gene products. SIVnef+ replicates more rapidly than nef-defective viruses in both human and rhesus peripheral blood mononuclear cells (PBMCs). As previously described for HIV-1 Nef, SIV Nef also enhances virus infectivity within each cycle of virus replication. As a strategy for evaluating the in vivo contribution of HIV-1 nef alleles and long terminal repeat regulatory sequences to the pathogenesis of immunodeficiency disease, we constructed SIV-HIV chimeras in which the nef coding and U3 regulatory regions of SIVmac239 were replaced by the corresponding regions from HIV-1/R73 (SIVR7nef+). SIVR7nef+ displays enhanced infectivity and accelerated replication kinetics in primary human and rhesus PBMC infections compared to its nef-defective counterpart. Converse chimeras, containing SIV Nef in an HIV-1 background (R7SIVnef+) also exhibit greater infectivity than matched nef-defective viruses (R7SIV delta nef). These data indicate that SIV Nef, like that of HIV-1, does enhance virus replication in primary cells in tissue culture and that HIV-1 and SIV Nef are functionally interchangeable in the context of both HIV-1 and SIV.     

Requirement of human immunodeficiency virus type 1 nef for in vivo replication and pathogenicity.

The role of human immunodeficiency virus type 1 (HIV-1) accessory genes in pathogenesis has remained unclear because of the lack of a suitable in vivo model. The most controversial of these genes is nef. We investigated the requirement for Nef for in vivo replication and pathogenicity of two isolates of HIV-1 (HIV-1JR-CSF and HIV-1NL4-3) in human fetal thymus and liver implants in severe combined immunodeficient mice. HIV-1JR-CSF and HIV-1NL4-3 differ in their in vitro phenotypes in that HIV-1JR-CSF does not induce syncytia and is relatively noncytopathic, while HIV-1NL4-3 is highly cytopathic and readily induces syncytia. The nef mutants of both isolates grew with kinetics similar to those of parental virus strains in stimulated peripheral blood lymphocytes but demonstrated attenuated growth properties in vivo. HIV-1NL4-3 induced severe depletion of human thymocytes within 6 weeks of infection, whereas its nef mutant did not. Thus, HIV-1 Nef is required for efficient in vivo viral replication and pathogenicity.     

Human immunodeficiency virus type 1 induces apoptosis in CD4(+) but not in CD8(+) T cells in ex vivo-infected human lymphoid tissue

Progression of human immunodeficiency virus (HIV) disease is associated with massive death of CD4(+) T cells along with death and/or dysfunction of CD8(+) T cells. In vivo, both HIV infection per se and host factors may contribute to the death and/or dysfunction of CD4(+) and CD8(+) T cells. Progression of HIV disease is often characterized by a switch from R5 to X4 HIV type 1 (HIV-1) variants. In human lymphoid tissues ex vivo, it was shown that HIV infection is sufficient for CD4(+) T-cell depletion. Here we address the question of whether infection of human lymphoid tissue ex vivo with prototypic R5 or X4 HIV variants also depletes or impairs CD8(+) T cells. We report that whereas productive infection of lymphoid tissue ex vivo with R5 and X4 HIV-1 isolates induced apoptosis in CD4(+) T cells, neither viral isolate induced apoptosis in CD8(+) T cells. Moreover, in both infected and control tissues we found similar numbers of CD8(+) T cells and similar production of cytokines by these cells in response to phorbol myristate acetate or anti-CD3-anti-CD28 stimulation. Thus, whereas HIV-1 infection per se in human lymphoid tissue is sufficient to trigger apoptosis in CD4(+) T cells, the death of CD8(+) T cells apparently requires additional factors.     

CD4 down-modulation by human immunodeficiency virus type 1 Nef correlates with the efficiency of viral replication and with CD4(+) T-cell depletion in human lymphoid tissue ex vivo

The human immunodeficiency virus type 1 (HIV-1) Nef protein is an important virulence factor. Nef has several functions, including down-modulation of CD4 and class I major histocompatibility complex cell surface expression, enhancement of virion infectivity, and stimulation of viral replication in peripheral blood mononuclear cells. Nef also increases HIV-1 replication in human lymphoid tissue (HLT) ex vivo. We analyzed recombinant and primary nef alleles with highly divergent activity in different in vitro assays to clarify which of these Nef activities are functionally linked. Our results demonstrate that Nef activity in CD4 down-regulation correlates significantly with the efficiency of HIV-1 replication and with the severity of CD4(+) T-cell depletion in HLT. In conclusion, HIV-1 Nef variants with increased activity in CD4 down-modulation would cause severe depletion of CD4(+) T cells in lymphoid tissues and accelerate AIDS progression.     

Fusion-induced apoptosis contributes to thymocyte depletion by a pathogenic human immunodeficiency virus type 1 envelope in the human thymus

The mechanisms of CD4(+) T-cell depletion during human immunodeficiency virus type 1 (HIV-1) infection remain incompletely characterized. Of particular importance is how CD4(+) T cells are depleted within the lymphoid organs, including the lymph nodes and thymus. Herein we characterize the pathogenic mechanisms of an envelope from a rapid progressor (R3A Env) in the NL4-3 backbone (NL4-R3A) which is able to efficiently replicate and deplete CD4(+) thymocytes in the human fetal-thymus organ culture (HF-TOC). We demonstrate that uninterrupted replication is required for continual thymocyte depletion. During depletion, NL4-R3A induces an increase in thymocytes which uptake 7AAD, a marker of cell death, and which express active caspase-3, a marker of apoptosis. While 7AAD uptake is observed predominantly in uninfected thymocytes (p24(-)), active caspase-3 is expressed in both infected (p24(+)) and uninfected thymocytes (p24(-)). When added to HF-TOC with ongoing infection, the protease inhibitor saquinavir efficiently suppresses NL4-R3A replication. In contrast, the fusion inhibitors T20 and C34 allow for sustained HIV-1 production. Interestingly, T20 and C34 effectively prevent thymocyte depletion in spite of this sustained replication. Apoptosis of both p24(-) and p24(+) thymocytes appears to be envelope fusion dependent, as T20, but not saquinavir, is capable of reducing thymocyte apoptosis. Together, our data support a model whereby pathogenic envelope-dependent fusion contributes to thymocyte depletion in HIV-1-infected thymus, correlated with induction of apoptosis in both p24(+) and p24(-) thymocytes.     

Induction of AIDS in Rhesus Monkeys by a Recombinant Simian Immunodeficiency Virus Expressing nef of Human Immunodeficiency Virus Type 1

A nef gene is present in all primate lentiviruses, including human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus of macaque monkeys (SIVmac). However, the nef genes of HIV-1 and SIVmac exhibit minimal sequence identity, and not all properties are shared by the two. Nef sequences of SIVmac239 were replaced by four independent nef alleles of HIV-1 in a context that was optimal for expression. The sources of the HIV-1 nef sequences included NL 4-3, a variant NL 4-3 gene derived from a recombinant-infected rhesus monkey, a patient nef allele, and a nef consensus sequence. Of 16 rhesus monkeys infected with these SHIVnef chimeras, 9 maintained high viral loads for prolonged periods, as observed with the parental SIVmac239, and 6 have died with AIDS 52 to 110 weeks postinfection. Persistent high loads were observed at similar frequencies with the four different SIV recombinants that expressed these independent HIV-1 nef alleles. Infection with other recombinant SHIVnef constructions resulted in sequence changes in infected monkeys that either created an open nef reading frame or optimized the HIV-1 nef translational context. The HIV-1 nef gene was uniformly retained in all SHIVnef-infected monkeys. These results demonstrate that HIV-1 nef can substitute for SIVmac nef in vivo to produce a pathogenic infection. However, the model suffers from an inability to consistently obtain persisting high viral loads in 100% of the infected animals, as is observed with the parental SIVmac239.     

Context-dependent role of human immunodeficiency virus type 1 auxiliary genes in the establishment of chronic virus producers.

Two molecularly cloned viruses, human immunodeficiency virus type 1 (HIV-1)-NL4-3 (NL4-3) and HIV-1-HXB-2 (HXB-2), have been used to study the role of HIV-1 auxiliary genes in the establishment of chronic virus producers. NL4-3 encodes all known HIV-1 proteins, whereas HXB-2 is defective for three auxiliary genes: vpr, vpu, and nef. Studies were done in H9 cells, a T-cell line unusually permissive for the establishment of chronic virus producers. NL4-3 and HXB-2 undergo lytic phases of infection in H9 cultures with HXB-2, but not NL4-3, supporting the efficient establishment of chronic virus producers. Tests of mutant NL4-3 genomes containing various combinations of defective auxiliary genes revealed that both vpr and nef limited the ability of NL4-3 to establish chronic virus producers. Tests of a series of recombinants between NL4-3 and HXB-2 revealed that 5' internal sequences as well as fragments containing defective auxiliary genes affected the establishment of chronic virus producers. Viral envelope sequences and levels of virus production did not correlate with the ability to establish chronic virus producers. These results suggest that complex interactions of viral auxiliary and nonauxiliary gene functions with the host cell determine the ability to establish chronic virus producers.     

Nef-mediated enhancement of virion infectivity and stimulation of viral replication are fundamental properties of primate lentiviruses

Nef is a multifunctional accessory protein of primate lentiviruses. Recently, it has been shown that the ability of Nef to downmodulate CD4, CD28, and class I major histocompatibility complex is highly conserved between most or all primate lentiviruses, whereas Nef-mediated downregulation of T-cell receptor-CD3 was lost in the lineage that gave rise to human immunodeficiency virus type 1 (HIV-1). Whether or not other Nef activities are preserved between different groups of primate lentiviruses remained to be determined. Here, we show that nef genes from a large variety of HIVs and simian immunodeficiency viruses (SIVs) enhance virion infectivity and stimulate viral replication in human cells and/or in ex vivo infected human lymphoid tissue (HLT). Notably, nef alleles from unpassaged SIVcpz and SIVsmm enhanced viral infectivity, replication, and cytopathicity in cell culture and in ex vivo infected HLT as efficiently as those from HIV-1 and HIV-2, their human counterparts. Furthermore, nef genes from several highly divergent SIVs that have not been found in humans were also highly active in human cells and/or tissues. Thus, most primate lentiviral Nefs enhance virion infectivity and stimulate viral replication. Moreover, our data show that SIVcpz and SIVsmm Nefs do not require adaptive changes to perform these functions in human cells or tissues and support the idea that nef alleles from other primate lentiviruses would also be capable of promoting efficient virus spread in humans.     

Human immunodeficiency virus type 1 (HIV-1) non-B subtypes are similar to HIV-1 subtype B in that coreceptor specificity is a determinant of cytopathicity in human lymphoid tissue infected ex vivo

We sought to determine the relationship between virus-mediated CD4(+) T-lymphocyte cytopathicity and viral coreceptor preference among various human immunodeficiency virus type 1 (HIV-1) subtypes in an ex vivo-infected human lymphoid tissue model. Our data show that all R5 HIV-1 infections resulted in mild depletion of CD4(+) T lymphocytes, whereas all X4 HIV-1 infections caused severe depletion of CD4(+) T lymphocytes regardless of their subtype origin. Thus, at least for the viruses within subtypes A, B, C, and E that were tested, coreceptor specificity is a critical factor that determines the ability of HIV-1 to deplete CD4(+) T cells in human lymphoid tissue infected ex vivo.     

Vpr and Vpu are important for efficient human immunodeficiency virus type 1 replication and CD4+ T-cell depletion in human lymphoid tissue ex vivo

The relevance of the accessory vpr, vpu, and nef genes for human immunodeficiency virus type 1 (HIV-1) replication in human lymphoid tissue (HLT), the major site of viral replication in vivo, is largely unknown. Here, we show that an individual deletion of nef, vpr, or vpu significantly decreases HIV-1 replication and prevents CD4+ T-cell depletion in ex vivo HLT. However, only combined defects in all three accessory genes entirely disrupt the replicative capacity of HIV-1. Our results demonstrate that nef, vpr, and vpu are all essential for efficient viral spread in HLT, suggesting an important role in AIDS pathogenesis.     

Noninfectious X4 but not R5 human immunodeficiency virus type 1 virions inhibit humoral immune responses in human lymphoid tissue ex vivo

Ex vivo human immunodeficiency virus type 1 (HIV-1) infection of human lymphoid tissue recapitulates some aspects of in vivo HIV-1 infection, including a severe depletion of CD4(+) T cells and suppression of humoral immune responses to recall antigens or to polyclonal stimuli. These effects are induced by infection with X4 HIV-1 variants, whereas infection with R5 variants results in only mild depletion of CD4(+) T cells and no suppression of immune responses. To study the mechanisms of suppression of immune responses in this ex vivo system, we used aldrithiol-2 (AT-2)-inactivated virions that have functional envelope glycoproteins but are not infectious and do not deplete CD4(+) T cells in human lymphoid tissues ex vivo. Nevertheless, AT-2-inactivated X4 (but not R5) HIV-1 virions, even with only a brief exposure, inhibit antibody responses in human lymphoid tissue ex vivo, similarly to infectious virus. This phenomenon is mediated by soluble immunosuppressive factor(s) secreted by tissue exposed to virus.     

cis expression of the F12 human immunodeficiency virus (HIV) Nef allele transforms the highly productive NL4-3 HIV type 1 to a replication-defective strain: involvement of both Env gp41 and CD4 intracytoplasmic tails

F12 human immunodeficiency virus type 1 (HIV-1) nef is a naturally occurring nef mutant cloned from the provirus of a nonproductive, nondefective, and interfering HIV-1 variant (F12-HIV). We have already shown that cells stably transfected with a vector expressing the F12-HIV nef allele do not downregulate CD4 receptors and, more peculiarly, become resistant to the replication of wild type (wt) HIV. In order to investigate the mechanism of action of such an HIV inhibition, the F12-HIV nef gene was expressed in the context of the NL4-3 HIV-1 infectious molecular clone by replacing the wt nef gene (NL4-3/chi). Through this experimental approach we established the following. First, NL4-3/chi and nef-defective (Deltanef) NL4-3 viral particles behave very similarly in terms of viral entry and HIV protein production during the first replicative cycle. Second, no viral particles were produced from cells infected with NL4-3/chi virions, whatever the multiplicity of infection used. The viral inhibition apparently occurs at level of viral assembling and/or release. Third, this block could not be relieved by in-trans expression of wt nef. Finally, NL4-3/chi reverts to a producer HIV strain when F12-HIV Nef is deprived of its myristoyl residue. Through a CD4 downregulation competition assay, we demonstrated that F12-HIV Nef protein potently inhibits the CD4 downregulation induced by wt Nef. Moreover, we observed a redistribution of CD4 receptors at the cell margin induced by F12-HIV Nef. These observations strongly suggest that F12-HIV Nef maintains the ability to interact with the intracytoplasmic tail of the CD4 receptor molecule. Remarkably, we distinguished the intracytoplasmic tails of Env gp41 and CD4 as, respectively, viral and cellular targets of the F12-HIV Nef-induced viral retention. For the first time, the inhibition of the viral life cycle by means of in-cis expression of a Nef mutant is here reported. Delineation of the F12-HIV Nef mechanism of action may offer additional approaches to interference with the propagation of HIV infection.     

Nef enhances human immunodeficiency virus type 1 infectivity and replication independently of viral coreceptor tropism

We investigated the infectivities and replicative capacities of a large panel of variants of the molecular human immunodeficiency virus type 1 (HIV-1) NL4-3 clone that differ exclusively in the V3 region of the viral envelope glycoprotein and the nef gene. Our results demonstrate that Nef enhances virion infectivity and HIV-1 replication independently of the viral coreceptor tropism.     

Deletion of nef slows but does not prevent CD4-positive T-cell depletion in human immunodeficiency virus type 1-infected human-PBL-SCID mice.

The pathogenicity of four human immunodeficiency virus type 1 (HIV-1) isolates with nef deleted for SCID mice repopulated with human peripheral blood leukocytes (hu-PBL-SCID mice) was studied. Deletion of nef led to a substantial reduction in CD4-positive T-cell depletion and delayed kinetics of plasma viremia in infected hu-PBL-SCID mice. Deletion of the nef gene impacts both the efficiency of primary infection and the cytopathicity of virus for infected CD4-positive T cells in this animal model of HIV-1 infection.     

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.     

Pathogenic effects of human herpesvirus 6 in human lymphoid tissue ex vivo

Human herpesvirus 6 (HHV-6) is a potentially immunosuppressive agent that has been suggested to act as a cofactor in the progression of human immunodeficiency virus disease. However, the lack of suitable experimental models has hampered the elucidation of the mechanisms of HHV-6-mediated immune suppression. Here, we used ex vivo lymphoid tissue to investigate the cellular tropism and pathogenic mechanisms of HHV-6. Viral strains belonging to both HHV-6 subgroups (A and B) were able to productively infect human tonsil tissue fragments in the absence of exogenous stimulation. The majority of viral antigen-expressing cells were CD4(+) T lymphocytes expressing a nonnaive phenotype, while CD8(+) T cells were efficiently infected only with HHV-6A. Accordingly, HHV-6A infection resulted in the depletion of both CD4(+) and CD8(+) T cells, whereas in HHV-6B-infected tissue CD4(+) T cells were predominantly depleted. The expression of different cellular antigens was dramatically altered in HHV-6-infected tissues: whereas CD4 was upregulated, both CD46, which serves as a cellular receptor for HHV-6, and CD3 were downmodulated. However, CD3 downmodulation was restricted to infected cells, while the loss of CD46 expression was generalized. Moreover, HHV-6 infection markedly enhanced the production of the CC chemokine RANTES, whereas other cytokines and chemokines were only marginally affected. These results provide the first evidence, in a physiologically relevant study model, that HHV-6 can severely affect the physiology of secondary lymphoid organs through direct infection of T lymphocytes and modulation of key membrane receptors and chemokines.