Macrophage tropism of human immunodeficiency virus type 1 isolates from brain and lymphoid tissues predicts neurotropism independent of coreceptor specificity

The viral determinants that underlie human immunodeficiency virus type 1 (HIV-1) neurotropism are unknown, due in part to limited studies on viruses isolated from brain. Previous studies suggest that brain-derived viruses are macrophage tropic (M-tropic) and principally use CCR5 for virus entry. To better understand HIV-1 neurotropism, we isolated primary viruses from autopsy brain, cerebral spinal fluid, blood, spleen, and lymph node samples from AIDS patients with dementia and HIV-1 encephalitis. Isolates were characterized to determine coreceptor usage and replication capacity in peripheral blood mononuclear cells (PBMC), monocyte-derived macrophages (MDM), and microglia. Env V1/V2 and V3 heteroduplex tracking assay and sequence analyses were performed to characterize distinct variants in viral quasispecies. Viruses isolated from brain, which consisted of variants that were distinct from those in lymphoid tissues, used CCR5 (R5), CXCR4 (X4), or both coreceptors (R5X4). Minor usage of CCR2b, CCR3, CCR8, and Apj was also observed. Primary brain and lymphoid isolates that replicated to high levels in MDM showed a similar capacity to replicate in microglia. Six of 11 R5 isolates that replicated efficiently in PBMC could not replicate in MDM or microglia due to a block in virus entry. CD4 overexpression in microglia transduced with retroviral vectors had no effect on the restricted replication of these virus strains. Furthermore, infection of transfected cells expressing different amounts of CD4 or CCR5 with M-tropic and non-M-tropic R5 isolates revealed a similar dependence on CD4 and CCR5 levels for entry, suggesting that the entry block was not due to low levels of either receptor. Studies using TAK-779 and AMD3100 showed that two highly M-tropic isolates entered microglia primarily via CXCR4. These results suggest that HIV-1 tropism for macrophages and microglia is restricted at the entry level by a mechanism independent of coreceptor specificity. These findings provide evidence that M-tropism rather than CCR5 usage predicts HIV-1 neurotropism.     

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.     

In vivo cellular tropism of human T-cell leukemia virus type 1.

To establish the phenotype of human T-cell leukemia virus type 1 (HTLV-1)-infected cells in peripheral blood, the polymerase chain reaction was used to detect and quantitate viral DNA in subpopulations of leukocytes obtained from patients with tropical spastic paraparesis and asymptomatic carriers. HTLV-1 could not be detected in peripheral blood mononuclear cells thoroughly depleted of T lymphocytes (E- CD3-), nor could it be detected in highly enriched populations of B lymphocytes (E- CD19+), monocytes (E- CD14+), or natural killer cells (E- CD16+). T lymphocytes were strongly positive for HTLV-1, and fractionation of this population revealed that 90 to 99% of the HTLV-1 DNA segregated with the CD4+ CD8- and CD45RO+ subsets. No difference between the cell type distribution of HTLV-1 in the asymptomatic carrier and the subjects with tropical spastic paraparesis was evident. Southern hybridization of genomic DNA prepared from the peripheral blood of HTLV-1 carriers indicated that up to 10% of circulating leukocytes may carry the HTLV-1 provirus.     

Macrophage tropism of human immunodeficiency virus type 1 and utilization of the CC-CKR5 coreceptor.

The recent identification of the CC-CKR5 beta chemokine receptor as a major cofactor for entry of macrophage-tropic isolates of human immunodeficiency virus type 1 (HIV-1) raises the question of whether macrophage tropism is determined by utilization of this chemokine receptor. We observe that in addition to macrophage-tropic isolates of clades A, B, and E, macrophage-tropic isolates of clade F also utilize the CC-CKR5 molecule for entry. However, using single-round replication-competent reporter viruses carrying the envelope genes of T-cell line-tropic or macrophage-tropic phenotypic recombinant and mutant HIV-1 strains in infection of stable cell lines that coexpress the CD4 and chemokine receptors, we were unable to establish a strict correlation between macrophage tropism and utilization of the CC-CKR5 chemokine receptor. This latter finding suggests that a cofactor other than CC-CKR5 serves to determine entry into primary macrophages.     

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.     

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.     

Primary infection by a human immunodeficiency virus with atypical coreceptor tropism

The great majority of human immunodeficiency virus type 1 (HIV-1) strains enter CD4+ target cells by interacting with one of two coreceptors, CCR5 or CXCR4. Here we describe a transmitted/founder (T/F) virus (ZP6248) that was profoundly impaired in its ability to utilize CCR5 and CXCR4 coreceptors on multiple CD4+ cell lines as well as primary human CD4+ T cells and macrophages in vitro yet replicated to very high titers (>80 million RNA copies/ml) in an acutely infected individual. Interestingly, the envelope (Env) glycoprotein of this clade B virus had a rare GPEK sequence in the crown of its third variable loop (V3) rather than the consensus GPGR sequence. Extensive sequencing of sequential plasma samples showed that the GPEK sequence was present in virtually all Envs, including those from the earliest time points after infection. The molecularly cloned (single) T/F virus was able to replicate, albeit poorly, in cells obtained from ccr5Δ32 homozygous donors. The ZP6248 T/F virus could also infect cell lines overexpressing the alternative coreceptors GPR15, APJ, and FPRL-1. A single mutation in the V3 crown sequence (GPEK->GPGK) of ZP6248 restored its infectivity in CCR5+ cells but reduced its ability to replicate in GPR15+ cells, indicating that the V3 crown motif played an important role in usage of this alternative coreceptor. These results suggest that the ZP6248 T/F virus established an acute in vivo infection by using coreceptor(s) other than CCR5 or CXCR4 or that the CCR5 coreceptor existed in an unusual conformation in this individual.     

Human immunodeficiency virus type 1 T-cell tropism is determined by events prior to provirus formation.

Different strains of human immunodeficiency virus type 1 (HIV-1) vary in the ability to replicate in cells that bear the HIV-1 receptor, CD4. The mechanism responsible for these cell tropism differences is unknown. We examined different isolates of HIV-1 with regard to replication in specific tumor-derived CD4-positive T-cell lines and normal peripheral blood lymphocytes. To investigate early events in the virus life cycle at low multiplicities of infection, we used a modification of the polymerase chain reaction method. Use of a molecularly cloned primary HIV-1 isolate, HIV-1 JR-CSF, restricted for replication in T-cell lines, demonstrated that little or no viral DNA or RNA was synthesized in nonpermissive cells after infection. However, transfection of proviral DNA resulted in efficient transient virus production from these cells. Therefore, we conclude that at least one block to infection for HIV-1 strains in nonpermissive T cells occurs at a point in entry or uncoating before provirus formation.     

Intrinsic obstacles to human immunodeficiency virus type 1 coreceptor switching

The natural evolution of human immunodeficiency virus type 1 infection often includes a switch in coreceptor preference late in infection from CCR5 to CXCR4, a change associated with expanded target cell range and worsened clinical prognosis. Why coreceptor switching takes so long is puzzling, since it requires as few as one to two mutations. Here we report three obstacles that impede the CCR5-to-CXCR4 switch. Coreceptor switch variants were selected by target cell replacement in vitro. Most switch variants showed diminished replication compared to their parental R5 isolate. Transitional intermediates were more sensitive to both CCR5 and CXCR4 inhibitors than either the parental R5 virus or the final R5X4 (or rare X4) variant. The small number of mutations in viruses selected for CXCR4 use were distinctly nonrandom, with a dominance of charged amino acid substitutions encoded by G-to-A transitions, changes in N-linked glycosylation sites, and isolate-specific mutation patterns. Diminished replication fitness, less-efficient coreceptor use, and unique mutational pathways may explain the long delay from primary infection until the emergence of CXCR4-using viruses.     

Cytotoxic T-cell abundance and virus load in human immunodeficiency virus type 1 and human T-cell leukaemia virus type 1.

The correlation between virus load and specific cytotoxic T-lymphocyte (CTL) frequency during the chronic phase in human immunodeficiency virus type 1 (HIV-1) infection has been found to be negative in cross-sectional studies. We report here that, in infection with the related retrovirus human T-cell leukaemia virus type 1 (HTLV-1), the correlation is positive in asymptomatic carriers and zero in patients with the associated inflammatory disease HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). We demonstrate that the direction of the correlation may depend on the efficacy of the CTL response using mathematical models. We conclude that the CTL response is effective in asymptomatic carriers of HTLV-1, but ineffective in patients with HAM/TSP. Virus-mediated impairment of specific CTL production in HIV-1 infection can account for the negative correlation observed.     

Cytopathicity of human immunodeficiency virus type 2 (HIV-2) in human lymphoid tissue is coreceptor dependent and comparable to that of HIV-1

Epidemiological studies have shown that human immunodeficiency virus type 2 (HIV-2) is markedly less pathogenic than HIV-1 in vivo. Individuals infected with HIV-2 exhibit a remarkably slow rate of disease development, and these clinical properties have been attributed presumptively to an "attenuated" phenotype of HIV-2 itself. Here, we investigated the impact of coreceptor usage on the cytopathicity of HIV-2 and compared its pathogenic potential with that of HIV-1 in a unique human lymphoid histoculture model. We found that HIV-2 strains, as well as closely related simian immunodeficiency viruses (SIV), displayed mildly or highly aggressive cytopathic phenotypes depending on their abilities to use the coreceptor CCR5 or CXCR4, respectively. A side-by-side comparison of primary X4 HIV-1 and HIV-2 strains revealed similar, high degrees of cytopathicity induced by both HIV types. Furthermore, we found that HIV-2 coreceptor specificity for CCR5 and CXCR4 determined the target cell population for T-cell depletion in lymphoid tissue. Finally, utilization of the alternate coreceptors BOB and Bonzo did not significantly increase the cytopathic properties of HIV-2. These findings demonstrate that coreceptor preference is a key regulator of target cell specificity and the cytopathic potential of HIV-2, with indistinguishable rules compared with HIV-1. Moreover, HIV-2 strains are not characterized by an intrinsically lower cytopathicity than HIV-1 strains. Therefore, direct cytopathic potential per se does not explain the unique behavior of HIV-2 in people, highlighting that other unknown factors need to be elucidated as the basis for their lesser virulence in vivo.     

Species-specific tropism determinants in the human immunodeficiency virus type 1 capsid

Retroviral tropism is determined in part by cellular restriction factors that block infection by targeting the incoming viral capsid. Indeed, human immunodeficiency virus type 1 (HIV-1) infection of many nonhuman primate cells is inhibited by one such factor, termed Lv1. In contrast, a restriction factor in humans, termed Ref1, does not inhibit HIV-1 infection unless nonnatural mutations are introduced into the HIV-1 capsid protein (CA). Here, we examined the infectivity of a panel of mutant HIV-1 strains carrying substitutions in the N-terminal CA domain in cells that exhibit restriction attributable to Lv1 or Ref1. Manipulation of HIV-1 CA could alter HIV-1 tropism, and several mutations were identified that increased or decreased HIV-1 infectivity in a target-cell-specific manner. Many residues that affected HIV-1 tropism were located in the three variable loops that lie on the outer surface of the modeled HIV-1 conical capsid. Some tropism determinants, including the CypA binding site, coincided with residues whose mutation conferred on HIV-1 CA the ability to saturate Ref1 in human cells. Notably, a mutation that reverses the infectivity defect in human cells induced by CypA binding site mutation inhibits recognition by Ref1. Overall, these findings demonstrate that exposed variable loops in CA and a partial CypA "coat" can modulate restriction and HIV-1 tropism and suggest a model in which the exposed surface of the incoming retroviral capsid is the target for inhibition by host cell-specific restriction factors.     

Severe CD4+ T-cell depletion in gut lymphoid tissue during primary human immunodeficiency virus type 1 infection and substantial delay in restoration following highly active antiretroviral therapy

Gut-associated lymphoid tissue (GALT) harbors the majority of T lymphocytes in the body and is an important target for human immunodeficiency virus type 1 (HIV-1). We analyzed longitudinal jejunal biopsy samples from HIV-1-infected patients, during both primary and chronic stages of HIV-1 infection, prior to and following the initiation of highly active antiretroviral therapy (HAART) to determine the onset of CD4(+) T-cell depletion and the effect of HAART on the restoration of CD4(+) T cells in GALT. Severe depletion of intestinal CD4(+) T cells occurred during primary HIV-1 infection. Our results showed that the restoration of intestinal CD4(+) T cells following HAART in chronically HIV-1-infected patients was substantially delayed and incomplete. In contrast, initiation of HAART during early stages of infection resulted in near-complete restoration of intestinal CD4(+) T cells, despite the delay in comparison to peripheral blood CD4(+) T-cell recovery. DNA microarray analysis of gene expression profiles and flow-cytometric analysis of lymphocyte homing and cell proliferation markers demonstrated that cell trafficking to GALT and not local proliferation contributed to CD4(+) T-cell restoration. Evaluation of jejunal biopsy samples from long-term HIV-1-infected nonprogressors showed maintenance of normal CD4(+) T-cell levels in both GALT and peripheral blood. Our results demonstrate that near-complete restoration of mucosal immune system can be achieved by initiating HAART early in HIV-1 infection. Monitoring of the restoration and/or maintenance of CD4(+) T cells in GALT provides a more accurate assessment of the efficacy of antiviral host immune responses as well as HAART.     

Human immunodeficiency virus pseudotypes with expanded cellular and species tropism.

One mechanism for expanding the cellular tropism of a virus is through the formation of phenotypically mixed particles or pseudotypes, a process commonly occurring during viral assembly in cells infected with two or more viruses. We report here that dual infection of cells with human immunodeficiency virus (HIV) and a murine amphotropic retrovirus leads to the production of HIV pseudotypes that have acquired the host range of the amphotropic retrovirus and are capable of infecting not only CD4- human cells but also mouse cells. The replication of the HIV pseudotypes in the various CD4- cells was determined by measuring the appearance of HIV antigens in the supernatants, by cocultivation of CD4+ CEM cells with the infected CD4- cells, and in some cases by assaying the culture supernatants directly for infectious virus. Of the cells tested, human foreskin fibroblasts were the best host cells, and by in situ cytohybridization, we were able to document that all cells in the culture were infected. In addition, the temporal appearance of HIV-specific proteins in the HIV pseudotype-infected fibroblasts was similar to that seen in CD4+ CEM cells. If the human fibroblasts were first infected with the amphotropic retrovirus, they demonstrated the property of superinfection exclusion and were resistant to subsequent infection by the HIV pseudotype. In other cell lines, including the human glioblastoma-derived cell line U373MG, HeLa cells, BALB/c mouse embryo cells, and SC-1 wild mouse cells, although the HIV pseudotype infection appeared to be less efficient, substantial amounts of HIV were nevertheless produced. These results indicate that the HIV (amphotropic retrovirus) pseudotypes may be useful for studying the molecular biology of HIV infections in a wide range of cells.     

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.     

Influence of gag on human immunodeficiency virus type 1 species-specific tropism

The narrow host range of human immunodeficiency virus type 1 (HIV-1) is due in part to dominant acting restriction factors in humans (Ref1) and monkeys (Lv1). Here we show that gag encodes determinants of species-specific lentiviral infection, related in part to such restriction factors. Interaction between capsid and host cyclophilin A (CypA) protects HIV-1 from restriction in human cells but is essential for maximal restriction in simian cells. We show that sequence variation between HIV-1 isolates leads to variation in sensitivity to restriction factors in human and simian cells. We present further evidence for the importance of target cell CypA over CypA packaged in virions, specifically in the context of gp160 pseudotyped HIV-1 vectors. We also show that sensitivity to restriction is controlled by an H87Q mutation in the capsid, implicated in the immune control of HIV-1, possibly linking immune and innate control of HIV-1 infection.     

Macrophage tropism determinants of human immunodeficiency virus type 1 in vivo.

Strains of human immunodeficiency virus type 1 differ in their abilities to infect and replicate in primary human macrophages. Chimeric clones were constructed from a provirus unable to infect macrophages (NLHX) and envelope sequences (V3 loop) of viruses derived without cultivation from brain (YU2 and w1-1c1) or spleen (w2-1b4) tissues. The substituted V3 loop sequences in each case were sufficient to confer upon NLHX the ability to infect macrophages. Furthermore, an envelope domain immediately N terminal to the V3 loop also was found to modulate the level of replication in macrophages. These results demonstrate that an envelope determinant derived directly from patients with AIDS confers HIV-1 tropism for macrophages.     

Conserved changes in envelope function during human immunodeficiency virus type 1 coreceptor switching

We studied the evolution of human immunodeficiency virus type 1 (HIV-1) envelope function during the process of coreceptor switching from CCR5 to CXCR4. Site-directed mutagenesis was used to introduce most of the possible intermediate mutations in the envelope for four distinct coreceptor switch mutants, each with a unique pattern of CCR5 and CXCR4 utilization that extended from highly efficient use of both coreceptors to sole use of CXCR4. Mutated envelopes with some preservation of entry function on either CCR5- or CXCR4-expressing target cells were further characterized for their sensitivity to CCR5 or CXCR4 inhibitors, soluble CD4, and the neutralizing antibodies b12-IgG and 4E10. A subset of mutated envelopes was also studied in direct CD4 or CCR5 binding assays and in envelope-mediated fusion reactions. Coreceptor switch intermediates displayed increased sensitivity to CCR5 inhibitors (except for a few envelopes with mutations in V2 or C2) that correlated with a loss in CCR5 binding. As use of CXCR4 improved, infection mediated by the mutated envelopes became more resistant to soluble CD4 inhibition and direct binding to CD4 increased. These changes were accompanied by increasing resistance to the CXCR4 inhibitor AMD3100. Sensitivity to neutralizing antibody was more variable, although infection of CXCR4-expressing targets was generally more sensitive to neutralization by both b12-IgG and 4E10 than infection of CCR5-expressing target cells. These changes in envelope function were uniform in all four series of envelope mutations and thus were independent of the final use of CCR5 and CXCR4. Decreased CCR5 and increased CD4 binding appear to be common features of coreceptor switch intermediates.