Virus immunocapture provides evidence of CD8 lymphocyte-derived HIV-1 in vivo.

OBJECTIVES: To demonstrate that HIV-1 immunocapture with an antibody against CD8 specifically captures virions derived from infected CD8 T cells, and to determine the proportion of HIV-1 derived from CD8 lymphocytes in plasma samples from HIV-infected individuals. METHODS: A virus capture method was developed to enable the detection of HIV-1 virions based upon the presence of certain cell-specific host-derived proteins (CD8, CD3, CD36) within the viral envelope. HIV-1 virions were captured using antibodies against these proteins and levels of bound virus were determined by quantitative reverse transcriptase-polymerase chain reaction. Highly pure CD8 and CD3+CD8- T-cell cultures were used as in-vitro models to determine the specificity of the virus capture technique. RESULTS: The in-vitro model demonstrates that incorporation of the CD8 molecule into released virions is specific to infection of CD8 T cells. Levels of HIV-1 immunocaptured from plasma of infected individuals using the anti-CD8 antibody indicate that up to 15% (range 10-33) of the plasma viral load is derived from CD8 lymphocytes. CONCLUSION: This study demonstrates for the first time that HIV-1-infected CD8 T cells can contribute substantially to levels of circulating virus during the course of infection. Levels of CD8-derived virus did not correlate with the level of infection of circulating CD8 T cells, but do show a significantly good fit to plasma viral loads based on a power model. The extensive infection of CD8 T cells implied by these results may contribute towards immune dysfunction and disease progression to AIDS.

Comparison of tissue distribution, persistence, and molecular epidemiology of parvovirus B19 and novel human parvoviruses PARV4 and human bocavirus.

BACKGROUND: PARV4 and human bocavirus (HBoV) are newly discovered human parvoviruses with poorly understood epidemiologies and disease associations. We investigated the frequencies of persistence, tissue distribution, and influence of immunosuppression on replication of these viruses. METHODS: At autopsy, bone marrow, lymphoid tissue, and brain tissue from human immunodeficiency virus (HIV)-infected individuals with acquired immunodeficiency syndrome (AIDS) and those without AIDS and from HIV-uninfected individuals were screened for parvovirus B19, PARV4, and HBoV DNA by means of quantitative polymerase chain reaction analyses. RESULTS: B19 DNA was detected both in HIV-infected study subjects (13 of 24) and in HIV-uninfected study subjects (8 of 8), whereas PARV4 DNA was detected only in HIV-infected study subjects (17 of 24). HBoV DNA was not detected in any study subjects. The degree of immunosuppression with HIV infection did not influence B19 or PARV4 viral loads. B19 or PARV4 plasma viremia was not detected in any study subjects (n=76; viral load <25 DNA copies/mL). A significantly older age distribution was found for study subjects infected with B19 genotype 2, compared with those infected with B19 genotype 1. Two genotypes of PARV4 were detected; study subjects carrying prototype PARV4 (genotype 1) were younger (all born after 1958) than those infected with genotype 2 (PARV5; study subjects born between 1949 and 1956). CONCLUSIONS: Tight immune control of replication of B19 and PARV4 was retained despite profound immunosuppression. Recent genotype replacement of PARV4, combined with absent sequence diversity among genotype 1 sequences, suggests a recent, epidemic spread in the United Kingdom, potentially through transmission routes shared by HIV.

Changes in the V3 region of gp120 contribute to unusually broad coreceptor usage of an HIV-1 isolate from a CCR5 Delta32 heterozygote.

Heterozygosity for the CCR5 Delta32 allele is associated with delayed progression to AIDS in human immunodeficiency virus type 1 (HIV-1) infection. Here we describe an unusual HIV-1 isolate from the blood of an asymptomatic individual who was heterozygous for the CCR5 Delta32 allele and had reduced levels of CCR5 expression. The primary virus used CCR5, CXCR4, and an unusually broad range of alternative coreceptors to enter transfected cells. However, only CXCR4 and CCR5 were used to enter primary T cells and monocyte-derived macrophages, respectively. Full-length Env clones had an unusually long V1/V2 region and rare amino acid variants in the V3 and C4 regions. Mutagenesis studies and structural models suggested that Y308, D321, and to a lesser extent K442 and E444, contribute to the broad coreceptor usage of these Envs, whereas I317 is likely to be a compensatory change. Furthermore, database analysis suggests that covariation can occur at positions 308/317 and 308/321 in vivo. Y308 and D321 reduced dependence on the extracellular loop 2 (ECL2) region of CCR5, while these residues along with Y330, K442, and E444 enhanced dependence on the CCR5 N-terminus compared to clade B consensus residues at these positions. These results suggest that expanded coreceptor usage of HIV-1 can occur in some individuals without rapid progression to AIDS as a consequence of changes in the V3 region that reduce dependence on the ECL2 region of CCR5 by enhancing interactions with conserved structural elements in G-protein-coupled receptors.

Inhibition of CCR5-mediated infection by diverse R5 and R5X4 HIV and SIV isolates using novel small molecule inhibitors of CCR5: effects of viral diversity, target cell and receptor density.

Highly active anti-retroviral therapy (HAART) has been very effective in reducing viral loads in human immunodeficiency virus (HIV)-1 patients. However, current therapies carry detrimental side effects, require complex drug regimes and are threatened by the emergence of drug-resistant variants. There is an urgent need for new anti-HIV drugs that target different stages of the replication cycle. Several synthetic small organic molecules that inhibit HIV infection by binding to the CCR5 coreceptor without causing cell activation have already been reported. Here, we have exploited a series of CCR5 antagonists to investigate their effects on diverse HIV and the simian counterpart (SIV) isolates for infection of a variety of cell types via different concentrations of cell surface CCR5. These inhibitors show no cross-reactivity against alternative HIV coreceptors including CCR3, CCR8, GPR1, APJ, CXCR4 and CXCR6. They are able to inhibit a diverse range of R5 and R5X4 HIV-1 isolates as well as HIV-2 and SIV strains. Inhibition was observed in cell lines as well as primary PBMCs and macrophages. The extent of inhibition was dependent on cell type and on cell surface CCR5 concentration. Our results underscore the potential of CCR5 inhibitors for clinical development.

Use of alternate coreceptors on primary cells by two HIV-1 isolates.

Two HIV-1 isolates (CM4 and CM9) able to use alternate HIV-1 coreceptors on transfected cell lines were tested for their sensitivity to inhibitors of HIV-1 entry on primary cells. CM4 was able to use CCR5 and Bob/GPR15 efficiently in transfected cells. The R5 isolate grew in Delta32/Delta32 CCR5 PBMC in the absence or presence of AMD3100, a CXCR4-specific inhibitor, indicating that it uses a receptor other than CCR5 or CXCR4 on primary cells. It was insensitive to the CCR5 entry inhibitors RANTES and PRO140, but was partially inhibited by vMIP-1, a chemokine that binds CCR3, CCR8, GPR15 and CXCR6. The coreceptor used by this isolate on primary cells is currently unknown. CM9 used CCR5, CXCR4, Bob/GPR15, CXCR6, CCR3, and CCR8 on transfected cells and was able to replicate in the absence or presence of AMD3100 in Delta32/Delta32 CCR5 PBMC. It was insensitive to eotaxin, vMIP-1 and I309 when tested individually, but was inhibited completely when vMIP-1 or I309 was combined with AMD3100. Both I309 and vMIP-1 bind CCR8, strongly suggesting that this isolate can use CCR8 on primary cells. Collectively, these data suggest that some HIV-1 isolates can use alternate coreceptors on primary cells, which may have implications for strategies that aim to block viral entry.

Identification of a subset of human immunodeficiency virus type 1 (HIV-1), HIV-2, and simian immunodeficiency virus strains able to exploit an alternative coreceptor on untransformed human brain and lymphoid cells.

The chemokine receptors CCR5 and CXCR4 are the major coreceptors for human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV). At least 12 other chemokine receptors or close relatives support infection by particular HIV and SIV strains on CD4(+) transformed indicator cell lines in vitro. However, the role of these alternative coreceptors in vivo is presently thought to be insignificant. Infection of cell lines expressing high levels of recombinant CD4 and coreceptors thus does not provide a true indication of coreceptor use in vivo. We therefore tested primary untransformed cell cultures that lack CCR5 and CXCR4, including astrocytes and brain microvascular endothelial cells (BMVECs), for naturally expressed alternative coreceptors functional for HIV and SIV infection. An adenovirus vector (Ad-CD4) was used to express CD4 in CD4(-) astrocytes and thus confer efficient infection if a functional coreceptor is present. Using a large panel of viruses with well-defined coreceptor usage, we identified a subset of HIV and SIV strains able to infect two astrocyte cultures derived from adult brain tissue. Astrocyte infection was partially inhibited by several chemokines, indicating a role for the chemokine receptor family in the observed infection. BMVECs were weakly positive for CD4 but negative for CCR5 and CXCR4 and were susceptible to infection by the same subset of isolates that infected astrocytes. BMVEC infection was efficiently inhibited by the chemokine vMIP-I, implicating one of its receptors as an alternative coreceptor for HIV and SIV infection. Furthermore, we tested whether the HIV type 1 and type 2 strains identified were able to infect peripheral blood mononuclear cells (PBMCs) via an alternative coreceptor. Several strains replicated in Delta32/Delta32 CCR5 PBMCs with CXCR4 blocked by AMD3100. This AMD3100-resistant replication was also sensitive to vMIP-I inhibition. The nature and potential role of this alternative coreceptor(s) in HIV infection in vivo is discussed.

Human Leydig cells are productively infected by some HIV-2 and SIV strains but not by HIV-1.

OBJECTIVES: With the use of highly active antiretroviral therapy, the identification of HIV reservoirs within the body has become an important issue. However, the testis has been largely ignored despite representing a pharmacologic sanctuary which could act as a viral reservoir. DESIGN: Because alterations in testosterone production have frequently been reported in HIV-infected individuals, we investigated whether the testosterone-producing Leydig cells could become directly infected by HIV-1, HIV-2 or SIV. METHODS: Purified Leydig cells were infected with a panel of HIV-1, HIV-2 and SIV strains and examined for expression of HIV/SIV receptors. Additionally, the impact of CD4 transduction on Leydig cell infection was determined. RESULTS: Leydig cells were unable to support productive infection of the seven HIV-1 isolates tested. No CD4, CXCR4 or CCR5 expression was evident on the surface of Leydig cells and transduction with a CD4 expressing adenovirus did not induce HIV-1 infection. In contrast, some primary and laboratory adapted CD4-independent HIV-2 and SIV strains were able to enter and replicate productively in Leydig cells. CONCLUSIONS: Our results suggest that Leydig cells do not represent a target for HIV-1 infection within the testis. In contrast, Leydig cells support HIV-2 and SIV infection and thus represent a potential target for infection. Receptor use and significance of HIV-2/SIV infection of Leydig cells remain to be determined.