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Macrophages express low levels of the CD4 receptor compared to T-cells. Macrophage-tropic HIV strains replicating in brain of untreated patients with HIV-associated dementia (HAD) express Envs that are adapted to overcome this restriction through mechanisms that are poorly understood. Here, bioinformatic analysis of env sequence datasets together with functional studies identified polymorphisms in the β3 strand of the HIV gp120 bridging sheet that increase M-tropism. D197, which results in loss of an N-glycan located near the HIV Env trimer apex, was detected in brain in some HAD patients, while position 200 was estimated to be under positive selection. D197 and T/V200 increased fusion and infection of cells expressing low CD4 by enhancing gp120 binding to CCR5. These results identify polymorphisms in the HIV gp120 bridging sheet that overcome the restriction to macrophage infection imposed by low CD4 through enhanced gp120-CCR5 interactions, thereby promoting infection of brain and other macrophage-rich tissues., (Copyright © 2015 Elsevier Inc. All rights reserved.)

HIV infects macrophages and microglia in the central nervous system (CNS). Mechanisms that enhance HIV macrophage/microglial tropism are not well understood. Here, we identify an HIV Env variant in the V4 region of gp120, Asp 386 (D386), that eliminates an N-linked glycosylation site at position 386, enhances viral replication in macrophages, and is present at a higher frequency in AIDS patients with HIV-associated dementia (HAD) compared with non-HAD patients. D386 enhances HIV entry and replication in macrophages but not in microglia or peripheral blood mononuclear cells, possibly due to differential glycosylation in these cell types. A D386N mutation in the UK1br Env, which restores the N-linked glycan site, reduced neutralization sensitivity to the IgG1b12 (b12) monoclonal antibody, which recognizes a conserved neutralization epitope that overlaps the CD4 binding site. Molecular modeling suggested that loss of the glycan at position 386 increases exposure of the CD4 and b12 binding sites on gp120. Loss of a glycan at 386 was more frequent in Envs from HAD patients (26%; n=185) compared with non-HAD patients (7%; n=99; p<0.001). The most significant association of these Env variants with HAD was in blood or lymphoid tissue rather than brain. These findings suggest that increased exposure of the b12 epitope overlapping the CD4 binding site via elimination of a glycan at position 386 is associated with enhanced HIV macrophage tropism, and provide evidence that determinants of macrophage and microglia tropism are overlapping but distinct.

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.

HIV infects macrophages and microglia in the central nervous system (CNS), which express lower levels of CD4 than CD4+ T cells in peripheral blood. To investigate mechanisms of HIV neurotropism, full-length env genes were cloned from autopsy brain and lymphoid tissues from 4 AIDS patients with HIV-associated dementia (HAD). Characterization of 55 functional Env clones demonstrated that Envs with reduced dependence on CD4 for fusion and viral entry are more frequent in brain compared to lymphoid tissue. Envs that mediated efficient entry into macrophages were frequent in brain but were also present in lymphoid tissue. For most Envs, entry into macrophages correlated with overall fusion activity at all levels of CD4 and CCR5. gp160 nucleotide sequences were compartmentalized in brain versus lymphoid tissue within each patient. Proline at position 308 in the V3 loop of gp120 was associated with brain compartmentalization in 3 patients, but mutagenesis studies suggested that P308 alone does not contribute to reduced CD4 dependence or macrophage-tropism. These results suggest that HIV adaptation to replicate in the CNS selects for Envs with reduced CD4 dependence and increased fusion activity. Macrophage-tropic Envs are frequent in brain but are also present in lymphoid tissues of AIDS patients with HAD, and entry into macrophages in the CNS and other tissues is dependent on the ability to use low receptor levels and overall efficiency of fusion.

The CD16+ subset of monocytes is dramatically expanded in peripheral blood during progression to AIDS, but its contribution to HIV pathogenesis is unknown. Here, we demonstrate that CD16+ but not CD16- monocytes promote high levels of HIV replication upon differentiation into macrophages and interaction with T cells. Conjugates formed between CD16+ monocyte-derived macrophages and T cells are major sites of viral replication. Furthermore, similar monocyte-T cell conjugates detected in peripheral blood of HIV-infected patients harbor HIV DNA. Thus, expansion of CD16+ monocytes during HIV infection and their subsequent recruitment into tissues such as lymph nodes, brain, and intestine may contribute to HIV dissemination and establishment of productive infection in T cells.

Plasma virus in human immunodeficiency virus type 1/simian immunodeficiency virus (HIV-1/SIV) infection most likely results from the combination of viruses produced in different tissues. As immunological pressure may be higher in effector sites than secondary lymphoid tissues, we investigated quantitative and qualitative changes in viral RNA in blood and tissues of 10 Mamu-A*01-positive SIV-infected macaques in parallel with the frequency of CD8+ T cells recognizing the dominant Gag181-189 CM9 epitope. The plasma virus level in these macaques directly correlated with the viral RNA levels in lymph nodes, spleen, lungs, colon, and jejunum. In contrast, the frequency of the Gag181-189 CM9 tetramer did not correlate with SIV RNA levels in any compartment. We investigated the presence of viral immune escape in RNA from several tissues. The complete substitution of wild-type genotype with viral immune-escape variant within the Gag181-189 CM9 epitope was associated with low tetramer response in all tissues and blood of two macaques. In one macaque, the replacement of wild type with an immune-escape mutant was asynchronous. While the mutant virus was prevalent in blood and effector tissues (lungs, jejunum, and colon), secondary lymphoid organs such as spleen and lymph nodes still retained 80% and 40%, respectively, of the wild-type virus. These results may imply that there are differences in the immunological pressure exerted by cytotoxic T lymphocytes (CTLs) in tissue compartments of SIVmac251-infected macaques.

Human immunodeficiency virus type 1 (HIV-1) fusion with its target cells is initiated by sequential interactions between its envelope glycoprotein, CD4, and a co-receptor, usually CCR5 or CXCR4. Small molecules that bind to CCR5 and prevent its use by R5 HIV-1 strains are now being developed clinically as antiviral drugs. To test whether a block to CCR5 promotes the replication of viruses that enter cells via CXCR4 and are associated with accelerated disease progression, we administered a small molecule CCR5 inhibitor, CMPD 167, to three macaques dual-infected with both R5 (SIVmac251) and X4 (SHIV-89.6P) viruses. CMPD 167 caused a rapid and substantial (on average, 50-fold) suppression of R5 virus replication in each animal. In two of the animals, but not in the third, a rapid, transient, 8- to 15-fold increase in the amount of plasma X4 virus occurred. In neither animal was the increase in X4 viral load sustained throughout therapy, however. These observations may have relevance for the development of CCR5 inhibitors for treatment of HIV-1 infection of humans., (Copyright 2004 Elsevier Inc.)

Primary strains of human immunodeficiency virus type 1 (HIV-1) are known to adapt to replication in cell lines in vitro by becoming sensitive to soluble CD4 (sCD4) and neutralizing antibodies (NAb). T-cell lines favor isolation of variants that use CXCR4 as a co-receptor, while primary isolates predominantly use CCR5. We have now studied how a primary R5 isolate, CC1/85, adapts to prolonged replication in primary human peripheral blood mononuclear cells (PBMC). After 19 passages, a variant virus, CCcon.19, had increased sensitivity to both sCD4 and NAb b12 that binds to a CD4-binding site (CD4BS)-associated epitope, but decreased sensitivity to anti-CD4 antibodies. CCcon.19 retains the R5 phenotype, its resistance to other NAbs was unaltered, its sensitivity to various entry inhibitors was unchanged, and its ability to replicate in macrophages was modestly increased. We define CCcon.19 as a primary T-cell adapted (PTCA) variant. Genetic sequence analysis combined with mutagenesis studies on clonal, chimeric viruses derived from CC1/85 and the PTCA variant showed that the most important changes were in the V1/V2 loop structure, one of them involving the loss of an N-linked glycosylation site. Monomeric gp120 proteins expressed from CC1/85 and the PTCA variant did not differ in their affinities for sCD4, suggesting that the structural consequences of the sequence changes were manifested at the level of the native, trimeric Env complex. Overall, the adaptation process probably involves selection for variants with higher CD4 affinity and hence greater fusion efficiency, but this also involves the loss of some resistance to neutralization by agents directed at or near to the CD4BS. The loss of neutralization resistance is of no relevance under in vitro conditions, but NAbs would presumably be a counter-selection pressure against such adaptive changes in vivo, at least when the humoral immune response is intact.

Structured treatment interruption (STI) of antiretroviral drugs has been proposed as an alternative approach for managing patients infected with HIV-1. While STI is thought to spare drug-related side effects and enhance the HIV-1-specific immune response, the long-lasting clinical benefit of this approach remains uncertain, particularly in patients with long-standing HIV-1 infection. Here, we investigated the basis of unabated virological replication following different STI regimens in rhesus macaques that expressed the MHC class I Mamu-A*01 molecule treated during acute and long-standing infection with SIVmac251. An amino acid change at the anchor residue within the immunodominant Mamu-A*01-restricted Gag(181-189) CM9 epitope (T --> A) in one of six macaques with acute SIVmac251 infection and in three of four macaques with long-standing SIVmac251 infection (T --> A; T --> S; S --> C) was found in the majority of plasma virus. These amino acid changes have been shown to severely decrease binding of the corresponding peptides to the Mamu-A*01 molecule and, in the case of the T --> A change, escape from CTL. In one macaque with long-standing SIVmac251 infection, a mutation emerged that conferred resistance to one of the antiretroviral drugs (PMPA) as well. These results provide insights into the mechanism underlying the limited capacity of repeated interruption of antiretroviral therapy as an approach to restrain viral replication. In addition, these data also suggest that interruption of therapy may be less effective in chronic infection because of preexisting immune escape and that immune escape is a risk of interruption of therapy.

A polymerase chain reaction-based analysis was used to define the structures of the mRNAs that encode human immunodeficiency virus type-1 (HIV-1) regulatory and structural proteins in infected H9 cells. Twenty alternatively spliced mRNAs encoding the vif, vpr, env, nef, tat, and rev proteins were characterized. An evaluation of the coding potentials of these transcripts recognized both leaky scanning and reinitiation at downstream initiation codons as mechanisms that may operate during translation of many of the polycistronic messages. Two new splice acceptor sites, one at nt 6018 defining a new mRNA coding for the env and vpu proteins and another at nt 8671 defining a novel tat-env fusion transcript, were characterized. The latter transcript expressed a novel protein p17tev that was immunoprecipitated by both polyclonal tat antibodies and monoclonals directed towards the C-terminal region of gp41. The p17tev protein was able to transactivate transcription from the HIV-1 LTR in transient transfection assays. The use of multiple alternative splice donor and acceptor sites and the generation of novel proteins may confer evolutionary advantages on the viral mutants encoding them and influence the course of clinical disease.

Human papillomaviruses (HPVs) infect specific human epithelial tissues. Because viral propagation in cultured cells has not been achieved, studies of HPV genetic activities have been difficult and rely largely on analyses of patient specimens by conventional biochemical methods. HPV type 6 and type 11 infections often result in genital warts (condylomata acuminata). Structural mapping of RNAs from such warts reveals that they use alternative promoters, splice sites, and polyadenylation sites to produce complex families of overlapping mRNAs that span multiple open reading frames. Based on the mRNA structures, we have developed message-specific subgenomic clones of HPV-6 and HPV-11 in pGEM vectors. Tritium-labeled, single-stranded RNA probes were synthesized in vitro and applied to serial thin sections of patient specimens for in situ hybridization. Our results reveal the qualitative and quantitative transcription patterns of different viral messages in relationship to one another, to viral DNA replication, and to cellular differentiation. The viral "E region" is transcribed before the onset of vegetative DNA replication and continues to be expressed in increasing amounts in the maturing epithelium. Even in mature epithelia, E region messengers are far more abundant than "L region" mRNAs. The L region messages encoding capsid proteins are truly late in that they appear concomitant with or after the onset of vegetative viral DNA replication and are only present in the superficial strata of the epithelium, which contain the oldest and most differentiated keratinocytes. Abundant intron material derived from processing E region transcripts accumulates in the nuclei. Strictly nuclear signals from the L region transcripts in the midepithelium suggest that regulation of their expression is at the level of transcription elongation.