Enhanced macrophage tropism of HIV in brain and lymphoid tissues is associated with sensitivity to the broadly neutralizing CD4 binding site antibody b12

Macrophages in the central nervous system (CNS) and other tissues are an important cellular reservoir for human immunodeficiency virus type 1 (HIV) infection, particularly in the later stages of disease. Macrophage-tropic HIV strains have an enhanced capacity to enter cells expressing low levels of CD4 through mechanisms that are not well understood. Here, we use a panel of primary HIV envelopes from brain and lymphoid tissues to examine the relationship between neutralization sensitivity to reagents targeting the CD4 binding site and virus entry into macrophages. Neutralization assays using pseudotyped viruses showed an association between the capacity of HIV to enter macrophages and increased sensitivity to the broadly neutralizing monoclonal antibody (mAb) b12, which recognizes a conserved epitope overlapping the CD4 binding site, but not sensitivity to soluble CD4 (sCD4) or b6, a non-neutralizing CD4 binding site mAb. Furthermore, loss of an N-linked glycosylation site at position 386 in the V4 region of Env enhanced macrophage tropism together with b12 sensitivity, but not neutralization by sCD4, b6, or a broadly neutralizing AIDS patient serum. These findings suggest that exposure of the b12 epitope, rather than exposure of the CD4 binding site per se, enhances HIV macrophage tropism, possibly by exposing a region on the outer domain of gp120 that is initially recognized by CD4. These findings suggest overlap between specific gp120 determinants in or near the b12 epitope and those conferring macrophage tropism

Asn 362 in gp120 contributes to enhanced fusogenicity by CCR5-restricted HIV-1 envelope glycoprotein variants from patients with AIDS

<p>Abstract</p> <p>Background</p> <p>CCR5-restricted (R5) human immunodeficiency virus type 1 (HIV-1) variants cause CD4+ T-cell loss in the majority of individuals who progress to AIDS, but mechanisms underlying the pathogenicity of R5 strains are poorly understood. To better understand envelope glycoprotein (Env) determinants contributing to pathogenicity of R5 viruses, we characterized 37 full-length R5 Envs from cross-sectional and longitudinal R5 viruses isolated from blood of patients with asymptomatic infection or AIDS, referred to as pre-AIDS (PA) and AIDS (A) R5 Envs, respectively.</p> <p>Results</p> <p>Compared to PA-R5 Envs, A-R5 Envs had enhanced fusogenicity in quantitative cell-cell fusion assays, and reduced sensitivity to inhibition by the fusion inhibitor T-20. Sequence analysis identified the presence of Asn 362 (N362), a potential N-linked glycosylation site immediately N-terminal to CD4-binding site (CD4bs) residues in the C3 region of gp120, more frequently in A-R5 Envs than PA-R5 Envs. N362 was associated with enhanced fusogenicity, faster entry kinetics, and increased sensitivity of Env-pseudotyped reporter viruses to neutralization by the CD4bs-directed Env mAb IgG1b12. Mutagenesis studies showed N362 contributes to enhanced fusogenicity of most A-R5 Envs. Molecular models indicate N362 is located adjacent to the CD4 binding loop of gp120, and suggest N362 may enhance fusogenicity by promoting greater exposure of the CD4bs and/or stabilizing the CD4-bound Env structure.</p> <p>Conclusion</p> <p>Enhanced fusogenicity is a phenotype of the A-R5 Envs studied, which was associated with the presence of N362, enhanced HIV-1 entry kinetics and increased CD4bs exposure in gp120. N362 contributes to fusogenicity of R5 Envs in a strain dependent manner. Our studies suggest enhanced fusogenicity of A-R5 Envs may contribute to CD4+ T-cell loss in subjects who progress to AIDS whilst harbouring R5 HIV-1 variants. N362 may contribute to this effect in some individuals.</p

Role of the B Allele of Influenza A Virus Segment 8 in Setting Mammalian Host Range and Pathogenicity.

UNLABELLED: Two alleles of segment 8 (NS) circulate in nonchiropteran influenza A viruses. The A allele is found in avian and mammalian viruses, but the B allele is viewed as being almost exclusively found in avian viruses. This might reflect the fact that one or both of its encoded proteins (NS1 and NEP) are maladapted for replication in mammalian hosts. To test this, a number of clade A and B avian virus-derived NS segments were introduced into human H1N1 and H3N2 viruses. In no case was the peak virus titer substantially reduced following infection of various mammalian cell types. Exemplar reassortant viruses also replicated to similar titers in mice, although mice infected with viruses with the avian virus-derived segment 8s had reduced weight loss compared to that achieved in mice infected with the A/Puerto Rico/8/1934 (H1N1) parent. In vitro, the viruses coped similarly with type I interferons. Temporal proteomics analysis of cellular responses to infection showed that the avian virus-derived NS segments provoked lower levels of expression of interferon-stimulated genes in cells than wild type-derived NS segments. Thus, neither the A nor the B allele of avian virus-derived NS segments necessarily attenuates virus replication in a mammalian host, although the alleles can attenuate disease. Phylogenetic analyses identified 32 independent incursions of an avian virus-derived A allele into mammals, whereas 6 introductions of a B allele were identified. However, A-allele isolates from birds outnumbered B-allele isolates, and the relative rates of Aves-to-Mammalia transmission were not significantly different. We conclude that while the introduction of an avian virus segment 8 into mammals is a relatively rare event, the dogma of the B allele being especially restricted is misleading, with implications in the assessment of the pandemic potential of avian influenza viruses. IMPORTANCE: Influenza A virus (IAV) can adapt to poultry and mammalian species, inflicting a great socioeconomic burden on farming and health care sectors. Host adaptation likely involves multiple viral factors. Here, we investigated the role of IAV segment 8. Segment 8 has evolved into two distinct clades: the A and B alleles. The B-allele genes have previously been suggested to be restricted to avian virus species. We introduced a selection of avian virus A- and B-allele segment 8s into human H1N1 and H3N2 virus backgrounds and found that these reassortant viruses were fully competent in mammalian host systems. We also analyzed the currently available public data on the segment 8 gene distribution and found surprisingly little evidence for specific avian host restriction of the B-clade segment. We conclude that B-allele segment 8 genes are, in fact, capable of supporting infection in mammals and that they should be considered during the assessment of the pandemic risk of zoonotic influenza A viruses.Wellcome Trust (Grant ID: 108070/Z/15/Z), Medical Research Council (Grant ID: MR/K000276/1), Biotechnology and Biological Sciences Research Council (Grant IDs: BB/J004324/1, BB/J01446X/1), Division of Intramural Research National Institute of Allergy and Infectious Diseases, University Of Edinburgh (Chancellor’s Fellowship)This is the final version of the article. It first appeared from the American Society for Microbiology via http://dx.doi.org/10.1128/JVI.01205-1

Macrophage entry mediated by HIV Envs from brain and lymphoid tissues is determined by the capacity to use low CD4 levels and overall efficiency of fusion

AbstractHIV 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

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

AbstractHeterozygosity for the CCR5 Δ32 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 Δ32 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

Enhanced macrophage tropism of HIV in brain and lymphoid tissues is associated with sensitivity to the broadly neutralizing CD4 binding site antibody b12

Abstract Macrophages in the central nervous system (CNS) and other tissues are an important cellular reservoir for human immunodeficiency virus type 1 (HIV) infection, particularly in the later stages of disease. Macrophage-tropic HIV strains have an enhanced capacity to enter cells expressing low levels of CD4 through mechanisms that are not well understood. Here, we use a panel of primary HIV envelopes from brain and lymphoid tissues to examine the relationship between neutralization sensitivity to reagents targeting the CD4 binding site and virus entry into macrophages. Neutralization assays using pseudotyped viruses showed an association between the capacity of HIV to enter macrophages and increased sensitivity to the broadly neutralizing monoclonal antibody (mAb) b12, which recognizes a conserved epitope overlapping the CD4 binding site, but not sensitivity to soluble CD4 (sCD4) or b6, a non-neutralizing CD4 binding site mAb. Furthermore, loss of an N-linked glycosylation site at position 386 in the V4 region of Env enhanced macrophage tropism together with b12 sensitivity, but not neutralization by sCD4, b6, or a broadly neutralizing AIDS patient serum. These findings suggest that exposure of the b12 epitope, rather than exposure of the CD4 binding site per se, enhances HIV macrophage tropism, possibly by exposing a region on the outer domain of gp120 that is initially recognized by CD4. These findings suggest overlap between specific gp120 determinants in or near the b12 epitope and those conferring macrophage tropism.</p

Asn 362 in gp120 contributes to enhanced fusogenicity by CCR5-restricted HIV-1 envelope glycoprotein variants from patients with AIDS-4

<p><b>Copyright information:</b></p><p>Taken from "Asn 362 in gp120 contributes to enhanced fusogenicity by CCR5-restricted HIV-1 envelope glycoprotein variants from patients with AIDS"</p><p>http://www.retrovirology.com/content/4/1/89</p><p>Retrovirology 2007;4():89-89.</p><p>Published online 12 Dec 2007</p><p>PMCID:PMC2225424.</p><p></p>ared to those from the highly fusogenic YU2 and ADA R5 Envs, the poorly fusogenic JR-CSF R5 Env, and the clade B consensus sequence. Dots indicate residues identical to the clade B consensus sequence, and dashes indicate gaps. Residues forming the CD4bs and the amino acid present at position 362 (numbered relative to the HXB2 reference sequence) are highlighted

Asn 362 in gp120 contributes to enhanced fusogenicity by CCR5-restricted HIV-1 envelope glycoprotein variants from patients with AIDS-0

<p><b>Copyright information:</b></p><p>Taken from "Asn 362 in gp120 contributes to enhanced fusogenicity by CCR5-restricted HIV-1 envelope glycoprotein variants from patients with AIDS"</p><p>http://www.retrovirology.com/content/4/1/89</p><p>Retrovirology 2007;4():89-89.</p><p>Published online 12 Dec 2007</p><p>PMCID:PMC2225424.</p><p></p>nal Envs cloned from the cross sectional (PA-R5 viruses NB23, NB24, NB25, NB27 and A-R5 viruses NB2, NB6, NB7, NB8) (B) or longitudinal (PA- and A-R5 viruses from subject IK1) (C) primary R5 HIV-1 isolates and 2 μg pSVL-Tat, as described in the Methods. Env expression at 72 h post-transfection was measured by Western blot analysis of cell lysates using rabbit anti-gp120 polyclonal antisera. Positions of gp160 and gp120 are shown on the right. C1, C2, C3 and C4 refer to independent Envs cloned from each virus