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. 2012 Jun;28(6):607-18.
doi: 10.1089/AID.2011.0021. Epub 2012 Jan 17.

Human Immunodeficiency Virus nef signature sequences are associated with pulmonary hypertension

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Human Immunodeficiency Virus nef signature sequences are associated with pulmonary hypertension

Sharilyn Almodovar et al. AIDS Res Hum Retroviruses. 2012 Jun.

Abstract

Severe pulmonary hypertension (PH) associated with vascular remodeling is a long-term complication of HIV infection (HIV-PH) affecting 1/200 infected individuals vs. 1/200,000 frequency in the uninfected population. Factors accounting for increased PH susceptibility in HIV-infected individuals are unknown. Rhesus macaques infected with chimeric SHIVnef virions but not with SIV display PH-like pulmonary vascular remodeling suggesting that HIV-Nef is associated with PH; these monkeys showed changes in nef sequences that correlated with pathogenesis after passage in vivo. We further examined whether HIV-nef alleles in HIV-PH subjects have signature sequences associated with the disease phenotype. We evaluated specimens from participants with and without HIV-PH from European Registries and validated results with samples collected as part of the Lung-HIV Studies in San Francisco. We found that 10 polymorphisms in nef were overrepresented in blood cells or lung tissue specimens from European HIV-PH individuals but significantly less frequent in HIV-infected individuals without PH. These polymorphisms mapped to known functional domains in Nef. In the validation cohort, 7/10 polymorphisms in the HIV-nef gene were confirmed; these polymorphisms arose independently from viral load, CD4(+) T cell counts, length of infection, and antiretroviral therapy status. Two out of 10 polymorphisms were previously reported in macaques with PH-like pulmonary vascular remodeling. Cloned recombinant Nef proteins from clinical samples down-regulated CD4, suggesting that these primary isolates are functional. This study offers new insights into the association between Nef polymorphisms in functional domains and the HIV-PH phenotype. The utility of these polymorphisms as predictors of PH should be examined in a larger population.

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Figures

FIG. 1.
FIG. 1.
HIV Nef polymorphisms overrepresented in subjects with HIV-associated PAH. Individual variations (using one-letter amino acid codes, numbered, colored, and indicated by arrows) are indicated in cartoons of Nef crystal structures. The Nef structure data are available as separate models. We used the models of (A) myristoylated Nef anchor domain, which includes Nef amino acids 2–57, and (B) Nef core to the C-terminus (amino acids 56–206). Alpha-helixes are colored in pink, beta-sheets are in orange. The N- and C-termini are indicated in each model. Nonconservative polymorphisms are indicated in yellow. Of the 10 polymorphisms identified in European subjects with HIV-associated PAH, seven were also found in the cohort from San Francisco (underlined). Refer to Supplementary Table S1 for frequency data. Symbol: ▾ indicates hydrophilic amino acid.
FIG. 2.
FIG. 2.
Variations in functional domains in HIV-PH vs. HIV-infected subjects with no PH. The number of variations in HIV-1 Nef functional domains was determined for each subject and is shown as a sum of variations in Nef domains. Each symbol represents a subject. Note that the HIV-PH group is characterized by two or more variations in Nef functional domains (odds ratio 10.29, 95% confidence interval, 2.76 to 38.38; p=0.0004).
FIG. 3.
FIG. 3.
Correlation between the number of polymorphisms in HIV Nef functional domains and length of HIV infection and age in the study group. The length of HIV infection and age (both in years) were plotted for each study subject (n=51). Linear regression analyses showed that there is no correlation between the number of polymorphisms in HIV-Nef functional domains and length of HIV infection (R2=0.142 (A) or age (R2=0.0089 (B).
FIG. 4.
FIG. 4.
HIV Nef sequence variations in blood compartments. HIV nef was genotyped from the peripheral blood mononuclear cells (PBMCs) and plasma from two HIV-infected individuals with PH and two normotensives of the San Francisco group. PBMCs and plasma samples analyzed were collected on the same day in all subjects except for Subject #4016, from whom the plasma sample was collected a month after the PBMCs. Sequences were aligned and translated; the frequency of polymorphisms in Nef functional domains was calculated for each case and plotted.
FIG. 5.
FIG. 5.
Homology modeling of HIV Nef isolates from HIV-PH subjects. Models of Nef isolates from the HIV-PH subjects were generated by the server PHYRE. (A) Model of the N-terminus/anchor domain of the Nef consensus B Sequence (Los Alamos Databases) (cyan) based on model 1 of the 1QA5 NMR structure. Nef point mutants R19K, R21Q, H40Y, and A53P are indicated in magenta. (B) Model of the consensus B sequence (cyan) with Nef point polymorphisms L58V, E63G, M79I, T80N, and Y81F (magenta) superimposed. P150X was not modeled by PHYRE. (C) Model of Nef M79I (magenta) superimposed on the structure of Nef with the FYN SH3 domain (cyan/1EFN). In the wild-type structure, the methionine side chain fits within the hydrophobic pocket formed between helixes α3 and α4. The larger side chain of isoleucine is flipped 180° to the methionine because it does not fit within the hydrophobic pocket. Even removed from the pocket HD13 makes significant van der Waals (VdW) clashes (–0.414 Å determined by Mol Probity) with HB3 of W124 (VdW radius indicated by spheres). The isoleucine is unfavorable for the observed positioning of the putative PKC site when Nef is bound to an SH3 domain-containing protein (Fyn). (D) Nef model of T80N and Y81F superimposed on the Consensus B 1EFN model (cyan) with possible T80 to G83 and Y81 to A190 hydrogen bonds shown (yellow dashed line). These hydrogen bonds potentially stabilize the positioning of the PKC site as well as the proline-rich helix for interaction with an SH3 domain. The T80N mutant not only is incapable of forming a hydrogen bonding to G83 within α3 but also could form a new hydrogen bond with the carbonyl of P78, which would increase the flexibility of the PKC site from α3.
FIG. 6.
FIG. 6.
Functional assay and immunoanalyses of Nef-HaloTag fusion constructs derived from HIV-PH individuals. Hela-CD4 cells were nucleofected with the indicated fusion constructs. Polymorphism information of each molecular construct (as per recovered from the subjects) is shown using one-letter amino acid codes; ▾ indicates hydrophilic amino acid. (A) The HaloTag portion of the construct was labeled with Oregon Green HaloTag ligand and CD4 receptor was labeled with an anti-CD4 antibody conjugated to allophycocyanin. Samples were acquired by flow cytometry; CD4 expression was assessed in mock-nucleofected and in cells nucleofected with truncated-Nef by gating in the whole cell population, while in the Nef-HaloTag-nucleofected cells, it was measured by gating in Nef-HaloTag positive cells. Baseline CD4 expression in percentage was determined using the mock-nucleofected cells. The histograms show the CD4 expression in cells nucleofected with each individual construct (tinted) compared to mock-nucleofected cells (open); percentages are shown. (B) Lysates from nucleofected cells were immunoblotted with anti-HaloTag polyclonal antibody and subsequently stripped and probed with antiactin monoclonal antibodies under denaturing/non-reducing conditions (left bottom panel). The panel at the lower right shows an immunoblot of the lysates with the anti-HaloTag polyclonal antibody under fully native conditions. Note the presence of multiple oligomeric forms in some Nef-HaloTag constructs.

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References

    1. Tuder RM. Groves B. Badesch DB. Voelkel NF. Exuberant endothelial cell growth and elements of inflammation are present in plexiform lesions of pulmonary hypertension. Am J Pathol. 1994;144:275–285. - PMC - PubMed
    1. Speich R. Jenni R. Opravil M. Pfab M. Russi EW. Primary pulmonary hypertension in HIV infection. Chest. 1991;100(5):1268–1271. - PubMed
    1. Petrosillo N. Chinello P. Cicalini S. Pulmonary hypertension in individuals with HIV infection. AIDS. 2006;20(16):2128–2129. - PubMed
    1. Sitbon O. Lascoux-Combe C. Delfraissy JF, et al. Prevalence of HIV-related pulmonary arterial hypertension in the current antiretroviral therapy era. Am J Respir Criti Care Med. 2008;177:108–113. - PubMed
    1. Ntsekhe M. Hakim J. Impact of human immunodeficiency virus infection on cardiovascular disease in Africa. Circulation. 2005;112(23):3602–3607. - PubMed

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