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Allosteric loss-of-function mutations in HIV-1 Nef from a long-term non-progressor.

ABSTRACT: Activation of Src family kinases by human immunodeficiency virus type 1 (HIV-1) Nef may play an important role in the pathogenesis of HIV/AIDS. Here we investigated whether diverse Nef sequences universally activate Hck, a Src family member expressed in macrophages and other HIV-1 target cells. In general, we observed that Hck activation is a highly conserved Nef function. However, we identified an unusual Nef variant from an HIV-positive individual that did not develop AIDS which failed to activate Hck despite the presence of conserved residues linked to Hck SH3 domain binding and kinase activation. Amino acid sequence alignment with active Nef proteins revealed differences in regions not previously implicated in Hck activation, including a large internal flexible loop absent from available Nef structures. Substitution of these residues in active Nef compromised Hck activation without affecting SH3 domain binding. These findings show that residues at a distance from the SH3 domain binding site influence Nef interactions allosterically with a key effector protein linked to AIDS progression.

SUBMITTER: Trible RP

PROVIDER: S-EPMC2117379 | biostudies-literature | 2007 Nov

REPOSITORIES: biostudies-literature

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Allosteric loss-of-function mutations in HIV-1 Nef from a long-term non-progressor.

Trible Ronald P RP Emert-Sedlak Lori L Wales Thomas E TE Ayyavoo Velpandi V Engen John R JR Smithgall Thomas E TE

Journal of molecular biology 20070911 1

Activation of Src family kinases by human immunodeficiency virus type 1 (HIV-1) Nef may play an important role in the pathogenesis of HIV/AIDS. Here we investigated whether diverse Nef sequences universally activate Hck, a Src family member expressed in macrophages and other HIV-1 target cells. In general, we observed that Hck activation is a highly conserved Nef function. However, we identified an unusual Nef variant from an HIV-positive individual that did not develop AIDS which failed to acti ...[more]

PMID: 17920628

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Project description:Certain host genetic variants, especially in the human leucocyte antigen (HLA) region, are associated with different progression of HIV-1-induced diseases and AIDS. Long term non progressors (LTNP) represent only the 2% of infected patients but are especially relevant because of their efficient HIV control. In this work we present a global analysis of genetic data in the large national multicenter cohort of Spanish LTNP, which is compared with seronegative individuals and HIV-positive patients. We have analyzed whether several single-nucleotide polymorphisms (SNPs) including in key genes and certain HLA-A and B alleles could be associated with a specific HIV phenotype. A total of 846 individuals, 398 HIV-1-positive patients (213 typical progressors, 55 AIDS patients, and 130 LTNPs) and 448 HIV-negative controls, were genotyped for 15 polymorphisms and HLA-A and B alleles. Significant differences in the allele frequencies among the studied populations identified 16 LTNP-associated genetic factors, 5 of which were defined for the first time as related to LTNP phenotype: the protective effect of HLA-B39, and the detrimental impact of HLA-B18, -A24, -B08 and -A29. The remaining eleven polymorphisms confirmed previous publications, including the protective alleles HLA-B57, rs2395029 (HCP5), HLA bw4 homozygosity, HLA-B52, HLA-B27, CCR2 V64I, rs9264942 (HLA-C) and HLA-A03; and the risk allele HLA bw6 homozygosity. Notably, individual Spanish HIV-negative individuals had an average of 0.12 protective HLA alleles and SNPs, compared with an average of 1.43 protective alleles per LTNP patient, strongly suggesting positive selection of LTNP. Finally, stratification of LTNP according to viral load showed a proportional relationship between the frequency of protective alleles with control of viral load. Interestingly, no differences in the frequency of protection/risk polymorphisms were found between elite controllers and LTNPs maintaining viral loads <2.000 copies/mL throughout the follow-up.

Project description:HIV-1 accessory proteins Nef and Vpu enhance viral pathogenesis through partially overlapping immune evasion activities. Attenuated Nef or Vpu functions have been reported in individuals who display slower disease progression, but few studies have assessed the relative impact of these proteins in non-B HIV-1 subtypes or examined paired proteins from the same individuals. Here, we examined the sequence and function of matched Nef and Vpu clones isolated from 29 long-term survivors (LTS) from Rwanda living with HIV-1 subtype A and compared our results to those of 104 Nef and 62 Vpu clones isolated from individuals living with chronic untreated HIV-1 subtype A from the same geographic area. Nef and vpu coding regions were amplified from plasma HIV RNA and cloned. The function of one intact, phylogenetically-validated Nef and Vpu clone per individual was then quantified by flow cytometry following transient expression in an immortalized CD4+ T-cell line. We measured the ability of each Nef clone to downregulate CD4 and HLA class I, and of each Vpu clone to downregulate CD4 and Tetherin, from the cell surface. Results were normalized to reference clones (Nef-SF2 and Vpu-NL4.3). We observed that Nef-mediated CD4 and HLA downregulation functions were lower in LTS compared to the control cohort (Mann-Whitney p=0.03 and p<0.0001, respectively). Moreover, we found a positive correlation between Nef-mediated CD4 downregulation function and plasma viral load in LTS and controls (Spearman ρ= 0.59, p=0.03 and ρ=0.30, p=0.005, respectively). In contrast, Vpu-mediated functions were similar between groups and did not correlate with clinical markers. Further analyses identified polymorphisms at Nef codon 184 and Vpu codons 60-62 that were associated with function, which were confirmed through mutagenesis. Overall, our results support attenuated function of Nef, but not Vpu, as a contributor to slower disease progression in this cohort of long-term survivors with HIV-1 subtype A.

Project description:BackgroundLoss-of-function mutations in the thyrotropin receptor (TSHR) gene lead to resistance to TSH (RTSH) presenting with either congenital hypothyroidism (CH) or subclinical hypothyroidism (SCH). Despite several reports of patients with TSHR mutations, data on the long-term outcome of this condition are limited, and no consensus exists on the need for hormone replacement therapy. The aim of the present study was to assess the long-term outcome in children and adolescents with RTSH due to TSHR mutations.MethodsThe TSHR gene was sequenced in 94 subjects (aged 3 days-21 years) with either nonautoimmune SCH or CH with RTSH.ResultsTwenty-seven subjects (29%) carried mutations in TSHR. Fifteen infants were identified by neonatal screening, and the other 79 patients were detected in the process of testing for various other conditions or because of family occurrence of thyroid test abnormalities. Six different mutations were identified: c.484C>G (p.P162A), c.202C>T (p.P68S), c.790C>T (p.P264S), c.269A>C (p.Q90P), c.1957C>G (p.L653V), and c.1347C>T (p.R450C). Twelve subjects were homozygous, three were compound heterozygous, and 12 were heterozygous. Mean serum TSH levels at diagnosis and at last visit were significantly higher in patients with TSHR mutations than in those without mutations (29.04 vs. 14.15, p=0.002; 31.73 vs. 6.19, p<0.0001, respectively). Homozygous patients had a more severe phenotype (TSH 53.6 vs. 9.24, p<0.0001). Mean serum free thyroxine (fT4) levels at the last visit were significantly lower than at the first visit in the homozygous individuals (p=0.05) for a follow-up period of as long as 11 years. Heterozygous subjects had only mild hyperthyrotropinemia with stable TSH levels. However, homozygous subjects showed a trend toward increased TSH and decreased fT4 with time.ConclusionSCH in heterozygotes with TSHR mutations is a stable compensated condition with an appropriately adjusted set point for pituitary-thyroid feedback that does not require replacement therapy. However, homozygous subjects, with incompletely compensated SCH, show reduced fT4 levels over time and may require levothyroxine treatment. Replacement therapy should be considered on an individual basis, and long-term follow up is recommended.

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Allosteric loss-of-function mutations in HIV-1 Nef from a long-term non-progressor.

Publications

Allosteric loss-of-function mutations in HIV-1 Nef from a long-term non-progressor.

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