HIV-1 Nef protein inhibits the in vitro induction of a specific antibody response to Candida albicans by an early up-regulation of IL-15 production

We have previously demonstrated that exogenous Nef protein induced activation of normal human T cells up-regulating IL-15 production by monocytes. Since HIV-1 infection results in the early impairment of immune functions we decided to evaluate if Nef is able to modulate the induction of a specific antibody response. Human peripheral blood mononuclear cells from healthy donors were induced in vitro to mount a specific antibody response to the Candida albicans antigen. We show that Nef inhibited, in a dose-dependent manner, the induction of the anti-C. albicans antibody response. The ability of an anti-Nef antibody to prevent such inhibition indicates that the effect was indeed Nef-specific. In the Nef-treated cultures an early increase of IL-15 production was observed and the addition of anti-IL-15 antibody abrogated the Nef-induced inhibitory effect. Moreover the addition of IL-15 to the cultures inhibited, as well as Nef, the induction of the specific antibody response. Thus, our results suggest that Nef may inhibit the induction of a specific antibody response by an early up-regulation of IL-15 production. A better comprehension of this phenomenon may be important for unravelling some aspects of the B cell defects in HIV infection.     

Nef Mutations in Long-term Non-progressors from Former Plasma Donors Infected with HIV-1 Subtype B in China

Objective To study the specific amino acid variation in Nef that may be related to disease progression after infection with HIV-1 subtype B, a predominant strain circulating in China, and to determine whether changes in Nef secondary structure may influence different stages of AIDS development based on the concept that the Nef gene of HIV infection dramatically alter the severity of viral infection and virus replication and disease progression, and that long-term non-progressors （LTNP） of HIV infection are commonly associated with either a deletion of the Nef gene or the defective Nef alleles. Methods The study subjects were divided into LTNPI（n=14）, LTNP2 （n=16） and slow progressor （SP, n=19） groups for mutational analysis of the Nef sequence. The data were obtained by using Bioedit, MEGA, Anthewin and SAS software. Results Residues in Nef TA48/49 and K151 occurred more frequently in the LTNP group while AA48/49 was more frequently observed in the SP group. Of the differences observed in the secondary structure comparison using Nef consensus sequences of these three groups, one was roughly corresponding to the Nef48/49 mutation site. Conclusion TA48/49, Kiss, and AA48/49 in the Nef gene might be associated with the different stages of HIV infection, and there may be a link between the Nef secondary structure and the progression of HIV- 1 infection.     

Selective downmodulation of HLA-A and -B by Nef alleles from different groups of primate lentiviruses

It has been demonstrated that the HIV-1 NL4-3 and IIIB Nef alleles downregulate HLA-A and -B but not -C or -E from the cell surface. It remained elusive, however, whether selective modulation of specific HLA molecules is conserved between different groups of human and simian immunodeficiency viruses, respectively. To address this, we analyzed a large panel of primate lentiviral Nef proteins and we found that this property is conserved among nef alleles from the M, N and O groups of HIV-1, as well as those from SIVcpz, the precursor of HIV-1, and a variety of other highly divergent primate lentiviruses. In conclusion, our data indicate that Nef's ability to selectively downregulate HLA-A and -B alleles to prevent CTL lysis and NK killing of virally infected cells is conserved among different primate lentiviral lineages and preceded the zoonotic transmission of SIVcpz from chimpanzees to humans.     

培美曲塞二钠的合成

对碘苯甲酸甲酯与烯丙醇缩合,与硝基甲烷反应得1-硝基-4-(甲氧基羰基苯基)-1-丁烯,继而与2,6-二氨基-4(3H)-嘧啶酮加成,再经Nef反应脱硝基、闭环、水解得4-[2-(2-氨基-4-氧代-4,7-二氢-3H-吡咯并[2,3-d]嘧啶-5-基)乙基]苯甲酸,最后与L-谷氨酸二乙酯缩合、水解、中和得培美曲塞二钠,总收率1.8%.     

Higher cross-subtype IFN-γ ELISpot responses to Gag and Nef peptides in Brazilian HIV-1 subtype B- and F1- than in C-infected subjects

HIV-1 diversity has been considered a huge challenge for the HIV-1 vaccine development. To overcome it, immunogens based on centralized sequences, as consensus, have been tested. In Brazil, the co-circulation of three subtypes offers a suitable scenario to test T cell cross-subtype responses to consensus sequences. Furthermore, we included peptides based on closest viral isolates (CVI) from each subtype analyzed to compare with T cell responses detected against the consensus sequences. The study included 32 subjects infected with HIV-1 subtype B (n = 13),C (n = 11), and F1 (n = 8). Gag and Nef-specific T cell responses were evaluated by IFN-γ-ELISpot assay. Peptides based on CVI sequences were similar to consensus in both reducing genetic distance and detecting T cell responses. A high cross-subtype response between B and F1 in both regions was observed in HIV-1 subtype B and F1-infected subjects. We also found no significant difference in responses to subtype B and C consensus peptides among subtype B-infected subjects. In contrast, the magnitude of T cell responses to consensus C peptides in the Gag region was higher than to consensus B peptides among HIV-1 subtype C-infected subjects. Regarding Nef, subtype C-infected subjects showed higher values to consensus C than to consensus F1 peptides. Moreover, subtype F1-infected subjects presented lower responses to subtype C peptides than to subtype F1 and B. A similar level of responses was detected with group M based peptides in subtype B and F1 infected subjects. However, among subtype C infected subjects, this set of peptides detected lower levels of response than consensus C. Overall, the level of cross-subtype response between subtypes B and F1 was higher than between subtype C and B or C and F1. Our data suggests that the barrier of genetic diversity in HIV-1 group M for vaccine design may be dependent on the subtypes involved.     

分泌性HIV-1 Nef-EGFP融合蛋白表达载体的构建与鉴定

目的： 构建含有人类免疫缺陷病毒I型（human immunodeficiency virus type I,HIV-1）负性调节因子（negative regulation factor,Nef）和增强型绿色荧光蛋白（enhanced green fluorescent protein,EGFP）基因的重组融合蛋白分泌性表达载体,并检测该融合蛋白在真核细胞中的表达及其在培养上清中分泌情况,为进一步研究Nef功能奠定基础。方法： 利用PCR扩增出HIV-1 Nef和EGFP基因,插入到分泌性表达载体pSecTag2B中。构建的pSecTag2B-Nef-EGFP融合蛋白表达质粒,转染293T细胞,荧光显微镜下观测细胞中Nef-EGFP融合蛋白的表达。收集细胞及上清液,蛋白质印迹和ELISA法分别检测Nef-EGFP融合蛋白的表达及其分泌。结果： 限制性内切酶酶切鉴定和核酸序列测定证实,成功构建了含有Nef-EGFP基因的分泌性表达载体,该质粒转染293T细胞后,蛋白质印迹法能够检测到Nef EGFP融合蛋白的表达条带,ELISA法检测上清液中目的蛋白分泌量为1.7 ng/ml。结论： 成功构建含有Nef-EGFP的融合蛋白表达质粒,融合蛋白Nef EGFP在293T细胞中获得表达,并能分泌到胞外。     

Consequences of HLA-associated mutations in HIV-1 subtype C Nef on HLA-I downregulation ability

Identification of CD8+ T lymphocyte (CTL) escape mutations that compromise the pathogenic functions of the Nef protein may be relevant for an HIV-1 attenuation-based vaccine. Previously, HLA-associated mutations 102H, 105R, 108D, and 199Y were individually statistically associated with decreased Nef-mediated HLA-I downregulation ability in a cohort of 298 HIV-1 subtype C infected individuals. In the present study, these mutations were introduced by site-directed mutagenesis into different patient-derived Nef sequence backgrounds of high similarity to the consensus C Nef sequence, and their ability to downregulate HLA-I was measured by flow cytometry in a CEM-derived T cell line. A substantial negative effect of 199Y on HLA-I downregulation and Nef expression was observed, while 102H and 105R displayed negative effects on HLA-I downregulation ability and Nef expression to a lesser extent. The total magnitude of CTL responses in individuals harboring the 199Y mutation was lower than those without the mutation, although this was not statistically significant. Overall, a modest positive relationship between Nef-mediated HLA-I downregulation ability and total magnitude of CTL responses was observed, suggesting that there is a higher requirement for HLA-I downregulation with increased CTL pressure. These results highlight a region of Nef that could be targeted by vaccine-induced CTL to reduce HLA-I downregulation and maximize CTL efficacy.     

Spotlight on HIV-1 Nef: SERINC3 and SERINC5 Identified as Restriction Factors Antagonized by the Pathogenesis Factor

The Nef protein is an accessory gene product encoded by human immunodeficiency virus types 1 and 2 (HIV-1/-2) and simian immunodeficiency virus (SIV) that boosts virus replication in the infected host and accelerates disease progression. Unlike the HIV-1 accessory proteins Vif, Vpr and Vpu, Nef was, until recently, not known to antagonize the antiviral activity of a host cell restriction factor. Two recent reports now describe the host cell proteins serine incorporator 3 and 5 (SERINC3 and SERINC5) as potent inhibitors of HIV-1 particle infectivity and demonstrate that Nef counteracts these effects. These findings establish SERINC3/5 as restrictions to HIV replication in human cells and define a novel activity for the HIV pathogenesis factor Nef.     

C3 Glomerulopathy: Ten Years' Experience at Mayo Clinic

Objective

To describe the clinicopathological features, complement abnormalities, triggers, treatment, and outcomes of C3 glomerulopathy.

HIV-1 Nef expression in microglia disrupts dopaminergic and immune functions with associated mania-like behaviors

BackgroundNeuropsychiatric disorders during HIV/AIDS are common although the contribution of HIV-1 infection within the brain, and in particular individual HIV-1 proteins, to the development of these brain disorders is unknown. Herein, an in vivo transgenic mouse model was generated in which the HIV-1 Nef protein was expressed in microglia cells, permitting investigation of neurobehavioral phenotypes and associated cellular and molecular properties.MethodsTransgenic (Tg) mice that expressed full length HIV-1 nef under the control of the c-fms promoter and wildtype (Wt) littermates were investigated using different measures of neurobehavioral performance including locomotory, forced swim (FST), elevated plus maze (EPM) and T-maze tests. Host gene and transgene expression were assessed by RT-PCR, immunoblotting, enzymatic activity and immunohistochemistry. Biogenic amine levels were measured by HPLC with electrochemical detection.ResultsTg animals exhibited Nef expression in brain microglia and cultured macrophages. Tg males displayed hyperactive behaviors including augmented locomotor activity, decreased immobility in the FST and increased open-arm EPM exploration compared to Wt littermates (p < 0.05). Tg animals showed increased CCL2 expression with concurrent IFN-α suppression in striatum compared with Wt littermates (p < 0.05). Dopamine levels, MAO activity and the dopamine transporter (DAT) expression were reduced in the striatum of Tg animals (p < 0.05).ConclusionsHIV-1 Nef expression in microglia induced CCL2 expression together with disrupting striatal dopaminergic transmission, resulting in hyperactive behaviors which are observed in mania and other psychiatric comorbidities among HIV-infected persons. These findings emphasize the selective effects of individual viral proteins in the brain and their participation in neuropathogenesis.