Nef Gene Research Articles

Characterization of a Novel HIV-1 Second-Generation Recombinant Form in Men Who Have Sex with Men in Beijing, China.

Cocirculation of multiple subtypes in the same population contributes significantly to the emergence of recombinant viruses. BJ2015EU19, a novel CCR5-tropic human immunodeficiency virus (HIV)-1, second-generation recombinant virus, was isolated from a man who has sex with men in Beijing, China. Phylogenetic analysis of the near full-length genome showed that BJ2015EU19 consisted of seven fragments from CRF01_AE and CRF07_BC. There were six recombinant breakpoints in the pol, vpu, env, and nef genes of BJ2015EU19, which were different from the other circulating recombinant forms and unique recombinant forms (URFs). The new emergence of the URF increases the complexity of the HIV epidemic in the Chinese homosexual group, and the ongoing generation of recombinant viruses may provide an important basis for future analysis of the recombination mechanism of HIV-1.

Near Full-Length Genome Sequence of a Novel HIV-1 Second-Generation Recombinant Form (CRF01_AE/CRF07_BC) Detected Among Blood Donors in North China.

In this study, a novel CRF01_AE/07_BC HIV-1 recombinant form, with five breakpoints in the pol, vpr, env, and nef gene regions, was identified from a voluntary blood donor in North China. Our study showed that this HIV-1 recombinant form might be a second-generation recombinant form from two predominant strains in the men who have sex with men (MSM) population, with the CRF07_BC fragments belonging to a CRF07_BC lineage specific to Chinese MSM and the CRF01_AE fragments belonging to the CRF01_AE lineage 5 common among MSM in northern China. Our findings supported the high-risk blood donation behaviors of MSM, implied the increasing complexity of the HIV-1 epidemic among the Chinese MSM population, and called more attention to the effectiveness of the screening test for the growing number of new HIV-1 recombinant forms.

The Efficiency of Tat Cell Penetrating Peptide for Intracellular Uptake of HIV-1 Nef Expressed in E. coli and Mammalian Cell.

Cell penetrating peptides (CPPs) or protein transduction domains (PTDs) have been known as a new field in cargo delivery. These peptides such as Tat, Pep-1 and Cady-2 are able to deliver genes and biologically active proteins to cytoplasmic compartments via the plasma membrane. In current study, the efficiency of pEGFP-N1 eukaryotic vector for expression of HIV-1 Tat- Nef fusion was evaluated in HEK-293T cells using TurboFect transfection reagent. In addition, the recombinant GST-Tat-Nef protein was generated in E. coli and transfected using two amphipathic CPPs (Pep-1 and Cady-2) into mammalian cells. The size and morphology of the CPP/GST-Tat-Nef complexes were evaluated by scanning electron microscopy (SEM), Zetasizer, and SDS-PAGE. The transfection of HIV-1 GST-Tat-Nef protein was also analyzed using SDS-PAGE and western blotting. Our data indicated that the recombinant GST-Tat-Nef protein generated in BL21 strain migrated as a clear band of ~ 52 kDa in SDS-PAGE. The results of SEM and Zetasizer confirmed the formation of protein/ Pep-1 or protein/ Cady-2 nanoparticles less than 200 nm in diameter. Tat CPP fused to Nef protein could deliver the recombinant Nef protein alone and notably by forming the noncovalent complexes with TurboFect, Pep-1 and Cady-2 as detected in western blotting. Moreover, intracellular uptake of Tat-Nef gene and subsequently its expression in mammalian cells was considerably higher than that for Nef gene. This data indicated that the Tat gene sequence could also increase the transfection of Nef gene in vitro. Generally, the Tat-Nef interaction led to enhance further gene expression and also protein delivery.

Comparative genetic variability in HIV-1 subtype C nef gene in early age groups of infants.

Targeting properties of vertically transmitted viruses in early infancy is important to understand disease progression. To investigate genotypic characteristics of transmitted viruses, blood samples were obtained from infants aged 6 weeks-18 months, categorized in two age groups, acute (<6 months) and early (>6-18 months). Nef having an important role in pathogenesis was selected to explore the viral characteristics. A total of 57 PCR positive samples, amplified by nef gene were sequenced. Analysis showed that 50 sequences belonged to subtype C. In one sequence of acute age group, a long insertion of 10 residues (AAERMRRAEP) in variable region and a 13 residues deletion (ATNNADCAWLEAQ) around proteolytic cleavage region of gene in another sequence was observed. Insertions were also observed in sequences of early age group, however, they ranged from two to eight residues only. In one sequence of early age group, 3/4 arginines at positions 19, 21, 22 of arginine cluster were mutated to glutamine, alanine, and glutamine, respectively. Entropy analysis of two age groups revealed presence of several residues with statistically significant differences in their variability. Among these, 15 (R18,R23,R24; A66,L68,Q71; E74,E77,E78; V87,M92; R119, P144, E167, and C176) belonged to functional motifs, out of which, 12 were in acute age group, suggesting that variability was greater in this group. Prediction of HLA binding peptide motif revealed that epitope LTFGWCFKL was present in >80% study sequences. This epitope was also present in maximum number of HLA types circulating in India and vaccine candidate sequences, suggesting that it may be helpful in designing an epitope-based vaccine.

Unusual Fusion Proteins of HIV-1.

Despite its small genome size, the Human Immunodeficiency Virus 1 (HIV-1) is one of the most successful pathogens and has infected more than 70 million people worldwide within the last decades. In total, HIV-1 expresses 16 canonical proteins from only nine genes within its 10 kb genome. Expression of the structural genes gag, pol, and env, the regulatory genes rev and tat and the accessory genes vpu, nef, vpr, and vif enables assembly of the viral particle, regulates viral gene transcription, and equips the virus to evade or counteract host immune responses. In addition to the canonically expressed proteins, a growing number of publications describe the existence of non-canonical fusion proteins in HIV-1 infected cells. Most of them are encoded by the tat-env-rev locus. While the majority of these fusion proteins (e.g., TNV/p28tev, p186Drev, Tat1-Rev2, Tat^8c, p17tev, or Ref) are the result of alternative splicing events, Tat-T/Vpt is produced upon programmed ribosomal frameshifting, and a Rev1-Vpu fusion protein is expressed due to a nucleotide polymorphism that is unique to certain HIV-1 clade A and C strains. A better understanding of the expression and activity of these non-canonical viral proteins will help to dissect their potential role in viral replication and reveal how HIV-1 optimized the coding potential of its genes. The goal of this review is to provide an overview of previously described HIV-1 fusion proteins and to summarize our current knowledge of their expression patterns and putative functions.

Single Genome Sequencing of Expressed and Proviral HIV-1 Envelope Glycoprotein 120 (gp120) and nef Genes.

The current study provides detailed protocols utilized to amplify the complete HIV-1 gp120 and nef genes from single copies of expressed or integrated HIV present in fresh-frozen autopsy tissues of patients who died while on combined antiretroviral therapy (cART) with no detectable plasma viral load (pVL) at death ( Lamers et al., 2016a and 2016b; Rose et al., 2016 ). This method optimizes protocols from previous publications ( Palmer et al., 2005 ; Norström et al., 2012 ; Lamers et al., 2015 ; 2016a and 2016b; Rife et al., 2016 ) to produce single distinct PCR products that can be directly sequenced and includes several cost-saving and time-efficient modifications.

Delivery of HIV-1 Nef linked to heat shock protein 27 using a cationic polymer is more effective than cationic lipid in mammalian cells.

Different adjuvants and delivery systems have been used to enhance the potency of DNA vaccines against viral diseases. Among them, heat shock proteins (HSPs) are stress proteins that have multiple roles such as chaperon activity and anti-apoptotic and adjuvant properties. The goal of this study was to compare the expression of HIV-1 Nef, Hsp27 and Hsp27-Nef genes transfected in HEK-293T mammalian cells by TurboFect and Lipofectamine as a cationic polymer and lipid, respectively. At first, the pEGFP eukaryotic vectors encoding HIV-1 Nef, Hsp27 and Hsp27-Nef genes were generated and transfected in HEK-293T using TurboFect and Lipofectamine delivery systems. Then, the expression of proteins was evaluated and compared using fluorescent microscopy, flow cytometry and western blotting 48 hr after transfection. The accuracy of the DNA constructs was confirmed on agarose gel electrophoresis to be ~ 720 bp, ~ 648 bp, and ~ 1368 bp bands for Hsp27, Nef, and Hsp-Nef, respectively. The expression analysis in the transfected cells showed that the delivery of genes using TurboFect was significantly higher than that using Lipofectamine. Furthermore, transfection of Hsp27 gene was more effective than that of Nef gene using both delivery systems. Hsp27 linked to Nef could also increase its delivery and expression in HEK-293T cells. Generally, Hsp27 can be used as a suitable carrier in DNA vaccine design against HIV-1 infections (Fig. 5, Ref. 28).

Improving the potency of DNA vaccine encoding HIV-1 Nef antigen using two endogenous adjuvants in mouse model.

DNA immunization can induce long-term immune responses, which are required to design an effective HIV vaccine. It was shown that antigen-expressing plasmids can increase the protective immunity against infectious diseases such as: influenza and malaria. However, DNA-based immunizations have poor immunogenicity, thus the use of potent immunoadjuvants can enhance their potency. In the current study, preparation of the recombinant HIV-1 Nef, Gp96 and HMGB1 DNA constructs was performed in bacterial system. Then, the immunogenicity of DNA construct harboring HIV-1 Nef gene (pcDNA-Nef) was studied using two endogenous adjuvants (pcDNA-HMGB1 and pcDNA-Gp96) in BALB/c mouse model. Our data showed that co-injection of pcDNA-Nef with pcDNA-HMGB1 effectively raised both humoral and cell-mediated immune responses in mice as compared to pcDNA-Nef adjuvanted with pcDNA-gp96. Indeed, co-immunization of HIV-1 Nef DNA with HMGB1 DNA significantly induced high levels of IgG2a and IFN-γ directed toward Th1 responses and also cytotoxic T lymphocytes (CTLs) activity in comparison with other immunized groups. These findings suggest that the full length of HMGB1 gene could be a more efficient adjuvant for improvement of therapeutic HIV DNA-based immunization compared to the full length of gp96 gene (Tab. 1, Fig. 3, Ref. 58).

Changes in the Plasticity of HIV-1 Nef RNA during the Evolution of the North American Epidemic.

Because of a high mutation rate, HIV exists as a viral swarm of many sequence variants evolving under various selective pressures from the human immune system. Although the Nef gene codes for the most immunogenic of HIV accessory proteins, which alone makes it of great interest to HIV research, it also encodes an RNA structure, whose contribution to HIV virulence has been largely unexplored. Nef RNA helps HIV escape RNA interference (RNAi) through nucleotide changes and alternative folding. This study examines Historic and Modern Datasets of patient HIV-1 Nef sequences during the evolution of the North American epidemic for local changes in RNA plasticity. By definition, RNA plasticity refers to an RNA molecule’s ability to take alternative folds (i.e., alternative conformations). Our most important finding is that an evolutionarily conserved region of the HIV-1 Nef gene, which we denote by R2, recently underwent a statistically significant increase in its RNA plasticity. Thus, our results indicate that Modern Nef R2 typically accommodates an alternative fold more readily than Historic Nef R2. Moreover, the increase in RNA plasticity resides mostly in synonymous nucleotide changes, which cannot be a response to selective pressures on the Nef protein. R2 may therefore be of interest in the development of antiviral RNAi therapies.

Enhancement of viral escape in HIV-1 Nef by STEP vaccination.

Objective:Properly priming cytotoxic T-lymphocyte (CTL) responses is an important task in HIV-1 vaccination. However, the STEP trial showed no efficacy even though the vaccine elicited HIV-specific CTL responses. Our study is to investigate whether or not the STEP vaccine enhanced viral escape in infected volunteers.Methods:The signature of viral escape, the presence of multiple escape variants, could be falsely represented by the existence of multiple founder viruses. Therefore, we use a mathematical model to designate STEP study patients with infections from a single founder virus. We then conduct permutation tests on each of 9988 Gag, Pol, and Nef overlapping peptides to identify epitopes with significant differences in diversity between the vaccine and placebo groups using previously published STEP trial sequence data.Results:We identify signatures of vaccine-enhanced viral escape within HIV-1 Nef from the STEP trial. Vaccine-treated patients showed a greater level of epitope diversity in one of the immunodomiant epitopes, EVGFPVRPQVPL (Nef65–76), compared with placebo-treated patients (P = 0.0038). In the other three Nef epitopes, there is a marginally significant difference in the epitope diversity between the vaccine and placebo group (P < 0.1). This greater epitope diversity was neither due to any difference in infection duration nor overall nef gene diversity between the two groups, suggesting that the increase in viral escape was likely mediated by vaccine-induced T-cell responses.Conclusion:Viral escape in Nef is elevated preferentially in STEP vaccine-treated individuals, suggesting that vaccination primarily modulated initial CTL responses. Our observations provide important insights into improving vaccine-primed first immune control.

Identification of a Novel HIV Type 1 Circulating Recombinant Form (CRF86_BC) Among Heterosexuals in Yunnan, China.

In recent years, multiple circulating recombinant forms (CRFs) and unique recombinant forms of human immunodeficiency virus type 1 (HIV-1) have been described in Yunnan, China. Here, we identified a novel HIV-1 CRF (CRF86_BC) isolated from three heterosexuals with no obvious epidemiologic linkage in western Yunnan (Baoshan prefecture) in China. CRF86_BC had a subtype C backbone with four subtype B fragments inserted into the pol, vpr, vpu, env, and nef gene regions, respectively. Furthermore, subregion tree analysis revealed that subtype C backbone originated from an Indian C lineage and subtype B segment inserted was from a Thai B lineage. They are different from previously documented B/C forms in its distinct backbone, inserted fragment size, and break points. This highlighted the importance of continual monitoring of genetic diversity and complexity of HIV-1 strains in this region.

Delivery of HIV-1 Nef Protein in Mammalian Cells Using Cell Penetrating Peptides as a Candidate Therapeutic Vaccine

The HIV-1 Nef protein expressed early in viral life cycle has been known as a potent candidate for therapeutic vaccine development. Due to different cell barriers, various cell penetrating peptides (CPPs) such as Pep-1 and CADY-2 have been known to deliver biologically active proteins to cytoplasmic compartments via the plasma membrane. In current study, we firstly evaluated the efficiency of lentiviral vector (pCDH-CMV-MCS-EF1-cGFP-T2A-puro) and eukaryotic expression vector (pEGFP-N1) for expression of HIV-1 Nef protein in HEK-293T cells using TurboFect transfection reagent. Our results showed that both vectors can effectively express the Nef proteins within the target cell. The pEGFP-N1 was more effective than pCDH-GFP for protein expression. Furthermore, Nef protein was expressed in E. coli as GST-Nef fusion and transfected by the amphipathic CPPs including Pep-1 and CADY-2 into HEK-293T cells. The size and morphology of the GST-Nef/CPP complexes were evaluated by scanning electron microscopy, and Zetasizer. Our data indicated that the recombinant GST-Nef protein generated in BL21 strain migrated as a clear band of ~50 kDa in SDS-PAGE. The CPP/GST-Nef nanoparticles were formed with a diameter of below 200 nm and notably delivered into HEK-293T cells. Generally, the Nef protein was expressed in prokaryotic and eukaryotic expression systems using different vectors and efficiently transfected in mammalian cells using various delivery systems. The in vitro efficient delivery of HIV-1 Nef gene and also its protein supports the potential of Nef DNA constructs and CPPs as potent carriers of Nef protein for HIV vaccine design in Future.

Near Full-Length Genome Identification of a Novel HIV-1 Unique (B/C) Recombinant Isolate in Sichuan, China.

A novel HIV-1 unique recombinant virus (XC2014EU20) was identified. It was isolated from an HIV-positive man who was infected through heterosexual sex in Sichuan, China. The near full-length genome analyses showed that the novel recombinant was composed of three subtype B regions in a subtype C backbone. Different from the other circulating recombinant forms and unique recombinant forms, six recombinant breakpoints of XC2014EU20 with seven fragments were observed in the pol, vpu, and nef genes, respectively. The emergence of B/C recombinant strain indicated the increasing complexity of the HIV-1 epidemic in Sichuan, China.

Study of Antigenic properties of Human immunodeficiency Virus NEF protein Recombinant Analog

Protein p27/p25 is a product of HIV nef gene and is a multifunctional factor of HIV pathogenicity. Antigenic properties of recombinant NEF polypeptide which includes N-terminal fragment of HIV-1 p27 protein fused to E. coli ß-galactosidase were studied by serological and virological methods. The interaction was shown between recombinant NEF antigen and serums of HIV-positive individuals by ELISA, Western blot and line immunoassay. There was no interaction with sera of healthy individuals. The antigen specificity of recombinant antigen was shown in the reactions with commercial HIV-1 p27/p25 protein analog and with polyclonal antibodies to synthetic peptides corresponding to N-, C-terminal regions of HIV-1 p27/p25 protein. The interaction between IgG of rabbits immunized with recombinant NEF antigen and viral antigens was shown by indirect immunofluorescence and neutralizing assays. Thus it was proven the possibility of using recombinant NEF protein as an antigen for diagnostic and experimental purposes.

Molecular Genotyping of HIV-1 Strains from Newly Infected Men Who Have Sex with Men in Harbin, China.

In this study, blood samples from newly HIV-1 infected men who have sex with men (MSM) were collected, and HIV-1 genotypes were identified based on gag p17-p24 and nef gene regions. We found that participants aged from 20 to 40 years old were the major infection group in Harbin. CRF01_AE was the predominant genotype, contributing to 84.7% of HIV-1 infections, followed by subtype B (4.7%), CRF07_BC (3.5%), and subtype B' (Thai B, 1.2%). Moreover, five unique recombinant forms (5.9%) were also identified, including genotypes 01B, 01C, and 01/02. The recombinant CRF01_AE/CRF02_AG was first reported in China. These results suggested that current HIV-1 genotype epidemic among MSM in Harbin is more complicated and that intersubtype recombinants have emerged. Therefore, timely regional epidemiological surveillance of HIV-1 genotype and development of prevention measures for new HIV-1 infections among MSM are quite important.

Genomic Characterization of a Novel HIV-1 Second-Generation Recombinant Form Originated from CRF01_AE and CRF08_BC in Dali Prefecture of Yunnan Province, China.

Yunnan seems to be a "hot spot" region of HIV-1 recombination. CRF01_AE and subtype CRF08_BC are two main HIV-1 clades circulating in Yunnan. We report here a novel HIV-1 second-generation recombinant form originated from CRF01_AE and CRF08_BC. The strain (12YN10551) was isolated from a HIV-positive male infected through heterosexual contact in Dali prefecture of Yunnan province, China. This is the first report of HIV-1 near full-length genomic sequence in Dali. Recombinant analysis shows that 12YN10551 was composed of two well-established circulating recombinant forms (CRF01_AE and CRF08_BC). Two CRF01_AE recombinant fragments were inserted into the CRF08_BC backbone genome in the pol/vif/vpr/tat/rev and nef gene regions, respectively. The discovery and characterization of this new recombinant indicate that intersubtype recombination is continuously generating new forms of HIV-1. More work is needed to better monitor the genetic diversity of HIV-1 in this region.

Mucosal Humoral Immune Response to SIVmac239∆nef Vaccination and Vaginal Challenge.

Live attenuated vaccines such as SIV with a deleted nef gene have provided the most robust protection against subsequent vaginal challenge with wild-type (WT) SIV in the SIV-rhesus macaque model of HIV-1 transmission to women. Hence, identifying correlates of this protection could enable design of an effective HIV-1 vaccine. One such prechallenge correlate of protection from vaginal challenge has recently been identified as a system with three components: 1) IgG Abs reacting with the viral envelope glycoprotein trimeric gp41; 2) produced by plasma cells in the submucosa and ectopic tertiary lymphoid follicles in the ectocervix and vagina; and 3) concentrated on the path of virus entry by the neonatal FcR in the overlying epithelium. We now examine the mucosal production of the Ab component of this system after vaginal challenge. We show that vaginal challenge immediately elicits striking increases in plasma cells not only in the female reproductive tract but also at other mucosal sites, and that these increases correlate with low but persistent replication at mucosal sites. We describe vaginal ectopic follicles that are structurally and functionally organized similar to follicles in secondary lymphoid organs, and we provide inferential evidence for a key role of the female reproductive tract epithelium in facilitating Ab production, affinity maturation, and class switch recombination. Vaccination thus accesses an epithelial-immune system axis in the female reproductive tract to respond to exposure to mucosal pathogens. Designing strategies to mimic this system could advance development of an effective HIV-1 vaccine.

Identification of a Novel CRF01_AE/CRF07_BC Recombinant Form in Men Who Have Sex with Men in Sichuan, China.

A novel second-generation CCR5-tropic HIV-1 recombinant virus (XC2014EU09) was identified here, which was isolated from a HIV-positive man who had sex with men (MSM) in Sichuan, China. Phylogenic analyses showed that XC2014EU09 was composed of two well-established circulating recombinant forms (CRF01_AE and CRF07_BC). Different from the other four reported CRF01_AE/CRF07_BC recombinant forms, three recombinant breakpoints of XC2014EU09 with four fragments were observed in the vpr, env, and nef genes, respectively, as follows: ICRF01_AE (636-5,843 nt), IICRF07_BC (5,844-8,393 nt), IIICRF01_AE (8,394-9,119 nt), and IVCRF07_BC (9,120-9,600 nt). The emergence of CRF01_AE/CRF07_BC recombinant strain indicates the increasing complexity of the HIV-1 epidemic among the MSM group in China.

Genetic defects in the nef gene are associated with Korean Red Ginseng intake: monitoring of nef sequence polymorphisms over 20 years

BackgroundThe presence of gross deletions in the human immunodeficiency virus nef gene (gΔnef) is associated with long-term nonprogression of infected patients. Here, we investigated how quickly genetic defects in the nef gene are associated with Korean Red Ginseng (KRG) intake in 10 long-term slow progressors. MethodsThis study was divided into three phases over a 20-yr period; baseline, KRG intake alone, and KRG plus highly active antiretroviral therapy (ART). nef gene amplicons were obtained using reverse transcription polymerase chain reaction (PCR) and nested PCR from 10 long-term slow progressors (n = 1,396), and nested PCR from 36 control patients (n = 198), and 28 ART patients (n = 157), and these were then sequenced. The proportion of gΔnef, premature stop codons, and not in-frame insertion or deletion of a nucleotide was compared between three phases, control, and ART patients. ResultsThe proportion of defective nef genes was significantly higher in on-KRG patients (15.6%) than in baseline (5.7%), control (5.6%), on-KRG plus ART phase (7.8%), and on-ART patients (6.6%; p < 0.01). Small in-frame deletions or insertions were significantly more frequent among patients treated with KRG alone compared with controls (p < 0.01). Significantly fewer instances of genetic defects were detected in samples taken during the KRG plus ART phase (7.8%; p < 0.01). The earliest defects detected were gΔnef and small in-frame deletions after 7 mo and 67 mo of KRG intake, respectively. ConclusionKRG treatment might induce genetic defects in the nef gene. This report provides new insight into the importance of genetic defects in the pathogenesis of AIDS.

Genetic and functional characterization of HIV-1 Nef gene from North Indian HIV-1 infected patients

Genetic and functional characterization of HIV-1 Nef gene from North Indian HIV-1 infected patients