[HIV nef gene and nef protein].
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Animals↗
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[The expression of a fragment of the HIV-1 Nef gene in Escherichia coli bacteria].
The presence of antibodies to p27, the product of gene Nef, may be an important diagnostic sign since some sera from subjects of the risk groups negative to HIV-1 structural proteins may contain antibody to p27. The study resulted in construction of a hybrid plasmid determining in E coli bacteria the synthesis of a hybrid protein the N-terminus part of which is represented by full-size beta-galactosidase and the C-terminus by a part of protein p27 with the main immunoreactive epitopes. The resulting polypeptide specifically interacts with sera of the infected subjects and may be used for detection of antibodies to the protein Nef in the blood of virus-carriers.
Epitopes↗
Presence of Intact vpu and nef genes in nonpathogenic SHIV is essential for acquisition of pathogenicity of this virus by serial passage in macaques.
Use of the macaque model of human immunodeficiency virus (HIV) pathogenesis has shown that the accessory genes nef and vpu are important in the pathogenicity of simian immunodeficiency virus (SIV) and simian-human immunodeficiency virus (SHIV). We examined the ability of two nonpathogenic SHIVs, SHIV(PPC) and DeltavpuDeltanefSHIV(PPC), to gain pathogenicity by rapid serial passage in macaques. In this study, each virus was passaged by blood intravenously four times at 4-week intervals in macaques. Animals were monitored for 40 weeks for levels of CD4 T cells and quantitative measures of virus infection. DeltavpuDeltanefSHIV(PPC) maintained a limited phase of productive replication in the four animals, with no loss of CD4(+) T cells, whereas SHIV(PPC) became more pathogenic in later passages, judging by plasma viral load and viral mRNA in lymph nodes, infectious peripheral blood mononuclear cells and CD4(+) T cell loss. The nef, LTR, and env of the SHIV(PPC) viruses underwent numerous mutations, compared to DeltavpuDeltanefSHIV(PPC). This study confirms the seminal role that nef, LTR, and vpu could play in regulation of pathogenesis of HIV infection.
Acquired Immunodeficiency Syndrome↗
Allelic variation in the effects of the nef gene on replication of human immunodeficiency virus type 1.
The effects of the viral gene nef on human immunodeficiency virus type 1 (HIV-1) replication in culture were investigated using nef alleles of the HIV-1 IIIB and ELI strains. The results demonstrate significant allelic variation in the effect of nef on virus replication in both an established human CD4+ T-cell line and primary human lymphocytes. In the context of the HXB2 virus, the ELI nef allele but not the IIIB nef allele permits initiation of efficient low-multiplicity infection in primary peripheral blood mononuclear cells, including unfractionated peripheral blood lymphocytes, T cells, and monocyte/macrophages. Within the same genetic context, the IIIB nef allele slightly retards replication of the virus in a T-cell line, whereas the ELI nef allele accelerates replication of the virus. Sequences in the IIIB and ELI genomes outside of nef also moderate the effects of nef on HIV-1 replication. nef did not appear to determine the host-cell preference of the virus. These studies may help to reconcile apparently conflicting reports on the role of nef in HIV-1 replication and suggest that HIV-1 nef may play an important role in viral pathogenesis.
Alleles↗
Protective effects of a live attenuated SIV vaccine with a deletion in the nef gene.
Vaccine protection against the human immunodeficiency virus (HIV) and the related simian immunodeficiency virus (SIV) in animal models is proving to be a difficult task. The difficulty is due in large part to the persistent, unrelenting nature of HIV and SIV infection once infection is initiated. SIV with a constructed deletion in the auxiliary gene nef replicates poorly in rhesus monkeys and appears to be nonpathogenic in this normally susceptible host. Rhesus monkeys vaccinated with live SIV deleted in nef were completely protected against challenge by intravenous inoculation of live, pathogenic SIV. Deletion of nef or of multiple genetic elements from HIV may provide the means for creating a safe, effective, live attenuated vaccine to protect against acquired immunodeficiency syndrome (AIDS).
Animals↗
Altered T cell activation and development in transgenic mice expressing the HIV-1 nef gene.
The nef gene, which encodes related cytoplasmic proteins in both human (HIV) and simian (SIV) immunodeficiency viruses is dispensable for viral replication in vitro. In contrast, in vivo experiments have revealed that SIV nef is required for efficient viral replication and development of AIDS in SIV infected rhesus monkeys, thus indicating that nef plays an essential role in the natural infection. We show that expression of the Nef protein from the HIV-1 NL43 isolate in transgenic mice perturbs development of CD4+ T cells in the thymus and elicits depletion of peripheral CD4+ T cells. Thymic T cells expressing NL43 Nef show altered activation responses. In contrast, Nef protein of the HIV-1 HxB3 isolate does not have an overt effect on T cells when expressed in transgenic animals. The differential effects of the two HIV-1 nef alleles in transgenic mice correlate with down-regulation of CD4 antigen expression on thymic T cells. The differential interactions of the NL43 and HxB3 nef alleles with CD4 were reproduced in a transient assay in human CD4+ CEM T cells. Down-regulation of CD4 by nef in both human and transgenic murine T cells indicates that the relevant interactions are conserved in these two systems and suggests that the consequences of Nef expression on the host cell function can be analyzed in vivo in the murine system. Our observations from transgenic mice suggest that nef-elicited perturbations in T cell signalling play an important role in the viral life cycle in vivo, perhaps resulting in elimination of infected CD4+ T cells.
Animals↗
Expression and biochemical characterization of human immunodeficiency virus type 1 nef gene product.
nef genes from human immunodeficiency virus type 1 isolates BH10 and LAV1 (lymphadenopathy-associated virus type 1) were expressed in Escherichia coli under the deo operon promoter. The two proteins found in the soluble compartment of the bacterial lysate were purified by ion-exchange column chromatography to apparent homogeneity. Determination of the amino-terminal sequence revealed glycine as the first amino acid in the Nef protein, indicating removal of the initiator methionine during expression in E. coli. Under native conditions, the recombinant Nef protein is a monomer of 23 kilodaltons. In denaturing polyacrylamide gels, however, BH10 and LAV1 Nef proteins migrate as 28 and 26 kilodaltons, respectively. GTP binding and GTPase activity were monitored during Nef protein purification. These activities did not copurify with the recombinant Nef protein from either the BH10 or the LAV1 isolate. Purified recombinant BH10 Nef protein was used as an immunogen to elicit mouse monoclonal antibodies. A series of monoclonal antibodies were obtained which reacted with sequences at either the amino or carboxy terminus of Nef. In addition, a conformational epitope reacting with native BH10, but not LAV1, Nef was isolated.
Amino Acid Sequence↗
Genetic characterization of the nef gene from human immunodeficiency virus type 1 group M strains representing genetic subtypes A, B, C, E, F, G, and H.
Most efforts to characterize sequence variation of HIV isolates has been directed toward the structural envelope gene. Few studies have evaluated the sequence variability of auxiliary genes such as nef. In this study 41 new HIV-1 strains, representing the majority of the described envelope subtypes of HIV-1 (A to H), were genetically characterized in the nef region. Phylogenetic analysis showed that 34 strains could be classified in the same subtype in nef and env, and 7 (19%) of the 41 new viruses were recombinants. For two of the seven strains, recombination occurred upstream of the nef gene, whereas for five of the seven strains recombination occurred within the nef gene with a crossover close to the 5' end of the LTR (long terminal repeat). The low intersubtype distance between subtype B and D in the nef gene confirms previous observations in the pol, env, and gag genes, which suggest a common ancestor for these subtypes. The majority of all the previously described functional domains in the nef gene were relatively conserved among the different subtypes, with only minor differences being observed. The myristoylation signal among the different subtypes, with only minor differences being observed. The myristoylation signal was less conserved for subtype C, with one or more amino acid changes being observed at positions 3, 4, and 5. The highly conserved acidic region (positions 62 to 65), critical for the enhancement of viral synthesis with an increased virus growth rate, was less conserved among the subtype G strains from our study. At least three epitopic regions of the nef gene have been defined and each can be recognized by CTLs under a variety of HLA restrictions; all were also relatively well conserved between the different genetic subtypes. Despite the relatively important genetic variation in nef sequences obtained among the different genetic subtypes, functional domains and CTL epitopes were relatively well conserved. In vitro and/or in vivo studies are necessary to study the relevance of the observed differences.
Amino Acid Sequence↗
Macaques infected with cloned simian immunodeficiency virus show recurring nef gene alterations.
We compared nef gene sequences isolated by polymerase chain reaction from peripheral blood lymphocyte DNA of macaques that had been inoculated with either biologically (E11S) or molecularly (clone 8) cloned SIV/Mne. Two samples from each animal obtained either early (weeks 2-8) or late (weeks 21-137) after infection were analyzed. Three substitutions in the predicted Nef amino acid sequence were seen in all animals at the late time point, and two other substitutions were seen in all except one. Two of the common exchanges are located approximately 40 residues apart in the Nef core sequence but are juxtaposed on the tertiary structure as judged by computer modeling using the structure of the HIV Nef core protein as a guide. Most recurrent in vivo changes replaced a residue found in the cloned Nef sequence with one present in a consensus derived by aligning the Nef sequences of the SIV/Sm clade. Recombinant virus containing a macaque-adapted (MA nef) nef on the clone 8 backbone was 3-fold more infectious on SMAGI cells than the original virus. A lymphocyte line infected with SIV-clone 8-MAnef contained a large proportion of cells carrying provirus with defective nef genes. These findings suggest that the nef gene of the cloned SIV/Mne had undergone attenuating mutations during propagation in tissue culture that were "corrected" in vivo.
Amino Acid Sequence↗
The expression of HIV-1 tat and nef genes induces cell-specific changes in growth properties and morphology of different types of rat cells.
Among the viral regulatory genes the tat and nef genes of HIV-1 encode the proteins playing a central role in viral replication and exerting pleiotropic effects on the survival and growth of the cells. These effects differ in various cell types, possibly due to the use of genes from different HIV-1 isolates. In this work, we studied the effects of the tat and nef genes on three types of cultured rat cells: primary embryo fibroblasts, pseudonormal Rat-2, and pheochromocytoma PC12. Both genes affected growth properties and morphology of cells, the effects being cell-specific. The proliferative activity of both Rat-2 and PC12 cells was considerably increased after transfection with the tat gene. In primary rat embryo fibroblasts the tat gene induced multilayered foci. More importantly, it was shown that the efficiency of transformation was higher in cells coexpressing tat and nef. The nef gene caused considerable suppression of Rat-2 cell proliferation, but no changes in their morphology. The nef gene transfection of PC12 cells also led to suppression of their proliferative activity. In addition, cellular agglomerates which were morphologically similar to multinuclear syncytial cells were detected in these cells for the first time.
Animals↗
One explanation of Nef gene behavior.
The Nef (negative factor) gene of primate retroviruses may serve an important evolutionary function. Selection pressures in the natural world, may at times, demand that retroviral infection not cause disease in a newly entered host species. Genetic alterations in the Nef gene may function to permit retroviral speciation by lowering retroviral replication rate. Slow viral replication may allow small numbers of the newly infected host species to avoid the effects of retroviral pathology.
Animals↗
Polymorphism of the Human Immunodeficiency Virus Type 1 in Brazil: genetic characterization of the nef gene and implications for vaccine design.
Most of the Brazilian HIV-1 samples have been characterized based on the structural genes (env, gag and pol) and no data concerning the variability of the accessory genes such as nef have been available so far. Considering the role of the nef on virus biology and the inclusion of this region in some HIV/AIDS vaccine products under testing, the purpose of this study was to document the genetic diversity of the nef gene in third-four HIV-1 Brazilian samples previously subtyped based on the env C2-V3 region. Although only few non-subtype B samples have already been analyzed so far, the cytotoxic Tlymphocyte epitopes encoded in this region were relatively conserved among the subtypes, with some amino acid signatures mainly in the subtype C samples. Considering the increasing of the non-B HIV-1 subtypes worldwide, in special the subtype C, more data should be generated concerning the genetic and antigenic variability of these subtypes, as well as the study of the impact of such polymorphism in HIV/AIDS vaccine design and testing.
AIDS Vaccines↗
Evaluation of genetic diversity of human immunodeficiency virus type 1 NEF gene associated with vertical transmission.
The NEF gene is conserved among members of human and simian immunodeficiency viruses and may play an important role in viral pathogenesis. To determine the evolutionary dynamics and conservation of functionality of the human immunodeficiency virus type 1 (HIV-1) NEF gene during maternal-fetal transmission, we analyzed NEF sequences from seven mother-infant pairs following perinatal transmission, including a mother with infected twin infants. The NEF open reading frame was maintained in mother-infant isolates with a frequency of 86.2% following vertical transmission. While there was a low degree of viral heterogeneity and estimates of genetic diversity and high population growth rates of NEF sequences from mother-infant isolates, the infants' NEF sequences were slightly higher with respect to these parameters compared with the mothers' sequences. Both the mothers' and infants' NEF sequences were under positive selection pressure, as determined by a new method of Nielsen and Yang [Genetics 148:929-936;1998]. Based on genetic distance and phylogenetic parameters, the epidemiologically linked NEF sequences from mother-infant pairs were closer to each other compared with epidemiologically unlinked sequences from individuals. The functional domains essential for Nef activity, including membrane binding, CD4 and MHC-I downmodulation, T cell activation and interaction with factors of the cellular protein trafficking machinery, were conserved in most of the sequences from mother-infant pairs. The maintenance of intact NEF open reading frames with conserved functional domains and a low degree of genetic variability following vertical transmission supports the notion that NEF plays an important role in HIV-1 infection and replication in mothers and their perinatally infected infants.
Amino Acid Sequence↗
Analysis of human immunodeficiency virus type 1 nef gene sequences present in vivo.
The nef genes of the human immunodeficiency viruses type 1 and 2 (HIV-1 and HIV-2) and the related simian immunodeficiency viruses (SIVs) encode a protein (Nef) whose role in virus replication and cytopathicity remains uncertain. As an attempt to elucidate the function of nef, we characterized the nucleotide and corresponding protein sequences of naturally occurring nef genes obtained from several HIV-1-infected individuals. A consensus Nef sequence was derived and used to identify several features that were highly conserved among the Nef sequences. These features included a nearly invariant myristylation signal, regions of sequence polymorphism and variable duplication, a region with an acidic charge, a (Pxx)4 repeat sequence, and a potential protein kinase C phosphorylation site. Clustering of premature stop codons at position 124 was noted in 6 of the 54 Nef sequences. Further analysis revealed four stretches of residues that were highly conserved not only among the patient-derived HIV-1 Nef sequences, but also among the Nef sequences of HIV-2 and the SIVs, suggesting that Nef proteins expressed by these retroviruses are functionally equivalent. The "Nef-defining" sequences were used to evaluate the sequence alignments of known proteins reported to share sequence similarity with Nef sequences and to conduct additional computer-based searches for similar protein sequences. A gene encoding the consensus Nef sequence was also generated. This gene encodes a full-length Nef protein that should be a valuable tool in further studies of Nef function.
Acquired Immunodeficiency Syndrome↗
The nef gene from a long-term HIV type 1 nonprogressor.
We examined the nef gene of HIV-1 in a long-term nonprogressor to look for evidence suggesting an attenuated virus. The nef gene was previously shown to be required for induction of AIDS. Simian immunodeficiency virus (SIV) deleted in nef, while infectious, fails to sustain the high viral loads necessary for the induction of AIDS in infected adult rhesus monkeys. The human subject of this report was found to harbor virus (HIV-1 Sur25) encoding open-nef reading frames. However, the nef genes of this subject bore a signature point mutation: a cysteine at amino acid 138. The sequence at this position was identical in all clones examined over a 3-year period. When this sequence was compared to the sequence database for AIDS and human retroviruses at Los Alamos, New Mexico, several isolates from other asymptomatic individuals were also found to encode nef genes with a cysteine at position 138. Furthermore, Cys-138 was found in chimpanzee immunodeficiency virus (CIV), a lentivirus that is similar to HIV but does not cause AIDS in chimpanzees. Multiple cysteines are also found in the nef gene of African green monkey virus, SVIagm, including cysteine at the position analogous to Cys-138. While seroprevalence of SIVagm is high in the wild, there is no known disease associated with this virus. The pathogenic virus isolated from Asian macaques, SIVmac, encodes a Nef protein that has few cysteines. Although the virus HIVSur25 encodes a completely open-nef gene, the virus from this individual is similar to attenuated SIVmac (SIVmac239/nef-deletion) as well as HIV deleted in nef in its growth properties in H9 cells. Nef containing a cysteine at position 138 was shown to be responsible for determining the ability to grow in H9.
Amino Acid Sequence↗
Genomic diversity in the regulatory nef gene sequences in Indian isolates of HIV type 1: emergence of a distinct subclade and predicted implications.
The regulatory functional nef gene is known to mediate a cascade of events during pathogenesis in HIV infection. Variability in the nef gene sequences of HIV-1 A and B subtypes has been well documented. Reasonable data are also available on the pattern of genomic changes in the nef gene of African strains of HIV-1 subtype C, but very little is known about heterogeneity in the nef gene of Indian strains of HIV-1 subtype C, which accounts for 90% of the estimated 5.2 million cases of HIV infection in India. This is a huge number and, therefore, it is important to reveal the extent of sequence variability in the nef gene of HIV-1 subtypes circulating in different parts of India. We carried out full-length nef gene (approximately 620 bp) sequencing on a large number of clinical isolates of HIV-1 circulating in different geographic regions of India. Comparative and phylogenetic analysis revealed 88% (38/43) of cases was HIV-1 subtype C; four cases were diagnosed as subtype A and only one as subtype B. Although most of the crucial functional motifs of the nef gene were conserved, we did observe a few important variations in juxtapositions to functional domains. Interestingly, analyzed nef sequences showed an evolving pattern of segregation away from those reported from other parts of the world, to form a distinct Indian subclade. Deduced amino acid (aa) sequences used to predict HLA binding epitopes for consensus nef gene sequences of Indian strains of HIV-1 revealed two HLA subtype binding domains, GAFDLSFFL (at aa 83) and LTFGWCFKL (at aa 136), in high frequency. The findings from the present study may encourage use of nef gene in molecular diagnostics/genotyping, keeping track of the evolutionary trend and pinpointing the emergence of recombinant strains, and in the future, designing a multiepitope HIV vaccine suitable for the Indian population.
Adolescent↗
Mutational analysis of human immunodeficiency virus type 1 (HIV-1) accessory genes: requirement of a site in the nef gene for HIV-1 replication in activated CD4+ T cells in vitro and in vivo.
Human immunodeficiency virus type 1 (HIV-1) accessory genes including nef, vif, and vpr are important factors that determine the replication and pathogenesis of HIV-1. The state of activation is also important for the replication of HIV-1. We evaluated the properties of nef-, vif-, and vpr-minus macrophage-tropic HIV-1(JR) CSF in primary CD4+ Th1- or Th2-like cell cultures which had been activated through CD3 molecules in the presence of interleukin-2 (IL-2) and IL-12 (Th1-like culture) or IL-4 (Th2-like culture), respectively. In activated Th1- or Th2-like cultures, replication of nef-minus HIV-1(JR-CSF) was markedly lower than that of wild-type HIV-1. Subsequent analysis by site-directed mutagenesis showed that (i) the presence of an acidic amino acid-rich domain (amino acid residues 72 to 75) in the Nef protein was critical for the enhancement of viral DNA synthesis, resulting in increased virus growth rate, and (ii) prolines that form part of Src homology 3 binding domain were not essential for viral replication. We also confirmed the importance of sites by using an HIV-1-infected animal model, the hu-PBL-SCID mouse system, representing HIV-1 replication and pathogenesis in activated CD4+ T cells in vivo. These results indicate that Nef accelerates viral replication in activated CD4+ T cells.
Amino Acid Sequence↗
High frequency of grossly deleted nef genes in HIV-1 infected long-term slow progressors treated with Korean red ginseng.
To investigate the association between Korean red ginseng (KRG) intake in HIV-1 infected patients and the occurrence of grossly deleted nef genes (gDeltanef), we characterized nef genes in 10 long-term slow progressors (LTSP) infected with HIV-1 subtype B and 34 control patients. LTSP was defined by the annual decrease in CD4 T cells being less than 20/microl over 10 years in the absence of antiretroviral therapy. They were treated with KRG for a prolonged period. Nef genes were amplified from peripheral blood mononuclear cells (PBMC) using nested PCR and the products were sequenced directly. It was observed that the patients CD4 T cell counts decreased from 444 +/- 207/microl to 294 +/- 177/microl over 136 +/- 23 months of KRG intake. This corresponds to an annual decrease in the level of CD4 T cells of 13.3/microl. A total of 479 nef genes were amplified from 137 PBMC samples. Nine out of the 10 patients, 47 (34.3%) out of the 137 samples, and 90 out of the 479 genes revealed gDeltanef. The deletion extended outside the nef gene in 25 gDeltanef obtained from 6 patients. The proportion of samples with gDeltanef (34.3%) was significantly higher than 4.8% in control patients (P < 0.001). In addition, it significantly increased as the duration of KRG intake prolongs (P < 0.01). These data suggest that the occurrence of gDeltanef might be associated with long-term intake of KRG.
Adult↗