Disulfide bond formation in the human immunodeficiency virus type 1 Nef protein.

Substitution of alanine for cysteine residues of the human immunodeficiency virus type 1 LAI (BRU) and ELI Nef proteins was used to determine pairing of the cysteine residues present in each protein. The results show that under nonreducing conditions, alternative pairing of the cysteines occurs. The preferred pairing of cysteine residues of the LAI and ELI proteins differs. In the experimental system used, viruses carrying the ELI nef allele are found to express Nef proteins which accelerate virus replication. Mutation in critical cysteine residues of the protein reduce the rate of virus replication. In the same system, viruses harboring the LAI nef allele fail to replicate. These observations raise the possibility that differences in the observed biological activity of nef alleles may be attributed, at least in part, to differences in the secondary structure of the proteins.     

Proline 78 is crucial for human immunodeficiency virus type 1 Nef to down-regulate class I human leukocyte antigen

Human immunodeficiency virus type 1 Nef down-regulates human leukocyte antigen class I (HLA-I) in T lymphocytes, and the down-regulation involves the Nef proline-rich domain (PRD) containing four prolines at positions 69, 72, 75, and 78. We used a Sendai virus vector with nef and examined regulation by Nef of HLA-I and CD4 in suspension cultures of cells such as T lymphocytes. Analyses of a series of PRD substitution mutants indicated that, because the substitution of Pro78 with Ala abolished down-regulation of HLA-I but not of CD4, Pro78 is important for HLA-I down-regulation in T lymphocytes.     

Nef protein of human immunodeficiency virus type 1 binds its own myristoylated N-terminus

HIV-1 Nef is a small protein (approx. 25 kDa) that is posttranslationally modified by myristoylation. To explain its complex activities, a 'Nef-cycle' is discussed, which postulates different molecular conformations of Nef. Using recombinant full-length non-myristoylated Nef and synthetic peptides, we demonstrate by fluorescence titration experiments that a peptide representing the myristoylated N-terminus of Nef is specifically bound by Nef. A non-myristoylated N-terminal fragment of Nef or a myristoylated control peptide does not bind to Nef. These results are the first direct experimental evidence of the existence of a myristate-binding pocket in Nef, a prerequisite of the postulated 'closed' Nef conformation.     

The Nef protein of human immunodeficiency virus type 1 enhances serine phosphorylation of the viral matrix.

The human immunodeficiency virus type 1 matrix (MA) protein is phosphorylated during virion maturation on its C-terminal tyrosine and on several serine residues. Whereas MA tyrosine phosphorylation facilitates viral nuclear import, the significance of MA serine phosphorylation remains unclear. Here, we report that MA serine but not tyrosine phosphorylation is strongly enhanced by Nef. Mutations that abrogated the membrane association of Nef and its ability to bind a cellular serine/threonine kinase greatly diminished the extent of virion MA serine phosphorylation. Correspondingly, a protein kinase coimmunoprecipitated with Nef could phosphorylate MA on serine in vitro, producing a phosphopeptide pattern reminiscent of that of virion MA. Recombinant p21-activated kinase hPAK65, a recently proposed relative of the Nef-associated kinase, achieved a comparable result. Taken together, these data suggest that MA is a target of the Nef-associated serine kinase.     

Direct in vitro binding of full-length human immunodeficiency virus type 1 Nef protein to CD4 cytoplasmic domain

The Nef protein of the simian and human immunodeficiency viruses is known to directly bind and downregulate the CD4 receptor. Although the molecular mechanism is well understood, direct binding of Nef and CD4 is difficult to demonstrate and is believed to be of low affinity. Applying nuclear magnetic resonance and fluorescence spectroscopy, we biophysically reevaluated the CD4-Nef complex and found the dissociation constant to be in the submicromolar range. We conclude that additional, so far disregarded residues in the N terminus of Nef are important for interaction with CD4.     

The human immunodeficiency virus integrase protein

The DNA integration step in the replication cycle of the human immunodeficiency virus (HIV) has been recognized as an important target in antiviral strategies. There are two main reasons for this. First, integration of HIV DNA into the human genome is required for replication of this retrovirus. Second, since the integration reaction does not have an obvious cellular counterpart, drugs that specifically inhibit integration may not be toxic for the cell. Here, we focus on the only protein known to be required for retroviral integration, the integrase (IN) protein.     

Oligodendrocyte-specific expression of human immunodeficiency virus type 1 Nef in transgenic mice leads to vacuolar myelopathy and alters oligodendrocyte phenotype in vitro

Vacuolar myelopathy (VM) is a frequent central nervous system complication of human immunodeficiency virus type 1 (HIV-1) infection. We report here that transgenic (Tg) mice expressing even low levels of Nef in oligodendrocytes under the regulation of the myelin basic protein (MBP) promoter (MBP/HIV(Nef)) developed VM similar to the human disease in its appearance and topography. The spinal cords of these Tg mice showed lower levels of the myelin proteins MAG and CNPase and of the 21-kDa isoform of MBP prior to the development of vacuoles. In addition, Tg oligodendrocytes in primary in vitro cultures appeared morphologically more mature but, paradoxically, exhibited a less mature phenotype based on O4, O1, CNPase, and MBP staining. In particular, mature CNPase(+) MBP(+) Tg oligodendrocytes were less numerous than non-Tg oligodendrocytes. Therefore, Nef appears to affect the proper differentiation of oligodendrocytes. These data suggest that even low levels of Nef expression in human oligodendrocytes may be responsible for the development of VM in HIV-1-infected individuals.     

Simian and human immunodeficiency virus Nef proteins use different surfaces to downregulate class I major histocompatibility complex antigen expression

Simian immunodeficiency virus (SIV) and human immunodeficiency virus type 1 (HIV-1) Nef proteins are related regulatory proteins that share several functions, including the ability to downregulate class I major histocompatibility complex (MHC) and CD4 expression on the cell surface and to alter T-cell-receptor-initiated signal transduction in T cells. We compared the mechanisms used by SIV mac239 Nef and HIV-1 Nef to downregulate class I MHC and found that the ability of SIV Nef to downregulate class I MHC requires a unique C-terminal region of the SIV mac239 Nef molecule which is not found in HIV-1 Nef. Interestingly, mutation of the PxxP motif in SIV Nef, unlike in HIV-1 Nef, does not affect class I MHC downregulation. We also found that downregulation of class I MHC by SIV Nef requires a conserved tyrosine in the cytoplasmic domain of the class I MHC heavy chain and involves accelerated endocytosis of class I complexes, as previously found with HIV-1 Nef. Thus, while SIV and HIV-1 Nef proteins use a similar mechanism to downregulate class I MHC expression, they have evolved different surfaces for molecular interactions with cell factors that regulate class I MHC traffic. Mutations in the C-terminal domain of SIV mac239 Nef selectively disrupt class I MHC downregulation, having no detectable effect on other functions of Nef, such as the downregulation of CD4 and CD3 surface expression, the stimulation of SIV virion infectivity, and the induction of SIV replication from T cells infected in the absence of stimulation. The resulting mutants will be useful reagents for studying the importance of class I MHC downregulation for SIV replication and AIDS pathogenesis in infected rhesus macaques.     

Producer-cell modification of human immunodeficiency virus type 1: Nef is a virion protein.

Type 1 human immunodeficiency viruses encoding mutated nef reading frames are 10- to 30-fold less infectious than are isogenic viruses in which the nef gene is intact. This defect in infectivity causes nef-negative viruses to grow at an attenuated rate in vitro. To investigate the mechanism of Nef-mediated enhancement of viral growth rate and infectivity, a complementation analysis of nef mutant viruses was performed. To provide Nef in trans upon viral infection, a CEM derivative cell line (designated CLN) that expresses Nef under the control of the viral long terminal repeat was constructed. When nef-negative virus was grown in CLN cells, its growth rate was restored to wild-type levels. However, the output of nef-negative virus during the first 72 h after infection of CLN cells was not restored, suggesting that provision of Nef within the newly infected cell does not enhance the productivity of a nef-negative provirus. The genetically nef-negative virions produced by the CLN cells, however, were restored to wild-type levels of infectivity as measured in a syncytium formation assay in which CD4-expressing HeLa cells were targets. These trans-complemented, genetically nef-negative virions yielded wild-type levels of viral output following a single cycle of replication in primary CD4 T cells as well as in parental CEM cells. To define the determinants for producer cell modification of virions by Nef, the role of myristoylation was investigated. Virus that encodes a myristoylation-negative nef was as impaired in infectivity as was virus encoding a deleted nef gene. Because myristoylation is required for both membrane association of Nef and optimal viral infectivity, the possibility that Nef protein is included in the virion was investigated. Wild-type virions were purified by filtration and exclusion chromatography. A Western blot (immunoblot) of the eluate fractions revealed a correlation between peak Nef signal and peak levels of p24 antigen. Although virion-associated Nef was detected in part as the 27-kDa full-length protein, the majority of immunoreactive protein was detected as a 20-kDa isoform. nef-negative virus lacked both 27- and 20-kDa immunoreactive species. Production of wild-type virions in the presence of a specific inhibitor of the human immunodeficiency virus type 1 protease resulted in virions which contained only 27-kDa full-length Nef protein. These data indicate that Nef is a virion protein which is processed by the viral protease into a 20-kDa isoform within the virion particle.     

The Vpu protein of human immunodeficiency virus type 1 forms cation-selective ion channels.

Vpu is a small phosphorylated integral membrane protein encoded by the human immunodeficiency virus type 1 genome and found in the endoplasmic reticulum and Golgi membranes of infected cells. It has been linked to roles in virus particle budding and degradation of CD4 in the endoplasmic reticulum. However, the molecular mechanisms employed by Vpu in performance of these functions are unknown. Structural similarities between Vpu and the M2 protein of influenza A virus have raised the question of whether the two proteins are functionally analogous: M2 has been demonstrated to form cation-selective ion channels in phospholipid membranes. In this paper we provide evidence that Vpu, purified after expression in Escherichia coli, also forms ion channels in planar lipid bilayers. The channels are approximately five- to sixfold more permeable to sodium and potassium cations than to chloride or phosphate anions. A bacterial cross-feeding assay was used to demonstrate that Vpu can also form sodium-permeable channels in vivo in the E. coli plasma membrane.     

Leucine-specific, functional interactions between human immunodeficiency virus type 1 Nef and adaptor protein complexes

The human immunodeficiency virus type 1 virulence protein Nef interacts with the endosomal sorting machinery via a leucine-based motif. Similar sequences within the cytoplasmic domains of cellular transmembrane proteins bind to the adaptor protein (AP) complexes of coated vesicles to modulate protein traffic, but the molecular basis of the interactions between these motifs and the heterotetrameric complexes is controversial. To identify the target of the Nef leucine motif, the native sequence was replaced with either leucine- or tyrosine-based AP-binding sequences from cellular proteins, and the interactions with AP subunits were correlated with function. Tyrosine motifs predictably modulated the interactions between Nef and the mu subunits of AP-1, AP-2, and AP-3; heterologous leucine motifs caused little change in these interactions. Conversely, leucine motifs mediated a ternary interaction between Nef and hemicomplexes containing the sigma1 plus gamma subunits of AP-1 or the sigma3 plus delta subunits of AP-3, whereas tyrosine motifs did not. Similarly, only leucine motifs supported the Nef-mediated association of AP-1 and AP-3 with endosomal membranes in cells treated with brefeldin A. Functionally, Nef proteins containing leucine motifs down-regulated CD4 from the cell surface and enhanced viral replication, whereas those containing tyrosine motifs were inactive. Apparently, the interaction of Nef with the mu subunits of AP complexes is insufficient for function. A leucine-specific mode of interaction that likely involves AP hemicomplexes is further required for Nef activity. The mu and hemicomplex interactions may cooperate to yield high avidity binding of AP complexes to Nef. This binding likely underlies the unusual ability of Nef to induce the stabilization of these complexes on endosomal membranes, an activity that correlates with enhancement of viral replication.     

Modulation of different human immunodeficiency virus type 1 Nef functions during progression to AIDS

The human immunodeficiency virus type 1 (HIV-1) Nef protein has several independent functions that might contribute to efficient viral replication in vivo. Since HIV-1 adapts rapidly to its host environment, we investigated if different Nef properties are associated with disease progression. Functional analysis revealed that nef alleles obtained during late stages of infection did not efficiently downmodulate class I major histocompatibility complex but were highly active in the stimulation of viral replication. In comparison, functional activity in downregulation of CD4 and enhancement of HIV-1 infectivity were maintained or enhanced after AIDS progression. Our results demonstrate that various Nef activities are modulated during the course of HIV-1 infection to maintain high viral loads at different stages of disease progression. These findings suggest that all in vitro Nef functions investigated contribute to AIDS pathogenesis and indicate that nef variants with increased pathogenicity emerge in a significant number of HIV-1-infected individuals.     

Nef enhances human immunodeficiency virus replication and responsiveness to interleukin-2 in human lymphoid tissue ex vivo

The nef gene is important for the pathogenicity associated with simian immunodeficiency virus infection in rhesus monkeys and with human immunodeficiency virus type 1 (HIV-1) infection in humans. The mechanisms by which nef contributes to pathogenesis in vivo remain unclear. We investigated the contribution of nef to HIV-1 replication in human lymphoid tissue ex vivo by studying infection with parental HIV-1 strain NL4-3 and with a nef mutant (DeltanefNL4-3). In human tonsillar histocultures, NL4-3 replicated to higher levels than DeltanefNL4-3 did. Increased virus production with NL4-3 infection was associated with increased numbers of productively infected cells and greater loss of CD4(+) T cells over time. While the numbers of productively infected T cells were increased in the presence of nef, the levels of viral expression and production per infected T cell were similar whether the nef gene was present or not. Exogenous interleukin-2 (IL-2) increased HIV-1 production in NL4-3-infected tissue in a dose-dependent manner. In contrast, DeltanefNL4-3 production was enhanced only marginally by IL-2. Thus, Nef can facilitate HIV-1 replication in human lymphoid tissue ex vivo by increasing the numbers of productively infected cells and by increasing the responsiveness to IL-2 stimulation.     

The protein-body proteins phytohemagglutinin and tonoplast intrinsic protein are targeted to vacuoles in leaves of transgenic tobacco

To demonstrate the relationship between protein-bodies in seeds and vacuoles in other tissues, we expressed the coding sequences of two bean (Phaseolus vulgaris L.) protein-body proteins in tobacco (Nicotiana tabacum L.). We chose phytohemagglutinin-L (PHA-L) and tonoplast intrinsic protein (TIP) as representatives of the protein-body contents and protein-body membrane, respectively. The location of the two proteins in the leaves of transgenic tobacco was examined by immunocytochemistry and in preparations of isolated vacuoles. Tonoplast intrinsic protein accumulates primarily in tonoplasts in tobacco leaves, whereas PHA is found exclusively in the vacuolar sap, showing that the signals that target proteins to protein-bodies and their limiting membranes in seeds are correctly recognized in leaves. This observation provides further evidence that proteinbodies of dicotyledonous seeds should be considered as protein-storage vacuoles.Abbreviations TIP tonoplast intrinsic protein - PHA phytohemagglutinin - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis This work has been supported by a grant from the U.S. Department of Agriculture Competitive Research Grants Office to Maarten J. Chrispeels. Herman Höfte is a recipient of a Postdoctoral Fellow-ship from the European Molecular Biology Organization. Craig Dickinson is a recipient of a National Institutes of Health Postdoctoral Fellowship. Loîc Faye was on leave from the Centre National de la Recherche Scientifique, UA203, Université de Rouen, Mont Saint Aignan France and supported by a cooperative CNRS — National S cience Foundation grant. The mention of vendor or product in this paper does not imply that they are endorsed or recommended by the U.S. Department of Agriculture over vendors of similar products not mentioned. We thank Jürgen Denecke for providing plasmid pDE1001, Antje von Schaewen for the plant expression cassette and Marie-Theres Hauser for the modified vacuole preparation protocol.     

Functional characterization of the human immunodeficiency virus type 1 Nef acidic domain

The human immunodeficiency virus type 1 (HIV-1) accessory protein Nef downregulates major histocompatibility complex class I (MHC-I) from the cell surface. It has been proposed that the direct interaction of the acidic cluster (AC) of Nef, (62)EEEE(65), with the furin binding region (fbr) of PACS-1 is crucial for this Nef function. Contrary to this proposal, evidence is presented here that the four glutamates in Nef do not functionally engage the PACS-1 fbr. (i) The binding of Nef to the PACS-1 fbr in vitro is much weaker than the binding of the canonical furin AC to the PACS-1 fbr. (ii) The mutation of two of the four glutamates in Nef's AC to alanines does not alter Nef's ability to downregulate MHC-I, and triply mutated Nefs exhibit 50% activity. (iii) The introduction of lysine into the AC has little effect on Nef function. (iv) The mutation of all four glutamates to alanine does debilitate Nef MHC-I downregulation, but this quadruple mutation also impairs the ability of Nef to regulate p21-activated protein kinase and enhance viral particle infectivity. (v) The replacement of the Nef AC with the bona fide AC from furin results in the loss of the expected regulatory properties of the furin AC. (vi) The insertion of the conformation-disrupting amino acid proline into the Nef AC does not disrupt MHC-I downregulation. Our results are consistent with an alternative model in which (62)EEEE(65) plays a stabilizing role in the formation of a ternary complex between Nef, the MHC-I cytoplasmic domain, and AP-1.     

Nef enhances human immunodeficiency virus type 1 infectivity in the absence of matrix

Nef enhances the serine phosphorylation of the human immunodeficiency virus type 1 matrix (MA) protein, which suggests that MA may be a functional target of Nef. Using mutants that remain infectious despite the absence of most or all of MA, we show in the present study that the ability of Nef to enhance virus infectivity is not compromised even if MA is entirely replaced by a heterologous lipid anchor.     

Correct glycosylation,Golgi-processing,and targeting to protein bodies of the vacuolar protein phytohemagglutinin in transgenic tobacco

We used a heterologous system (transgenic Nicotiana tabacum L.) to investigate the processing, assembly and targeting of phytohemagglutinin (PHA), the lectin of the common bean, Phaseolus vulgaris L. In the bean, this glycoprotein accumulates in the protein bodies of the storage parenchyma cells in the cotyledons, and each polypeptide has a high-mannose glycan attached to Asn12 and a complex glycan on Asn60. The gene for PHA-L, dlec2, with 1200 basepairs (bp) 5 upstream and 1600 bp 3 downstream from the coding sequence was introduced into tobacco using Agrobacterium-mediated transformation (T. Voelker et al., 1987, EMBO J. 6, 3571–3577). Examination of thin sections of tobacco seeds by immunocytochemistry with antibodies against PHA showed that PHA-L accumulated in the amorphous matrix of the protein bodies in the embryo and endosperm. This localization was confirmed using a non-aqueous method to isolate the protein bodies from mature tobacco seeds. The biochemical analysis of tobacco PHA indicated that the signal peptide had been correctly removed, and that the polypeptides formed 6.4 S oligomers; tobacco PHA had a high-mannose glycan at Asn12 and a complex glycan at Asn60. The presence of the complex glycan shows that transport to the protein bodies was mediated by the Golgi complex. At seed maturity, a substantial portion of the PHA-L remained associated with the endoplasmic reticulum and the Golgi complex, as indicated by fractionation experiments using aqueous media and the presence of two high-mannose glycans on some of the polypeptides. Taken together, these data show that insertion of the nascent PHA into the endoplasmic reticulum, signal peptide processing, glycosylation, assembly into oligomers, glycan modification in the Golgi, and targeting of the protein occur faithfully in this heterologous system, although transport may not be as efficient as in bean cotyledons.Abbreviations Asn asparagine - Endo H endoglycosidase H - HPLC high-performance liquid chromatography - IgG immunoglobulin G - M_r relative molecular mass - PAGE polyacrylamide gel electrophoresis - PHA phytohemagglutinin - SDS sodium dodecylsulfate - TFMS trifluoromethanesulfonic acid     

Dynamic evolution of the human immunodeficiency virus type 1 pathogenic factor, Nef

The human immunodeficiency virus type 1 (HIV-1) early gene product Nef is a multifunctional protein that alters numerous pathways of T-cell function, including endocytosis, signal transduction, vesicular trafficking, and immune modulation, and is a major determinant of pathogenesis. Individual Nef functions include PAK-2 activation, CD4 downregulation, major histocompatibility complex (MHC) class I downregulation, and enhancement of viral particle infectivity. How Nef accomplishes its multiple tasks presents a difficult problem of mechanistic analysis because of the complications associated with multiple, overlapping functional domains in the context of significant sequence variability. To address these issues we determined the conservation of each Nef residue based on 1,643 subtype B Nef sequences. Mutational analysis based on conservative substitutions and Nef sequence data allowed us to search for amino acids on the surface of Nef that are specifically required for PAK-2 activation. We found residues 85, 89, and 191 to be highly significant determinants for Nef's PAK-2 activation function but functionally unlinked to CD4 and MHC class I downregulation or enhancement of infectivity. These residues are not conserved across HIV-1 subtypes but are confined to separate sets of surface elements within a subtype. Thus, L85/H89/F191 and F85/F89/R191 are dominant in subtype B and subtype E or C, respectively. Our results provide support for developing subtype-specific interventions in HIV-1 disease.