The influenza A virus genome contains 8 gene segments encoding 10 commonly recognized proteins. Additional protein products have been identified, including PB1-F2 and PA-X. We report the in-silico identification of novel isoforms of PB1-F2 and PA-X in influenza virus genomes sequenced from avian samples. The isoform observed in PA-X includes a mutated stop codon that should extend the protein product by 8 amino acids. The isoform observed in PB1-F2 includes two nonsense mutations that should truncate the N-terminal region of the protein product and remove the entire mitochondrial targeting domain. Both isoforms were uncovered during automatic annotation of CEIRS sequence data. Nominally termed PA-X8 and PB1-F2-Cterm, both predicted isoforms were subsequently found in other annotated influenza genomes previously deposited in GenBank. Both isoforms were noticed due to discrepant annotations output by two annotation engines, indicating a benefit of incorporating multiple algorithms during gene annotation.

MOTIVATION Understanding gene regulation and expression is the key to the advancement of biology. EST sequence assembly and analysis provide unique benefits in this regard. We have developed a standalone application, dCAS (Desktop cDNA Annotation System), which performs automated EST cleaning, clustering, assembly and annotation on a desktop computer. Compared with other available tools, dCAS provides a more convenient and user-friendly solution to biologists for extracting biological meaning from sequence data. AVAILABILITY The dCAS package is distributed freely. A cross-platform installer and associated sequence databases can be downloaded at: http://exon.niaid.nih.gov/applications.html.

BACKGROUND:Due to the complex and distributed nature of biological research, our current biological knowledge is spread over many redundant annotation databases maintained by many independent groups. Analysts usually need to visit many of these bioinformatics databases in order to integrate comprehensive annotation information for their genes, which becomes one of the bottlenecks, particularly for the analytic task associated with a large gene list. Thus, a highly centralized and ready-to-use gene-annotation knowledgebase is in demand for high throughput gene functional analysis.DESCRIPTION:The DAVID Knowledgebase is built around the DAVID Gene Concept, a single-linkage method to agglomerate tens of millions of gene/protein identifiers from a variety of public genomic resources into DAVID gene clusters. The grouping of such identifiers improves the cross-reference capability, particularly across NCBI and UniProt systems, enabling more than 40 publicly available functional annotation sources to be comprehensively integrated and centralized by the DAVID gene clusters. The simple, pair-wise, text format files which make up the DAVID Knowledgebase are freely downloadable for various data analysis uses. In addition, a well organized web interface allows users to query different types of heterogeneous annotations in a high-throughput manner.CONCLUSION:The DAVID Knowledgebase is designed to facilitate high throughput gene functional analysis. For a given gene list, it not only provides the quick accessibility to a wide range of heterogeneous annotation data in a centralized location, but also enriches the level of biological information for an individual gene. Moreover, the entire DAVID Knowledgebase is freely downloadable or searchable at http://david.abcc.ncifcrf.gov/knowledgebase/.

We investigated possible interactions between HIV-1 receptor (CD4) and the main coreceptors CXCR4 and CCR5. We found that CD4 and CXCR4 coexpressed in 293T cells form a complex that can be immunoprecipitated with antibodies directed against the extracellular domain of either protein. Mutagenesis revealed that the CD4/CXCR4 interaction maps to two previously uncharacterized basic motifs in the cytoplasmic domain of CD4. HIV-1 envelope glycoprotein-mediated membrane fusion was found to be independent of the ability of CD4 and CXCR4 to interact, whether fusion was studied in a virus-cell or a cell-cell model. However, this interaction might explain the adaptation of HIV-1 to CXCR4 as an alternative to CCR5. We found that CXCR4 also interacts with the cytoplasmic domain of CD8alpha in a way that is similar to the CD4/CXCR4 interaction. The CD4/CXCR4 and CD8alpha/CXCR4 interactions may thus be involved in cellular signaling pathways shared by the CD4 and CD8alpha molecules.

Two HIV-1 accessory proteins, Vpu and Vif, are notoriously difficult to express autonomously in the absence of the viral Tat and Rev proteins. We examined whether the codon bias observed in the vpu and vif genes relative to highly expressed human genes contributes to the Rev dependence and low expression level outside the context of the viral genome. The entire vpu gene as well as the 5′ half of the vif gene were codon optimized and the resulting open reading frames (ORFs) (vphu and hvif, respectively) were cloned in autonomous expression vectors under the transcriptional control of the CMV promoter. Codon optimization efficiently removed the expression block observed in the native genes and allowed high levels of Rev- and Tat-independent expression of Vpu and Vif. Most of the higher protein levels detected are accounted for by enhanced steady-state levels of the mRNA encoding the optimized species. Nuclear run-on experiments show for the first time that codon optimization has no effect on the rate of transcriptional initiation or elongation of the vphu mRNA. Instead, optimization of the vpu gene was found to stabilize the vphu mRNA in the nucleus and enhance its export to the cytoplasm. This was achieved by allowing the optimized mRNA to use a new CRM1-independent nuclear export pathway. This work provides a better understanding of the molecular mechanisms underlying the process of codon optimization and introduces novel tools to study the biological functions of the Vpu and Vif proteins independently of other viral proteins.

Sequence analysis predicted significant structural homology between the HIV-1 accessory protein Vpu and the N-terminal region of TASK-1, a mammalian background K(+) channel. If the homology resulted from molecular piracy during HIV-1 evolution, these two proteins may have important functional interactions. Here we demonstrate that TASK and Vpu physically interact in cultured cells and in AIDS lymphoid tissues. The functional consequences were potentially destructive for both components: Vpu abolished TASK-1 current, while overexpressing TASK led to a marked impairment of Vpu's ability to enhance viral particle release. Further, the first 40 amino acids of TASK-1 (part of the homology to Vpu) were capable of enhancing HIV-1 particle release. This virus-host interaction may influence HIV-1/AIDS progression, as well as electrical signaling in infected host tissues.

HIV accessory genes are expressed throughout the viral life cycle and regulate wide-ranging aspects of virus replication including viral infectivity (Vif and Nef), viral gene expression (Vpr) and progeny virion production (Vpu). While in many cases the molecular basis of accessory protein function is not fully understood, a consensus is emerging that these viral products are generally devoid of enzymatic activity and instead act as multifunctional adapters, subverting normal cellular processes to serve the needs of the virus. This review focuses on presenting our current knowledge of the HIV-1-specific Vpu protein and its essential role in regulating viral particle release, viral load and expression of the CD4 receptor.

Human cells resist viral infections by a variety of mechanisms. Viruses must overcome host cell restrictions to successfully reproduce their genetic material. Here, we identify a host restriction to viral replication that acts at the stage of particle assembly. Viral protein U (Vpu) is an HIV-1 accessory protein that enhances particle assembly and release in most human cells, but not in simian cells. By using human-simian cell heterokaryons, we show that the inhibition of assembly in human cells is dominant. Vpu overcomes the block to assembly in human cells and in human-simian heterokaryons. The HIV-1 vpu gene may have evolved to counteract an assembly restriction that is present in human cells.

The envelope glycoprotein of HIV-2 ROD10 has the intriguing ability to enhance the rate of viral particle release from infected cells. However, not all HIV-2 envelope glycoproteins are active in this regard. Indeed, we have previously noted that, despite a high degree of identity with that of ROD10, the envelope protein of the ROD14 isolate was unable to enhance virus production. In this study, site-directed mutagenesis was employed to reveal that a single naturally occurring alanine-to-threonine substitution at position 598, located in the extracellular part of the TM subunit, fully accounted for the lack of activity of the ROD14 Env in HeLa and 12D7 cells. A second mutation at position 422, substituting a lysine residue in ROD10 for an arginine in ROD14, was additionally required for efficient virus release from infected H9 cells, suggesting cell-type-specific requirements for this activity. Interestingly, the ROD14 Env protein exhibited a trans-dominant negative effect on particle release by ROD10 Env, suggesting that the viral release activity of the HIV-2 ROD envelope protein may be regulated by its ability to assemble into functional oligomeric structures.

Vpu is an 81‐residue accessory protein of HIV‐1. Because it is a membrane protein, it presents substantial technical challenges for the characterization of its structure and function, which are of considerable interest because the protein enhances the release of new virus particles from cells infected with HIV‐1 and induces the intracellular degradation of the CD4 receptor protein. The Vpu‐mediated enhancement of the virus release rate from HIV‐1‐infected cells is correlated with the expression of an ion channel activity associated with the transmembrane hydrophobic helical domain. Vpu‐induced CD4 degradation and, to a lesser extent, enhancement of particle release are both dependent on the phosphorylation of two highly conserved serine residues in the cytoplasmic domain of Vpu. To define the minimal folding units of Vpu and to identify their activities, we prepared three truncated forms of Vpu and compared their structural and functional properties to those of full‐length Vpu (residues 2–81). Vpu2–37 encompasses the N‐terminal transmembrane α‐helix; Vpu2–51 spans the N‐terminal transmembrane helix and the first cytoplasmic α‐helix; Vpu28–81 includes the entire cytoplasmic domain containing the two C‐terminal amphipathic α‐helices without the transmembrane helix. Uniformly isotopically labeled samples of the polypeptides derived from Vpu were prepared by expression of fusion proteins in E. coli and were studied in the model membrane environments of lipid micelles by solution NMR spectroscopy and oriented lipid bilayers by solid‐state NMR spectroscopy. The assignment of backbone resonances enabled the secondary structure of the constructs corresponding to the transmembrane and the cytoplasmic domains of Vpu to be defined in micelle samples by solution NMR spectroscopy. Solid‐state NMR spectra of the polypeptides in oriented lipid bilayers demonstrated that the topology of the domains is retained in the truncated polypeptides. The biological activities of the constructs of Vpu were evaluated. The ion channel activity is confined to the transmembrane α‐helix. The C‐terminal α‐helices modulate or promote the oligomerization of Vpu in the membrane and stabilize the conductive state of the channel, in addition to their involvement in CD4 degradation.

Human immunodeficiency virus (HIV) type 1 Vpu is an integral membrane protein with a unique affinity for (cid:3) TrCP (TrCP), a key member of the SkpI-Cullin-F-box E3 ubiquitin ligase complex that is involved in the regulated degradation of cellular proteins, including I (cid:2) B. Re-markably, Vpu is resistant to TrCP-mediated degradation and competitively inhibits TrCP-dependent degradation of I (cid:2) B, resulting in the suppression of nuclear factor (NF)- (cid:2) B activity in Vpu-expressing cells. We now report that Vpu, through its interaction with TrCP, potently contributes to the induction of apoptosis in HIV-infected T cells. Vpu-induced apoptosis is specific and independent of other viral proteins. Mutation of a TrCP-binding motif in Vpu abolishes its apoptogenic property, demonstrating a close correlation between this property of Vpu and its ability to inhibit NF- (cid:2) B activity. The involvement of NF- (cid:2) B in Vpu-induced apoptosis is further supported by the finding that the levels of antiapoptotic factors Bcl-xL, A1/ Bfl-1, and TNF receptor-associated factor (TRAF)1, all of which are expressed in an NF- (cid:2) B– dependent manner, are reduced and, at the same time, levels of active caspase-3 are elevated. Thus, Vpu induces apoptosis through activation of the caspase pathway by way of inhibiting the NF- (cid:2) B–dependent expression of antiapoptotic genes. a 30-min incubation at 4 (cid:10) C with a FITC-conjugated rabbit antiactive caspase-3 polyclonal antibody (BD PharMingen). Finally, the stained cells were fixed in 1% formaldehyde (in PBS) and analyzed for fluorescence intensity using a FACSort™ (Becton Dickinson). Western Blot Analysis. Western blot analyses were performed using the ECL detection system (Amersham Pharmacia Biotech) as described previously (16). The Abs used were as follows: anti– TNFR-associated factor (TRAF1) mAb (H-3; Santa Cruz Bio-technology, Inc.); anticaspase-8 polyclonal antibody (BD Phar-Mingen); anti–Bcl-xL mAb (2H12; BD PharMingen); anti-A1/ Bfl-1 polyclonal antibody (Santa Cruz Biotechnology, Inc.); anti– (cid:4) -tubulin mAb (DM 1A; Sigma-Aldrich); anti-Vpu polyclonal antibody (U2–3; reference 23); and anti-p24 capsid mAb (pro-vided by S. Zolla-Pazner, AIDS Research and Reference Re-agent Program, Division of AIDS, NIAID, NIH; reference 24).

ABSTRACT The human immunodeficiency virus type 1 (HIV-1) Vif protein plays a critical role in the production of infectious virions. Previous studies have demonstrated the presence of small amounts of Vif in virus particles. However, Vif packaging was assumed to be nonspecific, and its functional significance has been questioned. We now report that packaging of Vif is dependent on the packaging of viral genomic RNA in both permissive and restrictive HIV-1 target cells. Mutations in the nucleocapsid zinc finger domains that abrogate packaging of viral genomic RNA abolished packaging of Vif. Additionally, an RNA packaging-defective virus exhibited significantly reduced packaging of Vif. Finally, deletion of a putative RNA-interacting domain in Vif abolished packaging of Vif into virions. Virion-associated Vif was resistant to detergent extraction and copurified with components of the viral nucleoprotein complex and functional reverse transcription complexes. Thus, Vif is specifically packaged into virions as a component of the viral nucleoprotein complex. Our data suggest that the specific association of Vif with the viral nucleoprotein complex might be functionally significant and could be a critical requirement for infectivity of viruses produced from restrictive host cells.

Human immunodeficiency virus (HIV) type 1 Vpu is an integral membrane protein with a unique affinity for beta TrCP (TrCP), a key member of the SkpI-Cullin-F-box E3 ubiquitin ligase complex that is involved in the regulated degradation of cellular proteins, including I kappaB. Remarkably, Vpu is resistant to TrCP-mediated degradation and competitively inhibits TrCP-dependent degradation of I kappaB, resulting in the suppression of nuclear factor (NF)-kappaB activity in Vpu-expressing cells. We now report that Vpu, through its interaction with TrCP, potently contributes to the induction of apoptosis in HIV-infected T cells. Vpu-induced apoptosis is specific and independent of other viral proteins. Mutation of a TrCP-binding motif in Vpu abolishes its apoptogenic property, demonstrating a close correlation between this property of Vpu and its ability to inhibit NF-kappaB activity. The involvement of NF-kappaB in Vpu-induced apoptosis is further supported by the finding that the levels of antiapoptotic factors Bcl-xL, A1/Bfl-1, and TNF receptor-associated factor (TRAF)1, all of which are expressed in an NF-kappaB-dependent manner, are reduced and, at the same time, levels of active caspase-3 are elevated. Thus, Vpu induces apoptosis through activation of the caspase pathway by way of inhibiting the NF-kappaB-dependent expression of antiapoptotic genes.

The human immunodeficiency virus type 1 (HIV-1) Vpu protein binds to the CD4 receptor and induces its degradation by cytosolic proteasomes. This process involves the recruitment of human betaTrCP (TrCP), a key member of the SkpI-Cdc53-F-box E3 ubiquitin ligase complex that specifically interacts with phosphorylated Vpu molecules. Interestingly, Vpu itself, unlike other TrCP-interacting proteins, is not targeted for degradation by proteasomes. We now report that, by virtue of its affinity for TrCP and resistance to degradation, Vpu, but not a phosphorylation mutant unable to interact with TrCP, has a dominant negative effect on TrCP function. As a consequence, expression of Vpu in HIV-infected T cells or in HeLa cells inhibited TNF-alpha-induced degradation of IkappaB-alpha. Vpu did not inhibit TNF-alpha-mediated activation of the IkappaB kinase but instead interfered with the subsequent TrCP-dependent degradation of phosphorylated IkappaB-alpha. This resulted in a pronounced reduction of NF-kappaB activity. We also observed that in cells producing Vpu-defective virus, NF-kappaB activity was significantly increased even in the absence of cytokine stimulation. However, in the presence of Vpu, this HIV-mediated NF-kappaB activation was markedly reduced. These results suggest that Vpu modulates both virus- and cytokine-induced activation of NF-kappaB in HIV-1-infected cells.

One of the hallmarks of human immunodeficiency virus type I (HIV-1) infection is the rapid removal of the viral receptor CD4 from the cell surface. This remarkably efficient receptor interference requires the activity of three separate viral proteins: Env, Vpu, and Nef. We have investigated whether this unusually tight interference on cell surface CD4 expression had a more essential function during the viral life cycle than simply preventing superinfection. We now report that the removal of cell surface CD4 is required for optimal virus production by HIV-1. Indeed, maintenance of CD4 surface expression in infected cells lead to a 3–5-fold decrease in viral particle production. This effect was not due to the formation of intracellular complexes between CD4 and the gp160 viral envelope precursor but instead required the presence of CD4 at the cell surface and was specifically mediated by CD4 but not closely related plasma membrane receptors. The finding that CD4 had no significant effect on particle release by a Vpu-deficient variant indicates that CD4 acts by inhibiting the particle release-promoting activity of Vpu. Co-immunoprecipitation experiments further showed that CD4 and Vpu physically interact at the cell surface, suggesting that CD4 might inhibit Vpu activity by disrupting its oligomeric structure.

The human immunodeficiency virus type 1 (HIV-1) Vpu and Env proteins are expressed from a bicistronic mRNA. To address the biological significance of the coordinated expression of vpu and env, we compared the relative effects on particle release of HIV-1 isolates containing an intact vpu gene or carrying point mutations in its initiation codon or internal deletions, respectively. We found that the primary AD8 isolate, which is unable to express vpu due to a mutation in its translation initiation codon, was able to replicate in primary macrophages and peripheral blood mononuclear cells with efficiency similar to that of an isogenic variant expressing Vpu. Interestingly, AD8 lacking a vpu initiation codon produced higher levels of Env protein than its Vpu-expressing isogenic variant. In contrast, disabling Vpu without removing the vpu initiation codon did not alter Env expression but significantly reduced virus production. AD8 Env when provided in trans was capable of enhancing release not only of AD8 particles but also of viruses of the T-cell-tropic NL4-3 isolate. We conclude that AD8 Env encodes a Vpu-like activity similar to that previously reported for HIV-2 Env proteins and is thus able to augment virus secretion. When expressed at elevated levels, i.e., following mutation of the vpu initiation codon, AD8 Env was able to compensate for the lack of Vpu and thereby ensure efficient virus release. Thus, the ability to regulate virus release is redundant in AD8 and can be controlled by either Vpu or Env. Since Vpu controls several independent functions, including CD4 degradation, our results suggest that some HIV-1 isolates may have evolved a mechanism to regulate Vpu activity without compromising their ability to efficiently replicate in the host cells.

In addition to its role in receptor binding, the envelope glycoprotein of certain human immunodeficiency virus type 2 (HIV-2) isolates, including ROD10, exhibits a biological activity that enhances the release of HIV-2, HIV-1, and simian immunodeficiency virus particles from infected cells. The present study aims at better defining the functional domains involved in this biological activity. To this end, we have characterized the envelope protein of the ROD14 isolate of HIV-2, which, despite 95% homology with the ROD10 envelope at the amino acid level, is unable to enhance viral particle release. Site-directed mutagenesis showed that the presence of a truncation in the cytoplasmic tail of the ROD14 envelope was not responsible for the lack of activity, as previously reported for the HIV-2 ST isolate (G, D, Ritter, Jr,, G, Yamshchikov, S, J, Cohen, and M, J, Mulligan, J, Virol, 70:2669-2673, 1996), Similarly, several modifications of the length of the ROD10 envelope cytoplasmic tail did not impair its ability to enhance particle release, suggesting that, in the case of the HIV-2 ROD isolate, particle release activity is not regulated by the length of the cytoplasmic tail.

ABSTRACT The human immunodeficiency virus type 1 (HIV-1) vpugene encodes a type I anchored integral membrane phosphoprotein with two independent functions. First, it regulates virus release from a post-endoplasmic reticulum (ER) compartment by an ion channel activity mediated by its transmembrane anchor. Second, it induces the selective down regulation of host cell receptor proteins (CD4 and major histocompatibility complex class I molecules) in a process involving its phosphorylated cytoplasmic tail. In the present work, we show that the Vpu-induced proteolysis of nascent CD4 can be completely blocked by peptide aldehydes that act as competitive inhibitors of proteasome function and also by lactacystin, which blocks proteasome activity by covalently binding to the catalytic β subunits of proteasomes. The sensitivity of Vpu-induced CD4 degradation to proteasome inhibitors paralleled the inhibition of proteasome degradation of a model ubiquitinated substrate. Characterization of CD4-associated oligosaccharides indicated that CD4 rescued from Vpu-induced degradation by proteasome inhibitors is exported from the ER to the Golgi complex. This finding suggests that retranslocation of CD4 from the ER to the cytosol may be coupled to its proteasomal degradation. CD4 degradation mediated by Vpu does not require the ER chaperone calnexin and is dependent on an intact ubiquitin-conjugating system. This was demonstrated by inhibition of CD4 degradation (i) in cells expressing a thermally inactivated form of the ubiquitin-activating enzyme E1 or (ii) following expression of a mutant form of ubiquitin (Lys48 mutated to Arg48) known to compromise ubiquitin targeting by interfering with the formation of polyubiquitin complexes. CD4 degradation was also prevented by altering the four Lys residues in its cytosolic domain to Arg, suggesting a role for ubiquitination of one or more of these residues in the process of degradation. The results clearly demonstrate a role for the cytosolic ubiquitin-proteasome pathway in the process of Vpu-induced CD4 degradation. In contrast to other viral proteins (human cytomegalovirus US2 and US11), however, whose translocation of host ER molecules into the cytosol occurs in the presence of proteasome inhibitors, Vpu-targeted CD4 remains in the ER in a transport-competent form when proteasome activity is blocked.