Novel isoforms of influenza virus PA-X and PB1-F2 indicated by automatic annotation.

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.

dCAS: a desktop application for cDNA sequence 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.

DAVID Knowledgebase: a gene-centered database integrating heterogeneous gene annotation resources to facilitate high-throughput gene functional analysis.

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/.

Specific interaction of CXCR4 with CD4 and CD8alpha: functional analysis of the CD4/CXCR4 interaction in the context of HIV-1 envelope glycoprotein-mediated membrane fusion.

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.

Mutual functional destruction of HIV-1 Vpu and host TASK-1 channel.

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.

Codon optimization of the HIV-1 vpu and vif genes stabilizes their mRNA and allows for highly efficient Rev-independent expression.

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 CRM I-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.

Viral protein U counteracts a human host cell restriction that inhibits HIV-1 particle production.

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 HIV-1 Vpu protein: a multifunctional enhancer of viral particle release.

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.

Naturally occurring amino acid substitutions in the HIV-2 ROD envelope glycoprotein regulate its ability to augment viral particle release.

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.

Expression, purification, and activities of full-length and truncated versions of the integral membrane protein Vpu from HIV-1.

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). Vpu(2-37) encompasses the N-terminal transmembrane alpha-helix; Vpu(2-51) spans the N-terminal transmembrane helix and the first cytoplasmic alpha-helix; Vpu(28-81) includes the entire cytoplasmic domain containing the two C-terminal amphipathic alpha-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 alpha-helix. The C-terminal alpha-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.