Conservation of M Group Sequences across HIV-1 Proteins

<p>The full lengths of each of the viral proteins containing 1st and/or 2nd tier CE are shown. The histogram indicates the tier of conservation for each segment of at least eight AAs in length, with tier number shown on the <i>y</i>-axis. 1st tier segments correspond to segments at least eight AAs long in which the AA at each site is found in more than 98% of HIVDB database sequences. 2nd tier include sites at which the most common AA is found in less than 98% of sequences in the database, but at which two AAs together make up more than 99% of the AAs found at that site in the database. For comparison, additional tiers of potential use for vaccine design are shown. 3rd tier expands the set to include two variable sites, and 4th tier includes <i>n</i> variable sites, each of which satisfy the criteria of having two AAs that together make up more than 99% of the AAs in the database. The 5th tier corresponds to peptides that have <i>n</i> variable sites that satisfy the criteria for 4th tier, but the requirement for conservation encompassed by the two AAs at each site is relaxed to more than 98%.</p

Characterization of a new 5' splice site within the caprine arthritis encephalitis virus genome: evidence for a novel auxiliary protein-4

(pKCR3), Rev (pKcRev), or Rtm (pKcRtm and pKRtm) plasmids, as indicated (B). Cells were radiolabeled 5 h with [S]-methionine 48 h after transfection. Lysates were subjected to immunoprecipitation and fractionated on an SDS-10% polyacrylamide gel. Immunoprecipitations were performed using either anti-CD™ antibodies, a serum from CAEV-infected goat (anti-CAEV), or anti-Rtm peptide antibodies (anti-Rtm). , Immunoprecipitation of the Rtm protein from infected cells. GSM cells were either mock infected (-) or infected with CAEV-Cork strain (+). When cytopathic effects appeared in infected cell culture, cells were radiolabeled 5 h with [S]-methionine, and lysates were immunoprecipitated with either anti-Rtm or anti-CD™ antibodies, as indicated. Immunoprecipitated proteins were resolved on a SDS-15% polyacrylamide gel.<p><b>Copyright information:</b></p><p>Taken from "Characterization of a new 5' splice site within the caprine arthritis encephalitis virus genome: evidence for a novel auxiliary protein"</p><p>http://www.retrovirology.com/content/5/1/22</p><p>Retrovirology 2008;5():22-22.</p><p>Published online 29 Feb 2008</p><p>PMCID:PMC2291067.</p><p></p

Characterization of a new 5' splice site within the caprine arthritis encephalitis virus genome: evidence for a novel auxiliary protein-5

D six times in the presence of different ionic strengths of KCl, as indicated, and the bound proteins were subjected to 15% SDS-PAGE analysis and autoradiography.<p><b>Copyright information:</b></p><p>Taken from "Characterization of a new 5' splice site within the caprine arthritis encephalitis virus genome: evidence for a novel auxiliary protein"</p><p>http://www.retrovirology.com/content/5/1/22</p><p>Retrovirology 2008;5():22-22.</p><p>Published online 29 Feb 2008</p><p>PMCID:PMC2291067.</p><p></p

Characterization of a new 5' splice site within the caprine arthritis encephalitis virus genome: evidence for a novel auxiliary protein-7

Tained the β-globin intron flanked by its splicing sequences inserted between the early promoter and poly-A site of SV40. CAEV sequences are included in open boxes. In all constructs, the β-globin SD site was replaced by CAEV sequences containing the SD(grey box) and SD(hatched box) sites. In plasmids pKRmB1 and pKRB1, the β-globin SA site was substituted by the 3' end viral genome containing the SAsite. The positions of the primers used for PCR amplification of cDNA are indicated (horizontal arrows). The positions of probes MarN2 and MarS used in southern blot analysis are indicated. The MarN PCR primer used in experiment reported in Fig. 4 is indicated. , RT-PCR analysis of RNAs extracted from transfected 293T cells. cDNAs were PCR amplified using primer pairs PK5 and PK3, or PK5 and M3b, as indicated. PCR products were resolved on an agarose gel and visualized by ethidium bromide staining. Lane M, DNA size markers. , Southern blot analysis of transcripts from cells transfected with pKRmB1 and pKRB1 plasmids. PCR-amplified cDNAs were fractionated through a 2.5% agarose gel, blotted to nylon, and hybridized to probes MarN2 (left panel) and MarS (right panel).<p><b>Copyright information:</b></p><p>Taken from "Characterization of a new 5' splice site within the caprine arthritis encephalitis virus genome: evidence for a novel auxiliary protein"</p><p>http://www.retrovirology.com/content/5/1/22</p><p>Retrovirology 2008;5():22-22.</p><p>Published online 29 Feb 2008</p><p>PMCID:PMC2291067.</p><p></p

Characterization of a new 5' splice site within the caprine arthritis encephalitis virus genome: evidence for a novel auxiliary protein-0

Tained the β-globin intron flanked by its splicing sequences inserted between the early promoter and poly-A site of SV40. CAEV sequences are included in open boxes. In all constructs, the β-globin SD site was replaced by CAEV sequences containing the SD(grey box) and SD(hatched box) sites. In plasmids pKRmB1 and pKRB1, the β-globin SA site was substituted by the 3' end viral genome containing the SAsite. The positions of the primers used for PCR amplification of cDNA are indicated (horizontal arrows). The positions of probes MarN2 and MarS used in southern blot analysis are indicated. The MarN PCR primer used in experiment reported in Fig. 4 is indicated. , RT-PCR analysis of RNAs extracted from transfected 293T cells. cDNAs were PCR amplified using primer pairs PK5 and PK3, or PK5 and M3b, as indicated. PCR products were resolved on an agarose gel and visualized by ethidium bromide staining. Lane M, DNA size markers. , Southern blot analysis of transcripts from cells transfected with pKRmB1 and pKRB1 plasmids. PCR-amplified cDNAs were fractionated through a 2.5% agarose gel, blotted to nylon, and hybridized to probes MarN2 (left panel) and MarS (right panel).<p><b>Copyright information:</b></p><p>Taken from "Characterization of a new 5' splice site within the caprine arthritis encephalitis virus genome: evidence for a novel auxiliary protein"</p><p>http://www.retrovirology.com/content/5/1/22</p><p>Retrovirology 2008;5():22-22.</p><p>Published online 29 Feb 2008</p><p>PMCID:PMC2291067.</p><p></p

Characterization of a new 5' splice site within the caprine arthritis encephalitis virus genome: evidence for a novel auxiliary protein-1

Nd ORFs are shown. Env precursor and Rev derived domains are represented by open and shaded boxes, respectively. , Schematic representation of Rev and Rtm expression constructs. Plasmids pKcRev and pKcRtm are predicted to express singly-spliced mRNAs encoding the Rev and Rtm proteins, respectively. The pKRtm expression vector contains the cDNA generated by RT-PCR from cells transfected with pKcRtm. The approximate positions of PCR primers are indicated (horizontal arrows). , Coding capacity of the ORF. Transfected 293T cells were radiolabeled 5 h with [S]-methionine 48 h after transfection, and protein extracts were subjected to immunoprecipitation analysis using rabbit affinity-purified antibodies raised against either the first 38 amino acids of Env precursor (anti-NHEnv), the 110-amino acid cytoplasmic domain of TM (anti-CD™), or the 98-amino acid carboxy terminus of Rev (anti-Rev). Immunoprecipitated proteins were resolved by electrophoresis through a SDS-15% polyacrylamide gel and visualized by autoradiography. , Analysis of translation products of cDNA. [S]-methionine labeled polypeptides were synthesized in an coupled transcription-translation reaction with pGEM-1 (lanes 1 and 2) or cDNA (lanes 3 and 4). Crude products (lanes 1 and 3) and proteins immunoprecipitated with affinity-purified anti-CD™ antibodies (lanes 2 and 4) were analyzed as described above.<p><b>Copyright information:</b></p><p>Taken from "Characterization of a new 5' splice site within the caprine arthritis encephalitis virus genome: evidence for a novel auxiliary protein"</p><p>http://www.retrovirology.com/content/5/1/22</p><p>Retrovirology 2008;5():22-22.</p><p>Published online 29 Feb 2008</p><p>PMCID:PMC2291067.</p><p></p

Fitness Costs of Mutations at the HIV-1 Capsid Hexamerization Interface

<div><p>The recently available x-ray crystal structure of HIV-1 capsid hexamers has provided insight into the molecular interactions crucial for the virus’s mature capsid formation. Amino acid changes at these interaction points are likely to have a strong impact on capsid functionality and, hence, viral infectivity and replication fitness. To test this hypothesis, we introduced the most frequently observed single amino acid substitution at 30 sites: 12 at the capsid hexamerization interface and 18 at non-interface sites. Mutations at the interface sites were more likely to be lethal (Fisher’s exact test p = 0.027) and had greater negative impact on viral replication fitness (Wilcoxon rank sum test p = 0.040). Among the interface mutations studied, those located in the cluster of hydrophobic contacts at NTD-NTD interface and those that disrupted NTD-CTD inter-domain helix capping hydrogen bonds were the most detrimental, indicating that these interactions are particularly important for maintaining capsid structure and/or function. These functionally constrained sites provide potential targets for novel HIV drug development and vaccine immunogen design.</p></div

Relationship between sequence conservation and replication fitness.

<p>Relative fitness of all mutants evaluated as a function of database frequency of the amino acid found in the prototype COTM-CA sequence. Values shown are an average from two experiments, done in triplicate. The replication fitness of non-viable viruse is plotted as zero.</p

Structural localization of interface mutations.

<p><b>A</b>) Two CA chains are shown as gray/black and green ribbons. The interface residues evaluated in this study are highlighted in bold and red-orange-yellow color. Other previously studied residues <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066065#pone.0066065-vonSchwedler1" target="_blank">[7]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066065#pone.0066065-Bartonova1" target="_blank">[8]</a> are highlighted in cyan-blue-purple color. The fitness impact of mutations is represented by the color shade ranging from small (yellow/cyan), moderate (orange/blue) to lethal (red/purple). <b>B</b>) Prototype residues participating in inter-domain helix capping hydrogen bonds, represented by orange lines, are shown on left. The mutations that resulted in loss of hydrogen bonds are modeled and highlighted on the right.</p