HBV polymerase-derived peptide exerts an anti-HIV-1 effect by inhibiting the acetylation of viral integrase.

Here, we found that a 6-mer peptide, Poly6, derived from the hepatitis B virus (HBV), which overlaps with a polymerase corresponding to a preS1 deletion reported to contribute to liver disease progression, can elicit an antiviral effect against human immunodeficiency virus (HIV)-1 by inhibiting HIV-1 integrase (IN) activity of infected cells. Mechanistic studies revealed that the anti-HIV-1 effects of Poly6 occurred via the inhibition of integration, which resulted from the inhibition of acetylation of HIV-1 IN possibly by downregulation of p300 histone acetyltransferase. Our data suggest the potential therapeutic use of a 6-mer HBV-derived peptide, Poly6, as an anti-HIV-1 agent to suppress HIV-1 infection via inhibiting integrase activity.

Mapping of Functional Subdomains in the Terminal Protein Domain of Hepatitis B Virus Polymerase.

Hepatitis B virus (HBV) encodes a multifunction reverse transcriptase or polymerase (P), which is composed of several domains. The terminal protein (TP) domain is unique to HBV and related hepadnaviruses and is required for specifically binding to the viral pregenomic RNA (pgRNA). Subsequently, the TP domain is necessary for pgRNA packaging into viral nucleocapsids and the initiation of viral reverse transcription for conversion of the pgRNA to viral DNA. Uniquely, the HBV P protein initiates reverse transcription via a protein priming mechanism using the TP domain as a primer. No structural homologs or high-resolution structure exists for the TP domain. Secondary structure prediction identified three disordered loops in TP with highly conserved sequences. A meta-analysis of mutagenesis studies indicated these predicted loops are almost exclusively where functionally important residues are located. Newly constructed TP mutations revealed a priming loop in TP which plays a specific role in protein-primed DNA synthesis beyond simply harboring the site of priming. Substitutions of potential sites of phosphorylation surrounding the priming site demonstrated that these residues are involved in interactions critical for priming but are unlikely to be phosphorylated during viral replication. Furthermore, the first 13 and 66 TP residues were shown to be dispensable for protein priming and pgRNA binding, respectively. Combining current and previous mutagenesis work with sequence analysis has increased our understanding of TP structure and functions by mapping specific functions to distinct predicted secondary structures and will facilitate antiviral targeting of this unique domain. IMPORTANCE: HBV is a major cause of viral hepatitis, liver cirrhosis, and hepatocellular carcinoma. One important feature of this virus is its polymerase, the enzyme used to create the DNA genome from a specific viral RNA by reverse transcription. One region of this polymerase, the TP domain, is required for association with the viral RNA and production of the DNA genome. Targeting the TP domain for antiviral development is difficult due to the lack of homology to other proteins and high-resolution structure. This study mapped the TP functions according to predicted secondary structure, where it folds into alpha helices or unstructured loops. Three predicted loops were found to be the most important regions functionally and the most conserved evolutionarily. Identification of these functional subdomains in TP will facilitate its targeting for antiviral development.

Large-scale production and structural and biophysical characterizations of the human hepatitis B virus polymerase.

UNLABELLED: Hepatitis B virus (HBV) is a major human pathogen that causes serious liver disease and 600,000 deaths annually. Approved therapies for treating chronic HBV infections usually target the multifunctional viral polymerase (hPOL). Unfortunately, these therapies--broad-spectrum antivirals--are not general cures, have side effects, and cause viral resistance. While hPOL remains an attractive therapeutic target, it is notoriously difficult to express and purify in a soluble form at yields appropriate for structural studies. Thus, no empirical structural data exist for hPOL, and this impedes medicinal chemistry and rational lead discovery efforts targeting HBV. Here, we present an efficient strategy to overexpress recombinant hPOL domains in Escherichia coli, purifying them at high yield and solving their known aggregation tendencies. This allowed us to perform the first structural and biophysical characterizations of hPOL domains. Apo-hPOL domains adopt mainly α-helical structures with small amounts of ß-sheet structures. Our recombinant material exhibited metal-dependent, reverse transcriptase activity in vitro, with metal binding modulating the hPOL structure. Calcomine orange 2RS, a small molecule that inhibits duck HBV POL activity, also inhibited the in vitro priming activity of recombinant hPOL. Our work paves the way for structural and biophysical characterizations of hPOL and should facilitate high-throughput lead discovery for HBV. IMPORTANCE: The viral polymerase from human hepatitis B virus (hPOL) is a well-validated therapeutic target. However, recombinant hPOL has a well-deserved reputation for being extremely difficult to express in a soluble, active form in yields appropriate to the structural studies that usually play an important role in drug discovery programs. This has hindered the development of much-needed new antivirals for HBV. However, we have solved this problem and report here procedures for expressing recombinant hPOL domains in Escherichia coli and also methods for purifying them in soluble forms that have activity in vitro. We also present the first structural and biophysical characterizations of hPOL. Our work paves the way for new insights into hPOL structure and function, which should assist the discovery of novel antivirals for HBV.

Identification and classification of feline endogenous retroviruses in the cat genome using degenerate PCR and in silico data analysis.

The purpose of this study was to identify and classify endogenous retroviruses (ERVs) in the cat genome. Pooled DNA from five domestic cats was subjected to degenerate PCR with primers specific to the conserved retroviral pro/pol region. The 59 amplified retroviral sequences were used for in silico analysis of the cat genome (Felis_catus-6.2). We identified 219 ERV γ and ß elements from cat genome contigs, which were classified into 42 ERV γ and 4 ß families and further analysed. Among them, 99 γ and 5 ß ERV elements contained the complete retroviral structure. Furthermore, we identified 757 spuma-like ERV elements based on the sequence homology to murine (Mu)ERV-L and human (H)ERV-L. To the best of our knowledge, this is the first detailed genome-scale analysis examining Felis catus endogenous retroviruses (FcERV) and providing advanced insights into their structural characteristics, localization in the genome, and diversity.

Carbonyl J acid derivatives block protein priming of hepadnaviral P protein and DNA-dependent DNA synthesis activity of hepadnaviral nucleocapsids.

Current treatments for chronic hepatitis B are effective in only a fraction of patients. All approved directly antiviral agents are nucleos(t)ide analogs (NAs) that target the DNA polymerase activity of the hepatitis B virus (HBV) P protein; resistance and cross-resistance may limit their long-term applicability. P protein is an unusual reverse transcriptase that initiates reverse transcription by protein priming, by which a Tyr residue in the unique terminal protein domain acts as an acceptor of the first DNA nucleotide. Priming requires P protein binding to the ε stem-loop on the pregenomic RNA (pgRNA) template. This interaction also mediates pgRNA encapsidation and thus provides a particularly attractive target for intervention. Exploiting in vitro priming systems available for duck HBV (DHBV) but not HBV, we demonstrate that naphthylureas of the carbonyl J acid family, in particular KM-1, potently suppress protein priming by targeting P protein and interfering with the formation of P-DHBV ε initiation complexes. Quantitative evaluation revealed a significant increase in complex stability during maturation, yet even primed complexes remained sensitive to KM-1 concentrations below 10 µM. Furthermore, KM-1 inhibited the DNA-dependent DNA polymerase activity of both DHBV and HBV nucleocapsids, including from a lamivudine-resistant variant, directly demonstrating the sensitivity of human HBV to the compound. Activity against viral replication in cells was low, likely due to low intracellular availability. KM-1 is thus not yet a drug candidate, but its distinct mechanism of action suggests that it is a highly useful lead for developing improved, therapeutically applicable derivatives.

PAComplex: a web server to infer peptide antigen families and binding models from TCR-pMHC complexes.

One of the most adaptive immune responses is triggered by specific T-cell receptors (TCR) binding to peptide-major histocompatibility complexes (pMHC). Despite the availability of many prediction servers to identify peptides binding to MHC, these servers are often lacking in peptide-TCR interactions and detailed atomic interacting models. PAComplex is the first web server investigating both pMHC and peptide-TCR interfaces to infer peptide antigens and homologous peptide antigens of a query. This server first identifies significantly similar TCR-pMHC templates (joint Z-value ≥ 4.0) of the query by using antibody-antigen and protein-protein interacting scoring matrices for peptide-TCR and pMHC interfaces, respectively. PAComplex then identifies the homologous peptide antigens of these hit templates from complete pathogen genome databases (≥10(8) peptide candidates from 864,628 protein sequences of 389 pathogens) and experimental peptide databases (80,057 peptides in 2287 species). Finally, the server outputs peptide antigens and homologous peptide antigens of the query and displays detailed interacting models (e.g. hydrogen bonds and steric interactions in two interfaces) of hitTCR-pMHC templates. Experimental results demonstrate that the proposed server can achieve high prediction accuracy and offer potential peptide antigens across pathogens. We believe that the server is able to provide valuable insights for the peptide vaccine and MHC restriction. The PAComplex sever is available at http://PAcomplex.life.nctu.edu.tw.

Ultra-deep pyrosequencing analysis of the hepatitis B virus preCore region and main catalytic motif of the viral polymerase in the same viral genome.

Hepatitis B virus (HBV) pregenomic RNA contains a hairpin structure (ε) located in the preCore region, essential for viral replication. ε stability is enhanced by the presence of preCore variants and ε is recognized by the HBV polymerase (Pol). Mutations in the retrotranscriptase domain (YMDD) of Pol are associated with treatment resistance. The aim of this study was to analyze the preCore region and YMDD motif by ultra-deep pyrosequencing (UDPS). To evaluate the UDPS error rate, an internal control sequence was inserted in the amplicon. A newly developed technique enabled simultaneous analysis of the preCore region and Pol in the same viral genome, as well as the conserved sequence of the internal control. Nucleotide errors in HindIII yielded a UDPS error rate <0.05%. UDPS study confirmed the possibility of simultaneous detection of preCore and YMDD mutations, and demonstrated the complexity of the HBV quasispecies and cooperation between viruses. Thermodynamic stability of the ε signal was found to be the main constraint for selecting main preCore mutations. Analysis of ε-signal variability suggested the essential nature of the ε structural motif and that certain nucleotides may be involved in ε signal functions.

Foamy retrovirus integrase contains a Pol dimerization domain required for protease activation.

Unlike orthoretroviruses, foamy retroviruses (FV) synthesize Pol independently of Gag. The FV Pol precursor is cleaved only once between reverse transcriptase (RT) and integrase (IN) by the protease (PR), resulting in a PR-RT and an IN protein. Only the Pol precursor, not the cleaved subunits, is packaged into virions. Like orthoretroviral PRs, FV PR needs to dimerize to be active. Previously, we showed that a Pol mutant lacking IN has defects in PR activity and Pol packaging into virions. We now show that introduction of a leucine zipper (zip) dimerization motif in an IN truncation mutant can restore PR activity, leading to Pol processing in cells. However, these zip mutants neither cleave Gag nor incorporate Pol into virions. We propose that IN is required for Pol dimerization, which is necessary for the creation of a functional PR active site.

Mutational characterization of HBV reverse transcriptase gene and the genotype-phenotype correlation of antiviral resistance among Chinese chronic hepatitis B patients.

Background and Aims: The drug resistance of hepatitis B virus (HBV) originates from mutations within HBV reverse transcriptase (RT) region during the prolonged antiviral therapy. So far, the characteristics of how these mutations distribute and evolve in the process of therapy have not been clarified yet. Thus we aimed to investigate these characteristics and discuss their contributing factors. Methods: HBV RT region was direct-sequenced in 285 treatment-naive and 214 post-treatment patients. Mutational frequency and Shannon entropy were calculated to identify the specific mutations differing between genotypes or treatment status. A typical putative resistance mutation rtL229V was further studied using in-vitro susceptibility assays and molecular modeling. Results: The classical resistance mutations were rarely detected among treatment-naive individuals, while the putative resistance mutations were observed at 8 AA sites. rtV191I and rtA181T/V were the only resistance mutations identified as genotype-specific mutation. Selective pressure of drug usage not only contributed to the classical resistance mutations, but also induced the changes at a putative resistance mutation site rt229. rtL229V was the major substitution at the site of rt229. It contributed to the most potent suppression of viral replication and reduced the in-vitro drug susceptibility to entecavir (ETV) when coexisting with rtM204V, consistent with the hypothesis based on the molecular modeling and clinical data analysis. Conclusions: The analysis of mutations in RT region under the different circumstances of genotypes and therapy status might pave the way for a better understanding of resistance evolution, thus providing the basis for a rational administration of antiviral therapy.

Hepatitis B viruses: reverse transcription a different way.

Hepatitis B virus (HBV), the causative agent of B-type hepatitis in humans, is the type member of the Hepadnaviridae, hepatotropic DNA viruses that replicate via reverse transcription. Beyond long-established differences to retroviruses in gene expression and overall replication strategy newer work has uncovered additional distinctions in the mechanism of reverse transcription per se. These include protein-priming by the unique extra terminal protein domain of the reverse transcriptase (RT) utilizing an RNA hairpin for de novo initiation of first strand DNA synthesis, and the strict dependence of this process on cellular chaperones. Recent in vitro reconstitution systems enabled first biochemical insights into this multifactorial reaction, complemented by high resolution structural information on the RNA, though not yet the protein, level. Genetic approaches have revealed long-distance interactions in the nucleic acid templates as an important factor enabling the puzzling template switches required to produce the relaxed circular (RC) DNA found in infectious virions. Finally, the failure of even potent HBV RT inhibitors to eliminate nuclear covalently closed circular (ccc) DNA, the functional equivalent of integrated proviral DNA, has spurred a renewed interest in the mechanism of cccDNA generation. These new developments are in the focus of this review.

Analysis of HIV-1 pol sequences using Bayesian Networks: implications for drug resistance.

Human Immunodeficiency Virus-1 (HIV-1) antiviral resistance is a major cause of antiviral therapy failure and compromises future treatment options. As a consequence, resistance testing is the standard of care. Because of the high degree of HIV-1 natural variation and complex interactions, the role of resistance mutations is in many cases insufficiently understood. We applied a probabilistic model, Bayesian networks, to analyze direct influences between protein residues and exposure to treatment in clinical HIV-1 protease sequences from diverse subtypes. We can determine the specific role of many resistance mutations against the protease inhibitor nelfinavir, and determine relationships between resistance mutations and polymorphisms. We can show for example that in addition to the well-known major mutations 90M and 30N for nelfinavir resistance, 88S should not be treated as 88D but instead considered as a major mutation and explain the subtype-dependent prevalence of the 30N resistance pathway.

HIV type 1 vaccines for worldwide use: predicting in-clade and cross-clade breadth of immune responses.

One of the greatest challenges in HIV vaccine development is accommodating the worldwide sequence diversity of the HIV-1 virus. To understand how viral sequence diversity may affect the potential breadth of HIV-1 vaccines designed to elicit antiviral T cell immunity, we have developed novel approaches to assess sequence conservation at the amino acid level, where vaccine effects are exerted. Taking each sequence from the LANL 2004 amino acid alignments as a potential vaccine or as a challenge virus, all pairwise combinations of sequences were evaluated by two methods: first, a traditional comparison of aligned sequences, and second, by a new walking 9-mer algorithm chosen to emphasize the typical length of an MHC-I epitope. The rules for comparing mismatched 9-mer pairs between vaccine and challenge sequences were empirically deduced from an experiment on Nef-specific CD8 epitopes and the viral sequences from naturally HIV-1-infected patients. Results were weighted such that each clade contributed in proportion to its global prevalence. Cross-clade breadth of response is best maintained for vaccines encoding Pol and Gag, while commonly proposed Env- and Tat-based vaccines would be more clade sensitive. We evaluated the additional breadth that could be expected from multiclade vaccines including consensus and ancestral sequences. For more diverse proteins, adding a second strain can add a significant increase in breadth, although for three or more strains the intrinsic diversity of the protein leads to diminishing improvement.

Molecular and phylogenetic analyses of a new amphotropic murine leukemia virus (MuLV-1313).

BACKGROUND: The amphotropic murine leukemia viruses (MuLV-A's) are naturally occurring, exogenously acquired gammaretroviruses that are indigenous to the Southern California wild mice. These viruses replicate in a wide range of cell types including human cells in vitro and they can cause both hematological and neurological disorders in feral as well as in the inbred laboratory mice. Since MuLV-A's also exhibit discrete interference and neutralization properties, the envelope proteins of these viruses have been extremely useful for studying virus-host cell interactions and as vehicles for transfer of foreign genes into a variety of hosts including human cells. However, the genomic structure of any of the several known MuLV-A's has not been established and the evolutionary relationship of amphotropic retroviruses to the numerous exogenous or endogenous MuLV strains remains elusive. Herein we present a complete genetic structure of a novel amphotropic virus designated MuLV-1313 and demonstrate that this retrovirus together with other MuLV-A's belongs to a distinct molecular, biological and phylogenetic class among the MuLV strains isolated from a large number of the laboratory inbred or feral mice. RESULTS: The host range of MuLV-1313 is similar to the previously isolated MuLV-A's except that this virus replicates efficiently in mammalian as well as in chicken cells. Compared to ENV proteins of other MuLV-A's (4070A, 1504A and 10A-1), the gp70 protein of MuLV-1313 exhibits differences in its signal peptides and the proline-rich hinge regions. However, the MuLV-1313 envelope protein is totally unrelated to those present in a broad range of murine retroviruses that have been isolated from various inbred and feral mice globally. Genetic analysis of the entire MuLV-1313 genome by dot plot analyses, which compares each nucleotide of one genome with the corresponding nucleotide of another, revealed that the genome of this virus, with the exception of the env gene, is more closely related to the biologically distinct wild mouse ecotropic retrovirus (Cas-Br-E) isolated from another region of the Southern California, than to any of the 15 MuLV strains whose full-length sequences are present in the GenBank. This finding was corroborated by phylogenetic analyses and hierarchical clustering of the entire genomic sequence of MuLV-1313, which also placed all MULV-A's in a genetically distinct category among the large family of retroviruses isolated from numerous mouse strains globally. Likewise, construction of separate dendrograms for each of the Gag, Pol and Env proteins of MuLV-1313 demonstrated that the amphotropic retroviruses belong to a phylogenetically exclusive group of gammaretroviruses compared to all known MuLV strains. CONCLUSION: The molecular, biological and phylogenetic properties of amphotropic retroviruses including MuLV-1313 are distinct compared to a large family of exogenously- or endogenously-transmitted ecotropic, polytropic and xenotropic MuLV strains of the laboratory and feral mice. Further, both the naturally occurring amphotropic and a biologically discrete ecotropic retrovirus of the Southern California wild mice are more closely related to each other on the evolutionary tree than any other mammalian gammaretrovirus indicating a common origin of these viruses. This is the first report of a complete genomic analysis of a unique group of phylogenetically distinct amphotropic virus.

Different Variants in Reverse Transcriptase Domain Determined by Ultra-deep Sequencing in Treatment-naïve and Treated Indonesian Patients Infected with Hepatitis B Virus.

A nucleos(t)ide analog (NA) is the common antiviral drug available for directly treating hepatitis B virus (HBV) infection. However, its application has led to the emergence of NA-resistant mutations mostly in a conserved region of the reverse transcriptase domain of HBV polymerase. Harboring NA-resistant mutations decreases drug effectiveness and increases the frequency of end-stage liver disease. The invention of next-generation sequencing that can generate thousands of sequences from viral complex mixtures provides opportunities to detect minor changes and early viral evolution under drug stress. The present study used ultra-deep sequencing to evaluate discrepant quasispecies in the reverse transcriptase domain of HBV including NA-resistant hotspots between seven treatment-naïve Indonesian patients infected with HBV and five at the early phase of treatment. The most common sub-genotype was HBV B3 (83.34%). The substitution rate of variants determined among amino acids with a ratio of ≥ 1% changes was higher among the population in conserved regions (23.19% vs. 4.59%, P = 0.001) and in the inter-reverse transcriptase domain (23.95% vs. 2.94%, P = 0.002) in treatment naïve, than in treated patients. Nine hotspots of antiviral resistance were identified in both groups, and the mean frequency of changes in all patients was < 1%. The known rtM204I mutation was the most frequent in both groups. The lower rate of variants in HBV quasispecies in patients undergoing treatment could be associated with virus elimination and the extinction of sensitive species by NA therapy. The present findings imply that HBV quasispecies dynamically change during treatment.

Use of endogenous retroviral sequences (ERVs) and structural markers for retroviral phylogenetic inference and taxonomy.

BACKGROUND: Endogenous retroviral sequences (ERVs) are integral parts of most eukaryotic genomes and vastly outnumber exogenous retroviruses (XRVs). ERVs with a relatively complete structure were retrieved from the genetic archives of humans and chickens, diametrically opposite representatives of vertebrate retroviruses (over 3300 proviruses), and analyzed, using a bioinformatic program, RetroTector, developed by us. This rich source of proviral information, accumulated in a local database, and a collection of XRV sequences from the literature, allowed the reconstruction of a Pol based phylogenetic tree, more extensive than previously possible. The aim was to find traits useful for classification and evolutionary studies of retroviruses. Some of these traits have been used by others, but they are here tested in a wider context than before. RESULTS: In the ERV collection we found sequences similar to the XRV-based genera: alpha-, beta-, gamma-, epsilon- and spumaretroviruses. However, the occurrence of intermediates between them indicated an evolutionary continuum and suggested that taxonomic changes eventually will be necessary. No delta or lentivirus representatives were found among ERVs. Classification based on Pol similarity is congruent with a number of structural traits. Acquisition of dUTPase occurred three times in retroviral evolution. Loss of one or two NC zinc fingers appears to have occurred several times during evolution. Nucleotide biases have been described earlier for lenti-, delta- and betaretroviruses and were here confirmed in a larger context. CONCLUSION: Pol similarities and other structural traits contribute to a better understanding of retroviral phylogeny. "Global" genomic properties useful in phylogenies are i.) translational strategy, ii.) number of Gag NC zinc finger motifs, iii.) presence of Pro N-terminal dUTPase (dUTPasePro), iv.) presence of Pro C-terminal G-patch and v.) presence of a GPY/F motif in the Pol integrase (IN) C-terminal domain. "Local" retroviral genomic properties useful for delineation of lower level taxa are i.) host species range, ii.) nucleotide compositional bias and iii.) LTR lengths.

Multiple sclerosis and hepatitis B vaccination: could minute contamination of the vaccine by partial hepatitis B virus polymerase play a role through molecular mimicry?

Reports of multiple sclerosis developing after hepatitis B vaccination have led to the concern that this vaccine might be a cause of multiple sclerosis in previously healthy subjects. Some articles evidenced that minor Hepatitis B virus (HBV) polymerase proteins could be produced by alternative transcriptional or translational strategies. Their detection is very difficult because they are in minute concentration and probably enzymatically inactive, however, it was shown that they could be exposed on the outside of the virus particles and also be immunogenic. In addition, HBV polymerase shares significant amino acid similarities with the human myelin basic protein. We hypothesise that some of the apparent adverse reactions to the vaccine could be due to a process called of molecular mimicry, the HBV polymerase, which could be a contaminant in the recombinant or plasma-derived vaccines, could act as autoantigens and induce autoimmune demyelinating diseases such as multiple sclerosis.

Mechanism of Adefovir, Tenofovir and Entecavir Resistance: Molecular Modeling Studies of How A Novel Anti-HBV Agent (FMCA) Can Overcome the Drug Resistance.

Regardless of significant improvement in the area of anti-HBV therapy, resistance and cross-resistance against available therapeutic agents are the major consideration in drug discovery of new agents. The present study is to obtain the insight of the molecular basis of drug resistance conferred by the B and C domain mutations of HBV-polymerase on the binding affinity of four anti-HBV agents [Adefovir (ADV), Tenofovir (TNF), Entecavir (ETV) & 2'-Fluoro-6'-methylene-carbocyclic adenosine (FMCA)]. In this regard, homology modeled structure of HBV polymerase was used for minimization, conformational search and Glide XP docking followed by binding energy calculation on wild-type as well as on mutant HBV-polymerases (N236T, L180M+M204V+S202G & A194T). Our studies suggest a significant correlation between the fold resistances and the binding affinity of anti-HBV nucleosides. The domain B residue, L180 is indirectly associated with other active-site hydrophobic residues such as A87, F88 and M204, whereas the domain C residue, M204 is closely associated with sugar/pseudosugar ring positioning in the active site. These hydrophobic residues can directly influence the interaction of the incoming nucleoside triphosphates and change the binding efficacy. The carbohydrate ring part of natural substrate dATP, dGTP, FMCA and ETV, are occupied in similar passion in the grooves of HBV polymerase active site. The exocyclic double bond of Entecavir and FMCA occupies in the backside hydrophobic pocket (made by residues A87, F88, L180and M204), which enhances the overall binding affinity. Additional hydrogen bonding interaction of 2'-fluorine of FMCA with R41 residue of polymerase promotes a positive binding in wild-type as well as in ADVr, ETVr and TNFr with respect to that of entecavir.

Genomic divergence of an HIV-2 from a German AIDS patient probably infected in Mali.

The complete nucleotide sequence of an HIV-2 isolate derived from a German AIDS patient with predominantly neurological symptoms is reported. The HIV-2BEN sequence is highly divergent from those of previously described HIV-2 and SIV strains. Evolutionary tree analysis of eight HIV-2 sequences reveals the existence of three HIV-2 groups. HIV-2BEN belongs to a group with two isolates from Ghana and The Gambia. Based on a comparison of HIV-2BEN with six HIV-2 isolates, SIVsmm and SIVmac, the variability of the structural env and gag proteins is similar within the HIV-2/SIVsmm/mac and HIV-1 groups. In contrast, the regulatory HIV-1 proteins are more highly conserved than those from HIV-2 strains. Multiple sequence alignments reveal that some domains of the envelope and regulatory proteins are well conserved among HIV-1, HIV-2/SIVsmm/mac, SIVagm and SIVmnd. The identification of conserved domains within the external glycoprotein could help to develop broadly active vaccines.

Evolution and phylogeny of insect endogenous retroviruses.

BACKGROUND: The genome of invertebrates is rich in retroelements which are structurally reminiscent of the retroviruses of vertebrates. Those containing three open reading frames (ORFs), including an env-like gene, may well be considered as endogenous retroviruses. Further support to this similarity has been provided by the ability of the env-like gene of DmeGypV (the Gypsy endogenous retrovirus of Drosophila melanogaster) to promote infection of Drosophila cells by a pseudotyped vertebrate retrovirus vector. RESULTS: To gain insights into their evolutionary story, a sample of thirteen insect endogenous retroviruses, which represents the largest sample analysed until now, was studied by computer-assisted comparison of the translated products of their gag, pol and env genes, as well as their LTR structural features. We found that the three phylogenetic trees based respectively on Gag, Pol and Env common motifs are congruent, which suggest a monophyletic origin for these elements. CONCLUSIONS: We showed that most of the insect endogenous retroviruses belong to a major clade group which can be further divided into two main subgroups which also differ by the sequence of their primer binding sites (PBS). We propose to name IERV-K and IERV-S these two major subgroups of Insect Endogenous Retro Viruses (or Insect ERrantiVirus, according to the ICTV nomenclature) which respectively use Lys and Ser tRNAs to prime reverse transcription.

A two-domain model for the R domain of the cystic fibrosis transmembrane conductance regulator based on sequence similarities.

CFTR belongs to a group of proteins sharing the structural motif of six transmembrane helices and a nucleotide binding domain. Unique to CFTR is the R domain, a charged cytoplasmic domain. Comparison of R domain sequences from ten species revealed that the N-terminal third is highly conserved, while the C-terminal two-thirds is poorly conserved. The R domain shows no strong sequence similarity to known proteins; however, 14 viral pol proteins show limited similarity to fragments of the R domain. Analysis revealed a relationship between the N- and C-terminal fragments of the R domain and two discontinuous fragments of the pol protein. These observations support a two-domain model for the R domain.