Cysteine usage in Sulfolobus spindle-shaped virus 1 and extension to hyperthermophilic viruses in general

Fuselloviridae are ubiquitous crenarchaeal viruses found in high-temperature acidic hot springs worldwide. The type virus, Sulfolobus spindle-shaped virus 1 (SSV1), has a double-stranded DNA genome that contains 34 open reading frames (ORFs). Fuselloviral genomes show little similarity to other organisms, generally precluding functional predictions. However, tertiary protein structure can provide insight into protein function. We have thus undertaken a systematic investigation of the SSV1 proteome and report here on the F112 gene product. Biochemical, proteomic and structural studies reveal a monomeric intracellular protein that adopts a winged helix DNA binding fold. Notably, the structure contains an intrachain disulfide bond, prompting analysis of cysteine usage in this and other hyperthermophilic viral genomes. The analysis supports a general abundance of disulfide bonds in the intracellular proteins of hyperthermophilic viruses, and reveals decreased cysteine content in the membrane proteins of hyperthermophilic viruses infecting Sulfolobales. The evolutionary implications of the SSV1 distribution are discussed.     

A winged-helix protein from Sulfolobus turreted icosahedral virus points toward stabilizing disulfide bonds in the intracellular proteins of a hyperthermophilic virus

Sulfolobus turreted icosahedral virus (STIV) was the first non-tailed icosahedral virus to be isolated from an archaeal host. Like other archaeal viruses, its 37 open reading frames generally lack sequence similarity to genes with known function. The roles of the gene products in this and other archaeal viruses are thus largely unknown. However, a protein's three-dimensional structure may provide functional and evolutionary insight in cases of minimal sequence similarity. In this vein, the structure of STIV F93 reveals a homodimer with strong similarity to the winged-helix family of DNA-binding proteins. Importantly, an interchain disulfide bond is found at the dimer interface, prompting analysis of the cysteine distribution in the putative intracellular proteins of the viral proteome. The analysis suggests that intracellular disulfide bonds are common in cellular STIV proteins, where they enhance the thermostability of the viral proteome.     

Development of a genetic system for the archaeal virus Sulfolobus turreted icosahedral virus (STIV)

Our understanding of archaeal viruses has been limited by the lack of genetic systems for examining viral function. We describe the construction of an infectious clone for the archaeal virus Sulfolobus turreted icosahedral virus (STIV). STIV was isolated from a high temperature (82 °C) acidic (pH 2.2) hot spring in Yellowstone National Park and replicates in the archaeal model organism Sulfolobus solfataricus (Rice et al., 2004). While STIV is one of most studied archaeal viruses, little is known about its replication cycle. The development of an STIV infectious clone allows for directed gene disruptions and detailed genetic analysis of the virus. The utility of the STIV infectious clone was demonstrated by gene disruption of STIV open reading frame (ORF) B116 which resulted in crippled virus replication, while disruption of ORFs A197, C381 and B345 was lethal for virus replication.     

ATR-X mutations cause impaired nuclear location and altered DNA binding properties of the XNP/ATR-X protein

Mutations in the XNP/ATR-X gene, located in Xq13.3, are associated with several X linked mental retardation syndromes, the best known being α thalassaemia with mental retardation (ATR-X). The XNP/ATR-X protein belongs to the family of SWI/SNF DNA helicases and contains three C2-C2 type zinc fingers of unknown function. Previous studies have shown that 65% of mutations of XNP have been found within the zinc finger domain (encoded by exons 7, 8, and the beginning of exon 9) while 35% of the mutations have been found in the helicase domain extending over 3 kb at the C-terminus of the protein. Although different types of mutations have been identified, no specific genotype-phenotype correlation has been found, suggesting that gene alteration leads to a loss of function irrespective of mutation type. Our aims were to understand the function of the XNP/ATR-X protein better, with specific attention to the functional consequences of mutations to the zinc finger domain. We used monoclonal antibodies directed against the XNP/ATR-X protein and performed immunocytochemical and western blot analyses, which showed altered or absent XNP/ATR-X expression in cells of affected patients. In addition, we used in vitro experiments to show that the zinc finger domain can mediate double stranded DNA binding and found that the DNA binding capacity of mutant forms in ATR-X patients is severely reduced. These data provide insights into the understanding of the functional significance of XNP/ATR-X mutations.


Keywords: XNP/ATR-X; mutation; zinc finger; DNA binding activity     

A hypothetical new role for single-stranded DNA binding proteins in the immune system

The breadth of the host range of single-stranded DNA (ssDNA) viruses is roughly comparable to the host range of double-stranded DNA viruses (dsDNA). Yet, general ssDNA sensing receptors that activate the immune system have not been unequivocally identified while numerous dsDNA sensing receptors are known. Here, we hypothesize that some of the Single-Stranded DNA Binding (SSB) proteins may act as receptors that detect single-stranded DNA from pathogens and activate the innate immune system. As the first test of our hypothesis, we checked whether human genes that are known to bind to ssDNA are potentially interferon-regulated. Out of the 102 human genes that are known to have ssDNA binding ability 23 genes show a more than two-fold increase in gene expression upon interferon treatment. Single-stranded DNA viruses are pathogens of not only animals but also of plants and protozoans. We used this information to further prioritize our candidate list to ssDNA binding genes that are common between the model plant Arabidopsis thaliana and humans. Based on these strategies, we shortlist several promising candidate genes including the HMGB1 gene which could act as a ssDNA sensor that activates the immune system. Agreeably though we cannot establish a definitive role for these genes as ssDNA sensors of the immune system as yet, our preliminary analysis suggests the potential existence of ssDNA binding protein-like receptors (SLR's) that are worth investigating further.     

A single amino acid substitution in the central portion of the West Nile virus NS4B protein confers a highly attenuated phenotype in mice

West Nile virus (WNV) NS4B is a small hydrophobic nonstructural protein that is hypothesized to participate both in viral replication and evasion of host innate immune defenses. The protein has four cysteine residues (residues 102, 120, 227, and 237). Since cysteines are often critical for the function of proteins, each of the four cysteine residues found in WNV NS4B was mutated to serine by site-directed mutagenesis. While three of these substitutions had little effect on replication or mouse virulence phenotypes, the C102S mutation was associated with a temperature-sensitive phenotype at 41 degrees C as well as attenuation of the neuroinvasive and neurovirulence phenotypes in mice.     

A distinct single-stranded DNA-binding protein encoded by the Lactococcus lactis bacteriophage bIL67

Single-stranded binding proteins (SSBs) are found to participate in various processes of DNA metabolism in all known organisms. We describe here a SSB protein encoded by the Lactococcus lactis phage bIL67 orf14 gene. It is the first noted attempt at characterizing a SSB protein from a lactococcal phage. The purified Orf14(bIL67) binds unspecifically to ssDNA with the same high affinity as the canonical Bacillus subtilis SSB. Electrophoretic mobility-shift assays performed with mutagenized Orf14(bIL67) protein derivatives suggest that ssDNA-binding occurs via a putative OB-fold structure predicted by three-dimensional modeling. The native Orf14(bIL67) forms homotetramers as determined by gel filtration studies. These results allow distinguishing the first lactococcal phage protein with single-strand binding affinity, which defines a novel cluster of phage SSBs proteins. The possible role of Orf14(bIL67) in phage multiplication cycle is also discussed.     

Functional characterization of HgbB,a new hemoglobin binding protein of Pasteurella multocida

The biological function and role in pathogenesis of a Pasteurella multocida A:1 strain hemoglobin binding protein was investigated. The hgbB gene from the P. multocida A:1 strain, VP161, was cloned and characterized. hgbB was 2991 bp in length and encoded a mature length protein of 111 kDa. HgbB was predicted to be an outer membrane protein and shared 68 and 69% similarity to the hemoglobin/hemoglobin-haptoglobin binding protein, HI0712 from Haemophilus influenzae Rd and HgpC, from H. influenzae b, respectively. HgbB exhibited features typical of TonB dependent receptors, including seven conserved regions typical of these proteins, and conserved invariant residues. Escherichia coli expressing recombinant HgbB was found to bind hemoglobin in a solid phase dot blot binding assay. However, when a truncated form of the protein was expressed in E. coli, cells could no longer bind hemoglobin. Insertional inactivation of hgbB did not affect the ability of P. multocida to bind hemoglobin, nor its ability to produce disease in a mouse model. In addition, recombinant HgbB did not confer any protection against homologous or heterologous challenge.     

Association analysis between polymorphism rs2373115 of gene GRB-associated binding protein 2 and Mongolian Alzheimer patients

GRB-associated binding protein 2 (GAB2) may function as a risk factor of Alzheimer disease (AD) by affecting hyperphosphorylation of tau, causing neurofibrillary tangles. There had been genomewide analysis discovering polymorphism rs2373115 correlated with AD and in this study we performed an association analysis on this polymorphism in 107:100 Mongolian Alzheimer patients and controls in order to discover whether this correlation can be replicated in Chinese minority group. The results showed negative results, indicating GAB2 rs2373115 polymorphism was not a remarkable factor in developing Alzheimer disease among Mongolian.     

Naturally acquired inhibitory antibodies to Plasmodium vivax Duffy binding protein are short-lived and allele-specific following a single malaria infection

The Duffy binding protein of Plasmodium vivax (DBP) is a critical adhesion ligand that participates in merozoite invasion of human Duffy-positive erythrocytes. A small outbreak of P. vivax malaria, in a village located in a non-malarious area of Brazil, offered us an opportunity to investigate the DBP immune responses among individuals who had their first and brief exposure to malaria. Thirty-three individuals participated in the five cross-sectional surveys, 15 with confirmed P. vivax infection while residing in the outbreak area (cases) and 18 who had not experienced malaria (non-cases). In the present study, we found that only 20% (three of 15) of the individuals who experienced their first P. vivax infection developed an antibody response to DBP; a secondary boosting can be achieved with a recurrent P. vivax infection. DNA sequences from primary/recurrent P. vivax samples identified a single dbp allele among the samples from the outbreak area. To investigate inhibitory antibodies to the ligand domain of the DBP (cysteine-rich region II, DBP_II), we performed in vitro assays with mammalian cells expressing DBP_II sequences which were homologous or not to those from the outbreak isolate. In non-immune individuals, the results of a 12-month follow-up period provided evidence that naturally acquired inhibitory antibodies to DBP_II are short-lived and biased towards a specific allele.     

Protein and sequence requirements for the recruitment of the human origin recognition complex to the latent cycle origin of DNA replication of Epstein-Barr virus oriP

Initiation of DNA replication from within the Epstein-Barr virus (EBV) latent cycle origin oriP occurs once per cell cycle and is almost entirely dependent upon cellular proteins. The human origin recognition complex (ORC) is recruited to oriP and orchestrates the events that lead to the initiation of replication. EBNA-1, the sole viral protein required for oriP-plasmid replication, binds four sites within the replicator but the role(s) it plays in the replication of oriP plasmids has not been elucidated. We investigated the recruitment of ORC to oriP in vivo and show that the binding of EBNA-1 to the replicator is necessary for the association of the ORC subunit Orc2 with the replicator. The minimal replicator of oriP consists of two EBNA-1 binding sites flanked by perfect 14-bp inverted repeats (a and b), but these repeats are dispensable for the association of Orc2 with the replicator. A mutational analysis of the 14-bp repeats provided additional support for a role for the telomere repeat binding protein 2 in oriP replicator function. We show that nucleotide differences between the oriP replicator of the B95-8 and Raji EBV genomes are not solely responsible for the inefficient utilization of this origin in the Raji EBV genome.     

A novel Sulfolobus non-conjugative extrachromosomal genetic element capable of integration into the host genome and spreading in the presence of a fusellovirus

An integrative non-conjugative extrachromosomal genetic element, denoted as pSSVi, has been isolated from a Sulfolobus solfataricus P2 strain and was characterized. This genetic element is a double-stranded DNA of 5740 bp in size and contains eight open reading frames (ORFs). It resembles members of the pRN plasmid family in genome organization but shows only weak similarity to the latter in conserved regions. pSSVi has a copG gene similar to that of a pRN plasmid, encodes a large replication protein which, unlike a typical pRN RepA, contains no polymerase/primase domain, and lacks the plrA gene. Interestingly, pSSVi encodes an SSV-type integrase which probably catalyzes the integration of its genome into a specific site (a tRNA(Arg) gene) in the S. solfataricus P2 genome. Like pSSVx, pSSVi can be packaged into a spindle-like viral particle and spread with the help of SSV1 or SSV2. In addition, both SSV1 and SSV2 appeared to replicate more efficiently in the presence of pSSVi. Given the versatile genetic abilities, pSSVi appears to be well suited for a role in horizontal gene transfer.     

Mutational analysis of the West Nile virus NS4B protein

West Nile virus NS4B is a small hydrophobic nonstructural protein approximately 27 kDa in size whose function is poorly understood. Amino acid substitutions were introduced into the NS4B protein primarily targeting two distinct regions; the N-terminal domain (residues 35 through 60) and the central hydrophobic domain (residues 95 through 120). Only the NS4B P38G substitution was associated with both temperature-sensitive and small-plaque phenotypes. Importantly, this mutation was found to attenuate neuroinvasiveness greater than 10,000,000-fold and lower viremia titers compared to the wild-type NY99 virus in a mouse model. Full genome sequencing of the NS4B P38G mutant virus revealed two unexpected mutations at NS4B T116I and NS3 N480H (P38G/T116I/N480H), however, neither mutation alone was temperature sensitive or attenuated in mice. Following incubation of P38G/T116I/N480H at 41 °C, five mutants encoding compensatory substitutions in the NS4B protein exhibited a reduction in the temperature-sensitive phenotype and reversion to a virulent phenotype in the mouse model.     

Computer-assisted primary and secondary structure analyses of DNA polymerases of herpes simplex,Epstein-Barr and varicella zoster viruses reveal conserved domains with some homology to DNA-binding domain in E. coli DNA pol I

The primary and secondary structure of herpes simplex virus type 1 (HSV-1), varicella-zoster (VZV) and Epstein-Barr virus (EBV) DNA polymerases was calculated by means of computer programs. The comparison of HSV-1 polymerase (pol) sequence to the known primary and tertiary structure of E. coli DNA pol I revealed five short homologous sequences, one of which coincided with the -helical structure of the DNA-binding domain of E. coli DNA pol. Comparison by primary and secondary structure computer programs of the three DNA polymerases coded by herpesviruses HSV-1, VZV and EBV led to the identification of polypeptide sequences shared by the three DNA pols. In a similar way, the secondary structure of the DNA pol polypeptide in the vicinity of the mutation leading to PAA resistance in HSV-1 DNA pol helped to identify the role of this sequence in the binding of phosphate donated by the nucleoside triphosphate molecule which binds to the DNA pol. Although the computer secondary structure programs are about 60% accurate, it was possible to obtain new information on the properties of certain domains in the DNA polymerases of HSV-1, VZV and EBV.Abbreviations aa amino acids - EBV Epstein-Barr virus - HSV-1 herpes simplex virus type 1 - M_r molecular weight - PAA phosphonoacetic acid - PFA phosphonoformic acid - pol polymerase - VZV varicella-zoster virus Requests for reprints should be addressed to Professor Yechiel Becker, Department of Molecular Virology, Faculty of Medicine, The Hebrew University, P.O. Box 1172, 91010 Jerusalem, Israel.     

Protection against nasal colonization with Streptococcus pneumoniae by parenteral immunization with a DNA vaccine encoding PspA (Pneumococcal surface protein A)

Pneumococcal surface protein A (PspA) is an important candidate for a cost-effective vaccine with broad coverage against Streptococcus pneumoniae. We have previously shown that intramuscular immunization with PspA as a DNA vaccine induces an immune response characterized by the induction of a balanced IgG1/IgG2a antibody response in BALB/c mice, which was able to efficiently mediate complement deposition onto intact bacteria and to induce protection against an intraperitoneal challenge. We now confirm the results in C57BL/6 mice and further show that the response induced by the DNA vaccine expressing PspA is able to mediate protection against colonization of the nasopharyngeal mucosa even though immunization was given parenterally. Moreover, a positive correlation was observed between IgG1 and the numbers of CFU recovered, whereas an inverse correlation was observed between nasal CFU levels and IgG2a. A positive correlation was also found for IgG1/IgG2a antibody ratios with CFU recovered from the nasopharynx. Therefore, reduction of nasal colonization was strongly associated with increased levels of serum IgG2a complement fixing antibody and low levels of IgG1 antibody which has much less complement fixing activity. Passive transfer of serum from animals immunized with the DNA vaccine expressing PspA was also able to reduce the fraction of mice with high density of colonization of the nasopharynx. Secretion of IFN-γ, but not IL-17, was observed in splenocytes from mice immunized with the DNA vaccine.     

Role for a region of helically unstable DNA within the Epstein-Barr virus latent cycle origin of DNA replication oriP in origin function

The minimal replicator of the Epstein-Barr virus (EBV) latent cycle origin of DNA replication oriP is composed of two binding sites for the Epstein-Barr virus nuclear antigen-1 (EBNA-1) and flanking inverted repeats that bind the telomere repeat binding factor TRF2. Although not required for minimal replicator activity, additional binding sites for EBNA-1 and TRF2 and one or more auxiliary elements located to the right of the EBNA-1/TRF2 sites are required for the efficient replication of oriP plasmids. Another region of oriP that is predicted to be destabilized by DNA supercoiling is shown here to be an important functional component of oriP. The ability of DNA fragments of unrelated sequence and possessing supercoiled-induced DNA duplex destabilized (SIDD) structures, but not fragments characterized by helically stable DNA, to substitute for this component of oriP demonstrates a role for the SIDD region in the initiation of oriP-plasmid DNA replication.