Equine arteritis virus

Equine arteritis virus (EAV) is a small, enveloped, positive-stranded RNA virus, in the family Arteriviridae , W.H.ich can infect both horses and donkeys. While the majority of EAV infections are asymptomatic, acutely infected animals may develop a wide range of clinical signs, including pyrexia, limb and ventral edema, depression, rhinitis, and conjunctivitis. The virus may cause abortion and has caused mortality in neonates. After natural EAV infection, most horses develop a solid, long-term immunity to the disease. Marzz and geldings eliminate the virus within 60 days, but 30 to 60% of acutely infected stallions will become persistently infected. These persistently infected animals maintain EAV within the reproductive tract, shed virus continuously in the semen, and can transmit the virus venereally. Mares infected venereally may not have clinical signs, but they shed large amounts of virus in nasopharyngeal secretions and in urine, which may result in lateral spread of the infection by an aerosol route. The consequences of venereally acquired infection are minimal, with no known effects on conception rate, but mares infected at a late stages of gestation may abort. Identification of carrier stallions is crucial to control the dissemination of EAV. The stallions can be identified by serological screening using a virus neutralization (VN) test. If positive at a titer of >/= 1:4, the stallion should be tested for persistent infection by virus isolation from the sperm-rich fraction of the ejaculate, or by test mating Shedding stallions should not be used for breeding, or should be bred only to mares seropositive from a natural infection or from vaccination, the mares should be subsequently isolated from seronegative horses for three weeks after natural or artificial insemination. A live attenuated (ARVAC) and a formalin-inactivated (ARTERVAC) vaccine are available. Both vaccines induce virus-neutralizing antibodies, the presence of which correlates with protection from disease, abortion, and the development of a persistent infection. Serological investigations indicate that EAV has a worldwide distribution and that its prevalence is increasing. As a consequence, an increasing number of equine viral arteritis (EVA) outbreaks is being reported. This trend is likely to continue unless action is taken to slow or halt the transmission of this agent through semen.     

Primula subansirica G.D. Pal is not a Primula (Primulaceae), but rather belongs to Gesneriaceae

Examination of type specimens has shown that Primula subansirica G.D. Pal is a Gesneriad and conspecific with Beccarinda cordifolia (Anthony) B.L. Burtt (Gesneriaceae), thus a reduction is made and a new synonymy is proposed.     

A pea nuclear protein that is induced by dehydration belongs to the vicilin superfamily.

The purification to homogeneity of p16, a protein with an electrophoretic mobility compatible with an apparent molecular mass of 16 kDa, from nuclei of ungerminated pea embryonic axes is described. A cDNA clone of its gene, which was designated psp54, was also isolated. The psp54 cDNA contains an open reading frame coding for a 54.4-kDa polypeptide (p54). p16 corresponds to the C-terminal third of p54, although the mechanisms by which the primary polypeptide could be processed are not yet known. The sequence of p54 is 60% identical with that of the precursor of a sucrose-binding soybean protein, and, to a lesser extent (31-34%), it shares homology with some storage proteins. p16 is also 30% homologous with Nhp2p, a yeast nuclear protein. The psp54 gene, present in a single copy in pea genome, starts being expressed during seed desiccation. Soon after rehydration in seed germination, p54 mRNA disappears and is no longer detectable in vegetative tissues, except in response to hydric stress (exposure to abscisic acid, osmolites or desiccation). p16 can be recovered from nuclei cross-linked to histone H3, when the disulfide bridges that occur in vivo are preserved. On the other hand, p16 shares some properties with dehydrins, which are thought to protect cellular structures against desiccation. We propose that the possible precursor polypeptide p54 belongs to the vicilin superfamily, members of which play a variety of roles. The function of p16 may be related to the protection of chromatin structure against desiccation during seed development.     

Simian hemorrhagic fever virus: a new togavirus.

Using the prototype strain of SHF virus, we have confirmed the nature of the genome (RNA), the presence of an envelope (derived from an internal membrane), the virion size (45-50 nm), and probable cubic symmetry. We have also described four viral structural proteins and determined the phospholipid content of the virions. The known properties of SHF virus suggest that it should be classified in the Togavirus family, and possibly in the Flavivirus group.     

Type II restriction endonuclease R.Hpy188I belongs to the GIY-YIG nuclease superfamily,but exhibits an unusual active site

Background  

Catalytic domains of Type II restriction endonucleases (REases) belong to a few unrelated three-dimensional folds. While the PD-(D/E)XK fold is most common among these enzymes, crystal structures have been also determined for single representatives of two other folds: PLD (R.BfiI) and half-pipe (R.PabI). Bioinformatics analyses supported by mutagenesis experiments suggested that some REases belong to the HNH fold (e.g. R.KpnI), and that a small group represented by R.Eco29kI belongs to the GIY-YIG fold. However, for a large fraction of REases with known sequences, the three-dimensional fold and the architecture of the active site remain unknown, mostly due to extreme sequence divergence that hampers detection of homology to enzymes with known folds.     

Peptidase family U34 belongs to the superfamily of N-terminal nucleophile hydrolases

Peptidase family U34 consists of enzymes with unclear catalytic mechanism, for instance, dipeptidase A from Lactobacillus helveticus. Using extensive sequence similarity searches, we infer that U34 family members are homologous to penicillin V acylases (PVA) and thus potentially adopt the N-terminal nucleophile (Ntn) hydrolase fold. Comparative sequence and structural analysis reveals a cysteine as the catalytic nucleophile as well as other conserved residues important for catalysis. The PVA/U34 family is variable in sequence and exhibits great diversity in substrate specificity, to include enzymes such as choloyglycine hydrolases, acid ceramidases, isopenicillin N acyltransferases, and a subgroup of eukaryotic proteins with unclear function.     

Active foamy virus proteinase is essential for virus infectivity but not for formation of a Pol polyprotein.

To analyze proteolytic processing of foamy (spuma) retroviruses, two mutations were generated in the presumed active-site triplet Asp-Ser-Gly in the predicted proteinase (PR) region of the human foamy virus (HSRV). The mutations changed either the presumed catalytic aspartic acid residue to a catalytically incompetent alanine or the adjacent serine to a threonine found in most cellular and retroviral proteases at this position. Both mutations were cloned into the full-length infectious HSRV DNA clone. Wild-type and S/T mutant genomes directed the synthesis of particles with similar infectious titers, while the HSRV D/A PR mutant was noninfectious. Immunoblot analysis of transfected cells revealed identical patterns for the wild-type and for the S/T PR mutant. HSRV D/A mutant-transfected cells expressed only a single Gag polyprotein of 78 kDa instead of the 78-kDa-74-kDa doublet found in HSRV-infected or wild-type-transfected cells. Analysis with pol-specific antisera yielded a protein of approximately 120 kDa reactive with antisera against pol- but not gag-specific domains. No Gag-Pol polyprotein was detected in this study. Electron microscopy analysis of transfected cells showed heterogeneous particle morphology in the case of the D/A mutant, with particles of normal appearance and particles of aberrant size and shape. These results indicate that foamy viruses have an aspartic PR that is essential for infectivity but not for formation of the 120-kDa Pol polyprotein.     

Production and characterization of a thermostable alcohol dehydrogenase that belongs to the aldo-keto reductase superfamily

The gene encoding a novel alcohol dehydrogenase that belongs to the aldo-keto reductase superfamily has been identified in the hyperthermophilic archaeon Pyrococcus furiosus. The gene, referred to as adhD, was functionally expressed in Escherichia coli and subsequently purified to homogeneity. The enzyme has a monomeric conformation with a molecular mass of 32 kDa. The catalytic activity of the enzyme increases up to 100 degrees C, and a half-life value of 130 min at this temperature indicates its high thermostability. AdhD exhibits a broad substrate specificity with, in general, a preference for the reduction of ketones (pH optimum, 6.1) and the oxidation of secondary alcohols (pH optimum, 8.8). Maximal specific activities were detected with 2,3-butanediol (108.3 U/mg) and diacetyl-acetoin (22.5 U/mg) in the oxidative and reductive reactions, respectively. Gas chromatrography analysis indicated that AdhD produced mainly (S)-2-pentanol (enantiomeric excess, 89%) when 2-pentanone was used as substrate. The physiological role of AdhD is discussed.     

Coenzyme F390 synthetase from Methanobacterium thermoautotrophicum Marburg belongs to the superfamily of adenylate-forming enzymes.

Depending on the reduction-oxidation state of the cell, some methanogenic bacteria synthesize or hydrolyze 8-hydroxyadenylylated coenzyme F420 (coenzyme F390). These two reactions are catalyzed by coenzyme F390 synthetase and hydrolase, respectively. To gain more insight into the mechanism of the former reaction, coenzyme F390 synthetase from Methanobacterium thermoautotrophicum Marburg was purified 89-fold from cell extract to a specific activity of 0.75 mumol.min-1.mg of protein-1. The monomeric enzyme consisted of a polypeptide with an apparent molecular mass of 41 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. ftsA, the gene encoding coenzyme F390 synthetase, was cloned and sequenced. It encoded a protein of 377 amino acids with a predicted M(r) of 43,280. FtsA was found to be similar to domains found in the superfamily of peptide synthetases and adenylate-forming enzymes. FtsA was most similar to gramicidin S synthetase II (67% similarity in a 227-amino-acid region) and sigma-(L-alpha-aminoadipyl)-L-cysteine-D-valine synthetase (57% similarity in a 193-amino-acid region). Coenzyme F390 synthetase, however, holds an exceptional position in the superfamily of adenylate-forming enzymes in that it does not activate a carboxyl group of an amino or hydroxy acid but an aromatic hydroxyl group of coenzyme F420.     

hsp26 of Saccharomyces cerevisiae is related to the superfamily of small heat shock proteins but is without a demonstrable function.

Analysis of the cloned gene confirms that hsp26 of Saccharomyces cerevisiae is a member of the small heat shock protein superfamily. Previous mutational analysis failed to demonstrate any function for the protein. Further experiments presented here demonstrate that hsp26 has no obvious regulatory role and no major effect on thermotolerance. It is possible that the small heat shock protein genes originated as primitive viral or selfish DNA elements.     

Studies on hst, a transforming gene which belongs to a new superfamily of growth factors

The hst gene was originally identified in surgically obtained human gastric mucosae as a transforming gene which could transform NIH3T3 cells morphologically. The hst cDNA clone was synthesized from mRNA of one of the NIH3T3 transformants. A human leukocyte genomic library was screened with this cDNA clone, and an hst genomic fragment was obtained. This genomic fragment itself had transforming activity, and the protein coding sequences were proved to be completely identical to those of the cDNA clone prepared from mRNA of the NIH3T3 transformant. This fact suggests that rearrangement or other structural alterations in the coding sequence are not required for the activation of the hst gene. The predicted hst protein consists of 206 amino acids and has a significant homology (40-50%) to fibroblast growth factors and int-2 protein. They together make up a new superfamily of growth factors and transforming genes.     

Passage through mitosis is required for oncoretroviruses but not for the human immunodeficiency virus.

The human immunodeficiency virus productively infects and integrates into cells that have been arrested in the cell cycle with either gamma irradiation or aphidicolin. Integration by oncoretroviruses such as the murine leukemia virus (MuLV), on the other hand, depends on cell proliferation. Although the entire cell cycle is not necessary for MuLV infection, it is essential that the infected cells pass through mitosis. The long terminal repeat circle junction, a marker for nuclear entry, is first observed in MuLV-infected cells immediately after mitosis. These results suggest that mitosis is necessary for nuclear entry of MuLV, but not human immunodeficiency virus, unintegrated proviral DNA.     

Hepatitis B virus large surface protein is not secreted but is immunogenic when selectively expressed by recombinant vaccinia virus.

The envelope region of the hepatitis B virus (HBV) genome contains an open reading frame that begins upstream of the major surface protein gene. The two minor proteins that are initiated within this pre-s segment are immunogenic and may be involved in virus attachment to hepatocytes. We have constructed a recombinant vaccinia virus that contains the predicted coding segment for the large surface protein (LS) under control of a vaccinia virus that contains the predicted coding segment for the large surface protein (LS) under control of a vaccinia virus promoter. Cells infected with the recombinant virus synthesized HBV polypeptides of 39 and 42 kilodaltons, corresponding to the unglycosylated and glycosylated forms of LS, respectively. The presence of pre-s epitopes in the 39- and 42-kilodalton polypeptides was demonstrated by binding of antibody prepared against a synthetic peptide. Synthesis of the 42-kilodalton species was specifically inhibited by tunicamycin, suggesting that it is N-glycosylated. Despite apparent glycosylation, LS was not secreted into the medium of infected cells. Nevertheless, rabbits vaccinated with the purified recombinant virus made antibodies that recognized s and pre-s epitopes. Antibody to the NH2 terminus of LS appeared before or simultaneously with antibody that bound to the major surface protein. The additional immunogenicity provided by expression of LS may be advantageous for the development of an HBV vaccine.     

CENP-C is necessary but not sufficient to induce formation of a functional centromere.

CENP-C is an evolutionarily conserved centromeric protein. We have used the chicken DT40 cell line to test the idea that CENP-C is sufficient as well as necessary for the formation of a functional centromere. We have compared the effects of disrupting the localization of CENP-C with those of inducibly overexpressing the protein. Removing CENP-C from the centromere causes disassembly of the centromere protein complex and blocks cells at the metaphase-anaphase junction. Overexpressed CENP-C is associated with an increase in errors of chromosome segregation and inhibits the completion of mitosis. However, the excess CENP-C does not disrupt the native centromeres detectably and does not associate with another conserved centromere protein, ZW10. The distribution of the excess CENP-C changes during the cell cycle. In metaphase, the excess CENP-C coats the chromosome arms. At the metaphase-anaphase transition, the excess CENP-C clusters, and during interphase it is present in large bodies which form around pre-existing centromeres which are also clustered. These results indicate that CENP-C is necessary but not sufficient for the formation of a functional centromere and suggest that the structure of CENP-C may be regulated during the cell cycle.     

Cannabis demasculinizes rats but is not estrogenic.

Cannabis resin (54% THC) caused dose-related decreases in the development of testis, prostate and seminal vesicles when administered in the diet to immature male rats. Bioassays and estrogen-receptor studies showed no estrogenic activity in cannabis resin. Demasculinazation and possible feminization by cannabis are not due to simple estrogen-like action on male tissues.     

Envelope glycoprotein gp50 of pseudorabies virus is essential for virus entry but is not required for viral spread in mice.

Phenotypically complemented pseudorabies virus gp50 null mutants are able to produce plaques on noncomplementing cell lines despite the fact that progeny virions are noninfectious. To determine whether gp50 null mutants and gp50+gp63 null mutants are also able to replicate and spread in animals, mice were infected subcutaneously or intraperitoneally. Surprisingly, both gp50 mutants and gp50+gp63 double mutants proved to be lethal for mice. In comparison with the wild-type virus, gp50 mutants were still highly virulent, whereas the virulence of gp50+gp63 mutants was significantly reduced. Severe signs of neurological disorders, notably pruritus, were apparent in animals infected with the wild-type virus or a gp50 mutant but were much less pronounced in animals infected with a gp50+gp63 or gp63 mutant. Immunohistochemical examination of infected animals showed that all viruses were able to reach, and replicate in, the brain. Examination of visceral organs of intraperitoneally infected animals showed that viral antigen was predominantly present in peripheral nerves, suggesting that the viruses reached the central nervous system by means of retrograde axonal transport. Infectious virus could not be recovered from the brains and organs of animals infected with gp50 or gp50+gp63 mutants, indicating that progeny virions produced in vivo are noninfectious. Virions that lacked gp50 in their envelopes, and a phenotypically complemented pseudorabies virus gII mutant (which is unable to produce plaques in tissue culture cells), proved to be nonvirulent for mice. Together, these results show that gp50 is required for the primary infection but not for subsequent replication and viral spread in vivo. These results furthermore indicate that transsynaptic transport of the virus is independent of gp50. Since progeny virions produced by gp50 mutants are noninfectious, they are unable to spread from one animal to another. Therefore, such mutants may be used for the development of a new generation of safer (carrier) vaccines.