Genetic and functional analysis of the human immunodeficiency virus (HIV) type 1-inhibiting F12-HIVnef allele.

The primary human immunodeficiency virus type 1 (HIV-1) Nef mutant F12-HIVNef is characterized by three rare amino acid substitutions, G(140)E, V(153)L and E(177)G. It was reported previously that the expression of F12-HIVNef in the context of the highly productive NL4-3 HIV-1 strain blocks virus replication at the level of virus assembly and/or release by a mechanism depending on the presence of the CD4 intracytoplasmic tail. Here, it is reported that NL4-3 HIV-1 strains expressing F12-HIVnef alleles that were back-mutated in each amino acid substitution readily replicated in CD4(+) cells.

The Chryseobacterium meningosepticum PafA enzyme: prototype of a new enzyme family of prokaryotic phosphate-irrepressible alkaline phosphatases?

Chryseobacterium meningosepticum is an aerobic Gram-negative rod widely distributed in natural environments. Unlike many bacteria, it produces a phosphate-irrepressible periplasmic alkaline phosphatase (AP). This work describes cloning of the gene encoding that enzyme from C.

Porins from Salmonella enterica serovar Typhimurium induce TNF-alpha, IL-6 and IL-8 release by CD14-independent and CD11a/CD18-dependent mechanisms.

Lipopolysaccharide (LPS) of Gram-negative bacteria and several surface components of Gram-positive bacteria utilize CD14 and CD11a/18 as cellular receptors to induce expression and release of cytokines. Of the surface components of Gram-negative bacteria, porins exhibit a biological activity similar to that of LPS. The results in this paper show that the mechanism of stimulation by porins of THP-1 cells enriched in CD14 receptor after treatment with 1,25-dihydroxyvitamin D(3) (vitamin D(3)) is independent of this receptor, but is partially dependent on CD11a/18 integrins.

Generation of infectious and transmissible virions from a GB virus B full-length consensus clone in tamarins.

The strong similarity between GB virus B (GBV-B) and hepatitis C virus (HCV) makes tamarins infected by GBV-B an acceptable surrogate animal model for HCV infection. Even more attractive, for drug discovery purposes, is the idea of constructing chimeric viruses by inserting HCV genes of interest into a GBV-B genome frame. To accomplish this, infectious cDNA clones of both viruses must be available.

Mutagenesis of hepatitis C virus E1 protein affects its membrane-permeabilizing activity.

The E1 glycoprotein of hepatitis C virus is a transmembrane glycoprotein with a C-terminal anchor domain. When expressed in Escherichia coli, E1 induces a change in membrane permeability that is toxic to the bacterial cell. The C-terminal hydrophobic region (aa 331-383) of E1 is mainly responsible for membrane association and for inducing changes in membrane permeability.

Alteration in cellular fatty acid composition as a response to salt, acid, oxidative and thermal stresses in Lactobacillus helveticus.

The fundamental question in this study is concerned with whether the increase of unsaturated fatty acids in the cell membrane is a general response of certain thermotolerant strains or species when exposed to superoptimal temperatures, and in combination with other stresses, especially oxidative stress. A strain of Lactobacillus helveticus, a species widely used as a starter in the dairy industry and able to tolerate high temperature and NaCl concentrations as well as acidic conditions, was chosen for this study. Cells of strain CNBL 1156, grown in its natural medium (i.

Complete nucleotide sequence and genome organization of Grapevine fleck virus.

The complete nucleotide sequence of Grapevine fleck virus (GFkV) genomic RNA was determined. The genome is 7564 nt in size, excluding the 3'-terminal poly(A) tail, is characterized by an extremely high cytosine content (ca. 50%), and contains four putative open reading frames and untranslated regions of 291 and 35 nt at the 5' and 3' ends, respectively.

Human herpesvirus-8 (Kaposi's sarcoma-associated herpesvirus) ORF50 interacts synergistically with the tat gene product in transactivating the human immunodeficiency virus type 1 LTR.

Human herpesvirus-8 (HHV-8) is a lymphotropic virus associated with several AIDS-related neoplasms. Two ORFs play a critical role in the regulation of virus replication: ORF50, encoding an immediate-early transcriptional activator, and ORF57, encoding a post-transcriptional regulator. We analysed their effects on the activation of the human immunodeficiency virus type 1 (HIV-1) LTR.

The acid-stress response in Lactobacillus sanfranciscensis CB1.

Lactobacillus sanfranciscensis CB1, an important sourdough lactic acid bacterium, can withstand low pH after initial exposure to sublethal acidic conditions. The sensitivity to low pH varied according to the type of acid used. Treatment of LB: sanfranciscensis CB1 with chloramphenicol during acid adaptation almost completely eliminated the protective effect, suggesting that induction of protein synthesis was required for the acid-tolerance response.

Analysis of canine parvovirus sequences from wolves and dogs isolated in Italy.

The VP2 genes of Italian canine parvovirus (CPV) type 2 strains isolated from dogs and wolves were sequenced and a three-dimensional model of the VP2 capsid protein was constructed. Two mutations were detected in the VP2 sequences of the Italian strains: one at residue 297 and one at residue 265. Variant 297 is the predominant CPV isolate in Europe, whereas variant 265 has never been detected before.

A two-hybrid system based on chimeric operator recognition for studying protein homo/heterodimerization in Escherichia coli.

The development of a convenient and promising alternative to the various two-hybrid methods that are used to study protein-protein interactions is described. In this system, a lambdoid chimeric operator is recognized by a hybrid repressor formed by two chimeric monomers whose C-terminal domains are composed of heterologous proteins (or protein domains). Only if these proteins efficiently dimerize in vivo is a functional repressor formed able to bind the chimeric operator and shut off the synthesis of a downstream reporter gene.

Regulation of the p-hydroxybenzoic acid hydroxylase gene (pobA) in plant-growth-promoting Pseudomonas putida WCS358.

The regulation of the p-hydroxybenzoate hydroxylase gene (pobA) of Pseudomonas putida WCS358 involved in the catabolism of p-hydroxybenzoic acid (PHB) to the central intermediate protocatechuate was studied. Protocatechuic acid (PCA) is then degraded via the beta-ketoadipate pathway to form tricarboxylic acid intermediates. In several Gram-negative bacteria pobA has been found genetically linked to a regulator called pobR which activates pobA expression in response to PHB.

In vitro selection of human cytomegalovirus variants unable to transfer virus and virus products from infected cells to polymorphonuclear leukocytes and to grow in endothelial cells.

Four human cytomegalovirus (HCMV) isolates from different clinical sources were extensively propagated in human embryonic lung fibroblasts (HELF). Plaque isolates from each of the four virus strains were evaluated for their ability to be transferred to polymorphonuclear leukocytes (PMNL) and to grow in endothelial cells (EC). While all four of the clinical strains were found to be both PMNL- and EC-tropic, variants were identified from each of the four strains that lacked both biological properties, while three of the four parental strains lost their transfer capacity before passage 50 in HELF.

A bovine macrophage cell line supports bovine herpesvirus-4 persistent infection.

Although bovine herpesvirus-4 (BHV-4), a gammaherpesvirus lacking a clear disease association, has been demonstrated in many tissues during persistent BHV-4 infection, a likely site of virus persistence is in cells of the monocyte/macrophage lineage. To establish an in vitro model of persistent infection potentially useful for examining the molecular mechanisms of BHV-4 persistence/latency, we infected the bovine macrophage cell line BOMAC. Following extensive cell death, surviving cells were found to be persistently infected, maintaining the viral genome over many passages and producing low levels of infectious virus.

Activity of Toscana and Rift Valley fever virus transcription complexes on heterologous templates.

A transcription system for Toscana virus (TOSV) (a member of the family BUNYAVIRIDAE:, genus PHLEBOVIRUS:) was constructed. For in vivo expression, the TOSV transcription system uses the viral N and L proteins and an S-like RNA genome containing the chloramphenicol acetyltransferase reporter gene in the antisense orientation flanked by the viral genomic 5'- and 3'-terminal S sequences. It was found that the N and L proteins represent the minimal protein requirement for an active transcription complex.

The tegument protein ppUL25 of human cytomegalovirus (CMV) is a major target antigen for the anti-CMV antibody response.

A viral protein of approximately 82 kDa is the only structural protein of human cytomegalovirus (CMV) that is strongly immunogenic during natural infection and the corresponding gene of which is still unknown. In this work, strong evidence is presented that this protein is the product of UL25.

The open reading frame 1-encoded ('36K') protein of Carnation Italian ringspot virus localizes to mitochondria.

The localization of the 36 kDa ('36K') protein encoded by open reading frame 1 of Carnation Italian ringspot virus was studied in infected cells and in cells transiently expressing the 36K protein fused to green fluorescent protein (GFP). Subcellular fractionation demonstrated that the 36K protein accumulated in fractions containing mostly mitochondria. Fluorescence microscopy of transiently transformed cells showed that the 36K-GFP fusion protein accumulated in structures which could be stained with the mitochondrial-specific dye MitoTracker.

Identification and functional analysis of sequence rearrangements in the long control region of human papillomavirus type 16 Af-1 variants isolated from Ugandan penile carcinomas.

Human papillomavirus type 16 (HPV-16) is the predominant HPV isolate found in malignancies of male and female lower genital tracts. However, only a small percentage of individuals infected with high-risk HPVs develop a genital neoplasia, suggesting that additional events at both the cellular and the virus level are necessary for the progression to cancer, including genetic mutations/rearrangements of viral sequences involved in the oncogenic process. In this study, the genetic stability of the long control region (LCR) (nt 7289-114), which regulates expression levels of oncoproteins E6 and E7, was analysed in HPV-16 isolates from penile carcinoma (PC) biopsies of patients recruited from Uganda, one of the countries with the highest incidence of genital cancers in both men and women.

Processing of GB virus B non-structural proteins in cultured cells requires both NS3 protease and NS4A cofactor.

The identification of antivirals and vaccines against hepatitis C virus (HCV) infection is hampered by the lack of convenient animal models. The need to develop surrogate models has recently drawn attention to GB virus B (GBV-B), which produces hepatitis in small primates. In a previous study in vitro, it was shown that GBV-B NS3 protease shares substrate specificity with the HCV enzyme, known to be crucial for virus replication.

Polyomavirus persistence in lymphocytes: prevalence in lymphocytes from blood donors and healthy personnel of a blood transfusion centre.

BK and JC polyomaviruses (BKV and JCV) are widespread in humans and are thought to persist and reactivate under immune alterations. In addition to the kidney, lymphoid cells have been proposed as a site of latency. However, while this was shown to occur in immunocompromised patients, discordant data were published for healthy humans.

The acetyl xylan esterase of Bacillus pumilus belongs to a family of esterases with broad substrate specificity.

The Bacillus pumilus gene encoding acetyl xylan esterase (axe) was identified and characterized. The axe gene was expressed and the recombinant enzyme produced in Escherichia coli was purified and characterized. The recombinant enzyme displayed similar properties to the acetyl xylan esterase (AXE) purified from B.

Mu DNA reintegration upon excision: evidence for a possible involvement of nucleoid folding.

Mutations induced by the integration of a Mugem2ts prophage can revert at frequencies around 1x10(-6). In these revertant clones, the prophage excised from its original localization is not lost but reintegrated elsewhere in the host genome. One of the most intriguing aspects of this process is that the prophage reintegration is not randomly distributed: there is a strong correlation between the original site of insertion (the donor site) and the target site of the phage DNA migration (the receptor site).

A novel member of the subtilisin-like protease family from Bacillus subtilis.

aprX is a 1326 bp gene of Bacillus subtilis strain 168 that encodes a serine protease, probably intracellular, characterized by significant similarity with subtilisins, thermitases and pyrolysins. Transcription analysis, performed by RT-PCR and primer extension, allowed the localization of the active promoter and showed that aprX is expressed in stationary phase. The pattern of expression of aprX and its dependence on various transition state regulatory genes (degU, degQ, hpr, abrB, sinR), monitored by lacZ transcriptional fusions, are distinctive from those of subtilisin and other degradative enzymes.