Herpes simplex virus type 1 glycoprotein C-negative mutants exhibit multiple phenotypes, including secretion of truncated glycoproteins

A virus-neutralizing monoclonal antibody specific for glycoprotein C (gC) of herpes simplex virus type 1 strain KOS was used to select a number of neutralization-resistant mutants. A total of 103 of these mutants also were resistant to neutralization by a pool of gC-specific antibodies and thus were operationally defined as gC-. Analysis of mutant-infected cell mRNA showed that a 2.7-kilobase mRNA, comparable in size to the wild-type gC mRNA, was produced by nearly all mutants. However, six mutants, gC-5, gC-13, gC-21, gC-39, gC-46, and gC-98, did not produce the normal-size gC mRNA but rather synthesized a novel 1.1-kilobase RNA species. These mutants had deletions of 1.6 kilobases in the coding sequence of the gC structural gene, which explains their gC- phenotype. Despite the production of an apparently normal mRNA by the remaining 97 mutants, only 7 mutants produced a detectable gC polypeptide. In contrast to wild-type gC, which is a membrane-bound glycoprotein with an apparent molecular weight of 130,000 (130K), five of these mutants quantitatively secreted proteins of lower molecular weight into the culture medium. These were synLD70 (101K), gC-8 (109K), gC-49 (112K), gC-53 (108K), and gC-85 (106K). The mutant gC-3 secreted a protein that was indistinguishable in molecular weight from wild-type KOS gC. Another mutant, gC-44, produced a gC protein which also was indistinguishable from wild-type gC by molecular weight and which remained cell associated. Pulse-labeling of infected cells in the presence and absence of the glycosylation inhibitor tunicamycin demonstrated that these proteins were glycosylated and provided estimates of the molecular weights of the nonglycosylated primary translation products. The smallest of these proteins was produced by synLD70 and was 48K, about two-thirds the size of the wild-type polypeptide precursor (73K). Physical mapping of the mutations in synLD70 and gC-8 by marker rescue placed these mutations in the middle third of the gC coding sequence. Mapping of the mutations in other gC- mutants, including two in which no protein product was detected, also placed these mutations within or very close to the gC gene. The biochemical and genetic data available on mutants secreting gC gene products suggest that secretion is due to the lack of a functional transmembrane anchor sequence on these mutant glycoproteins.     

Herpes simplex virus type 1 DNA cleavage and encapsidation require the product of the UL28 gene: isolation and characterization of two UL28 deletion mutants.

The herpes simplex virus type 1 UL28 gene contains a 785-amino-acid open reading frame that codes for an essential protein. Studies with temperature-sensitive mutants which map to the UL28 gene indicate that the UL28 gene product (ICP18.5) is required for packaging of viral DNA and for expression of viral glycoproteins on the surface of infected cells (C. Addison, F. J. Rixon, and V. G. Preston, J. Gen. Virol. 71:2377-2384, 1990; B. A. Pancake, D. P. Aschman, and P. A. Schaffer, J. Virol. 47:568-585, 1983). In this study, we describe the isolation of two UL28 deletion mutants that were constructed and propagated in Vero cells transformed with the UL28 gene. The mutants, gCB and gC delta 7B, contained deletions of 1,881 and 537 bp, respectively, in the UL28 gene. Although the mutants synthesize viral DNA, they fail to form plaques or produce infectious virus in cells that do not express the UL28 gene. Transmission electron microscopy and Southern blot analysis demonstrated that both mutants are defective in cleavage and encapsidation of viral DNA. Analysis by cell surface immunofluorescence showed that the UL28 gene is not required for expression of viral glycoproteins on the surface of infected cells. A rabbit polyclonal antiserum was made against an Escherichia coli-expressed Cro-UL28 fusion protein. This antibody reacted with an infected-cell protein having an apparent molecular mass of 87 kDa. The 87-kDa protein was first detected at 6 h postinfection and was expressed as late as 24 h postinfection. No detectable UL28 protein was synthesized in gCB- or gC delta 7B-infected Vero cells.     

Hexon-only binding of VP26 reflects differences between the hexon and penton conformations of VP5, the major capsid protein of herpes simplex virus.

VP26 is a 12-kDa capsid protein of herpes simplex virus 1. Although VP26 is dispensable for assembly, the native capsid (a T=16 icosahedron) contains 900 copies: six on each of the 150 hexons of VP5 (149 kDa) but none on the 12 VP5 pentons at its vertices. We have investigated this interaction by expressing VP26 in Escherichia coli and studying the properties of the purified protein in solution and its binding to capsids. Circular dichroism spectroscopy reveals that the conformation of purified VP26 consists mainly of beta-sheets (approximately 80%), with a small alpha-helical component (approximately 15%). Its state of association was determined by analytical ultracentrifugation to be a reversible monomer-dimer equilibrium, with a dissociation constant of approximately 2 x 10(-5) M. Bacterially expressed VP26 binds to capsids in the normal amount, as determined by quantitative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Cryoelectron microscopy shows that the protein occupies its usual sites on hexons but does not bind to pentons, even when available in 100-fold molar excess. Quasi-equivalence requires that penton VP5 must differ in conformation from hexon VP5: our data show that in mature capsids, this difference is sufficiently pronounced to abrogate its ability to bind VP26.     

Phospholipid turnover and ultrastructural correlates during spontaneous germinal vesicle breakdown of the bovine oocyte: effects of a cyclic AMP phosphodiesterase inhibitor.

The turnover of [32P]orthophosphate in bovine oocyte phospholipids was studied during the early stages of spontaneous meiotic maturation, and during inhibition of this process by the cAMP phosphodiesterase inhibitor 3-isobutyl-1-methyl-xanthine (IBMX). Radioactive lipids were separated by TLC and the meiotic stage was determined cytogenetically. Ultrastructure of the nuclear membrane was examined using transmission EM. During the commitment period to meiotic resumption, which precedes germinal vesicle breakdown (GVBD), small localized convolutions appeared in the intact nuclear membrane. This was accompanied by a decrease in [32P]phosphatidic acid (PA) and an increase in [32P]-phosphatidylcholine (PC). This was followed by extensive convolutions, and subsequent dissociation, of the nuclear membrane, concomitant with a tremendous surge in [32P]PC and [32P]phosphatidylethanolamine (PE). The cAMP-mediated maintenance of meiotic arrest involved retention of entire nuclear envelope integrity and total inhibition of the surge in [32P]PC and [32P]PE which accompanied GVBD. The increase in [32P]phosphatidylinositol (PI) associated with all stages of early meiotic resumption was unaffected by IBMX. Microinjection of heparin inhibited GVBD, and injection of inositol 1,4,5-trisphosphate (IP3) overrode IBMX-maintained meiotic arrest in almost 40% of the oocytes. The results suggest that there may be several functions for phospholipid turnover in the regulation of spontaneous meiotic resumption in the bovine oocyte. The first precedes the commitment period, and involves IP3 generation to serve as the primary signal for meiotic resumption. The second occurs concomitant with the commitment period, is unaffected by the level of intracellular cAMP, and is associated with the general turnover of phospholipid. The third is associated with GVBD, and is cAMP-sensitive, and may represent stimulation of de novo synthesis of phospholipid, thereby permitting disruption of the nuclear membrane.     

Health risk assessment due to exposure of arsenic contamination in drinking water of district Shiekhupura,Punjab, Pakistan

Abstract

The present study was conducted to assess the magnitude and health impacts of As in drinking water. Drinking water samples (n?=?60) were collected from twenty different sites of Shiekhupura District (Pakistan). Health risk assessment through average daily dose (ADD), hazard indices (HI), hazard quotient (HQ), carcinogenic risk (CR), and cancer indices (CI) for dermal and oral exposure were determined. Results revealed that As concentration ranged from 2 to 900?µg?L^?1 in water samples, which was significantly greater than the safe limit of As (10?µg?L^?1) in water. Health risk assessment of As showed that ADD (1.07E?02–9.85E?04), HQ (1.06E+01–9.85E+00), and CR (1.60E?02–9.85E?04) for oral exposure and ADD (1.03E?05–9.69E?06), HQ (1.19E?02–7.96E?03), and CR (1.11E?05–8.98E?05) for dermal exposure which were exceeded the toxic risk index value. Comparison of the two exposure pathways indicated that the oral exposure is much higher risk than the dermal contact. Both values of HI and CI were greater than WHO limit. It is concluded that residents of study area are at higher risk of As induced diseases and carcinogenicity.     

Effectiveness of host cell protein removal using depth filtration with a filter containing diatomaceous earth

The increased cell density and product titer in biomanufacturing have led to greater use of depth filtration as part of the initial clarification of cell culture fluid, either as a stand-alone unit operation or after centrifugation. Several recent studies have shown that depth filters can also reduce the concentration of smaller impurities like host cell proteins (HCP) and DNA, decreasing the burden on subsequent chromatographic operations. The objective of this study was to evaluate the HCP removal properties of the Pall PDH4 depth filter media, a model depth filter containing diatomaceous earth, cellulose fibers, and a binder. Experiments were performed with both cell culture fluid (CCF) and a series of model proteins with defined pI, molecular weight, and hydrophobicity chosen to match the range of typical HCP. The location of adsorbed (fluorescently labeled) proteins within the depth filters was determined using confocal scanning laser microscopy. Protein binding was greater for proteins that were positively charged and more hydrophobic, consistent with adsorption to the negatively charged diatomaceous earth. The lowest degree of binding was seen with proteins near their pI, which were poorly removed by this filter. These results provide new mechanistic insights into the factors governing the filter capacity and performance characteristics of depth filters containing diatomaceous earth that are widely used in the clarification of CCF.     

Wolbachia host shifts: routes,mechanisms, constraints and evolutionary consequences

Wolbachia is one of the most abundant endosymbionts on earth, with a wide distribution especially in arthropods. Effective maternal transmission and the induction of various phenotypes in their hosts are two key features of this bacterium. Here, we review our current understanding of another central aspect of Wolbachia's success: their ability to switch from one host species to another. We build on the proposal that Wolbachia host shifts occur in four main steps: (i) physical transfer to a new species; (ii) proliferation within that host; (iii) successful maternal transmission; and (iv) spread within the host species. Host shift can fail at each of these steps, and the likelihood of ultimate success is influenced by many factors. Some stem from traits of Wolbachia (different strains have different abilities for host switching), others on host features such as genetic resemblance (e.g. host shifting is likely to be easier between closely related species), ecological connections (the donor and recipient host need to interact), or the resident microbiota. Host shifts have enabled Wolbachia to reach its enormous current incidence and global distribution among arthropods in an epidemiological process shaped by loss and acquisition events across host species. The ability of Wolbachia to transfer between species also forms the basis of ongoing endeavours to control pests and disease vectors, following artificial introduction into uninfected hosts such as mosquitoes. Throughout, we emphasise the many knowledge gaps in our understanding of Wolbachia host shifts, and question the effectiveness of current methodology to detect these events. We conclude by discussing an apparent paradox: how can Wolbachia maintain its ability to undergo host shifts given that its biology seems dominated by vertical transmission?