Partial resistance to gD-mediated interference conferred by mutations affecting herpes simplex virus type 1 gC and gK.

Cells expressing herpes simplex virus (HSV) gD can be resistant to HSV entry as a result of gD-mediated interference. HSV strains differ in sensitivity to this interference, which blocks viral penetration but not binding. Previous studies have shown that mutations or variations in virion-associated gD can confer resistance to gD-mediated interference. Here we show that HSV-1 mutants selected for enhanced ability to bind and penetrate in the presence of inhibitory concentrations of heparin were partially resistant to gD-mediated interference. The resistance was largely due to the presence of two mutations: one in gC (the major heparin-binding glycoprotein) resulting in the absence of gC expression and the other in gK resulting in a syncytial phenotype. The results imply that heparin selected for mutants with altered postbinding requirements for entry. Resistance to gD-mediated interference conferred by mutations affecting gC and gK has not been previously described.     

Human herpesvirus 8 glycoprotein B (gB), gH, and gL can mediate cell fusion

Herpesvirus entry into cells and herpesvirus-induced cell fusion are related processes in that virus penetration proceeds by fusion of the viral envelope and cell membrane. To characterize the human herpesvirus 8 (HHV-8) glycoproteins that can mediate cell fusion, a luciferase reporter gene activation assay was used. Chinese hamster ovary (CHO) cells expressing the HHV-8 glycoproteins of interest along with a luciferase reporter gene under the control of the T7 promoter were cocultivated with human cells transfected with T7 RNA polymerase. Because HHV-8 glycoprotein B (gB) expressed in CHO cells localizes to the perinuclear region, a truncated form of gB (designated gB(MUT)) that lacks putative endocytosis signals was constructed by deletion of the distal 58 amino acids of the cytoplasmic tail. HHV-8 gB(MUT) was expressed efficiently on the surface of CHO cells. HHV-8 gB, gH, and gL could mediate the fusion of CHO cells with two different human cell types, embryonic kidney cells and B lymphocytes. Substituting gB(MUT) for gB significantly enhanced the fusion of CHO cells with human embryonic kidney cells but not B lymphocytes. Thus, two human cell types known to be susceptible to HHV-8 entry were also suitable targets for cell fusion induced by HHV-8 gB, gH, and gL. For human embryonic kidney cells and B cells at least, optimal fusion was noted with the expression of all three HHV-8 glycoproteins.     

Vif counteracts a cyclophilin A-imposed inhibition of simian immunodeficiency viruses in human cells

Vif is a primate lentiviral accessory protein that is crucial for viral infectivity. Vif counteracts the antiviral activity of host deaminases such as APOBEC3G and APOBEC3F. We now report a novel function of African green monkey simian immunodeficiency virus (SIVagm) Vif that promotes replication of SIVagm in human cells lacking detectable deaminase activity. We found that cyclophilin A (CypA) was excluded from wild-type SIV particles but was efficiently packaged into vif-deficient SIVagm virions. The presence of CypA in vif-defective SIVagm was correlated with reduced viral replication. Infection of CypA knockout Jurkat cells or treatment of Jurkat cells with cyclosporine A eliminated the Vif-sensitive inhibition and resulted in replication profiles that were similar for wild-type and vif-deficient SIVagm. Importantly, the inhibitory effect of CypA was restricted to virus-producing cells and was TRIM5alpha independent. The abilities of SIVagm Vif to inhibit encapsidation of CypA and to increase viral infectivity were shared by rhesus macaque SIV Vif and thus seem to be general properties of SIV Vif proteins. Exclusion of CypA from SIVagm particles was not associated with intracellular degradation, suggesting a mode of Vif action distinct from that proposed for APOBEC3G. This is the first report of a novel vif-sensitive antiviral activity of human CypA that may limit zoonotic transmission of SIV and the first demonstration of CypA encapsidation into a virus other than human immunodeficiency virus type 1.     

IFITM3 inhibits influenza A virus infection by preventing cytosolic entry

To replicate, viruses must gain access to the host cell's resources. Interferon (IFN) regulates the actions of a large complement of interferon effector genes (IEGs) that prevent viral replication. The interferon inducible transmembrane protein family members, IFITM1, 2 and 3, are IEGs required for inhibition of influenza A virus, dengue virus, and West Nile virus replication in vitro. Here we report that IFN prevents emergence of viral genomes from the endosomal pathway, and that IFITM3 is both necessary and sufficient for this function. Notably, viral pseudoparticles were inhibited from transferring their contents into the host cell cytosol by IFN, and IFITM3 was required and sufficient for this action. We further demonstrate that IFN expands Rab7 and LAMP1-containing structures, and that IFITM3 overexpression is sufficient for this phenotype. Moreover, IFITM3 partially resides in late endosomal and lysosomal structures, placing it in the path of invading viruses. Collectively our data are consistent with the prediction that viruses that fuse in the late endosomes or lysosomes are vulnerable to IFITM3's actions, while viruses that enter at the cell surface or in the early endosomes may avoid inhibition. Multiple viruses enter host cells through the late endocytic pathway, and many of these invaders are attenuated by IFN. Therefore these findings are likely to have significance for the intrinsic immune system's neutralization of a diverse array of threats.     

Isoproterenol induces actin depolymerization in human airway smooth muscle cells via activation of an Src kinase and GS

In a previous study, we showed that isoproterenol induced actin depolymerization in human airway smooth muscle cells by both protein kinase A (PKA)-dependent and -independent signaling pathways. We now investigate the signaling pathway of PKA-independent actin depolymerization induced by isoproterenol in these cells. Cells were briefly exposed to isoproterenol or PGE(1) in the presence and absence of specific inhibitors of Src-family tyrosine kinases, phosphatidylinositol-3-kinase (PI3 kinase), or MAP kinase, and actin depolymerization was measured by concomitant staining of filamentous actin with FITC-phalloidin and globular actin with Texas red DNase I. Isoproterenol, cholera toxin, and PGE(1) induced actin depolymerization, indicated by a decrease in the intensity of filamentous/globular fluorescent staining. Pretreatment with the Src kinase inhibitors 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyriimidine (PP2) or geldanamycin or the PKA inhibitor Rp-cAMPS only partly inhibited isoproterenol- or PGE(1)-induced actin depolymerization. In contrast, PP2 and geldanamycin did not inhibit forskolin-induced actin depolymerization, and AG-213 (an EGF receptor tyrosine kinase inhibitor) did not inhibit isoproterenol- or PGE(1)-induced actin depolymerization. PI3 kinase or MAP kinase inhibition did not inhibit isoproterenol-induced actin depolymerization. Moreover, isoproterenol but not forskolin induced tyrosine phosphorylation of an Src family member at position 416. These results further confirm that both PKA-dependent and PKA-independent pathways mediate actin depolymerization in human airway smooth muscle cells and that the PKA-independent pathway by which isoproterenol induces actin depolymerization in human airway smooth muscle cells involves Src protein tyrosine kinases and the G(s) protein.     

Biological controls upon the physical taphonomy of exceptionally preserved salamanders from the Miocene of Rubielos de Mora,northeast Spain

McNamara, M.E., Orr, P.J., Manzocchi, T., Alcalá, L., Anadón, P. & Peñalver, E. 2011: Biological controls upon the physical taphonomy of exceptionally preserved salamanders from the Miocene of Rubielos de Mora, northeast Spain. Lethaia, Vol. 45, pp. 210–226. The middle Miocene Rubielos de Mora Konservat‐Lagerstätte of northeast Spain is hosted within profundal, finely laminated, lacustrine mudstones. The diverse biota includes abundant salamanders. Most individuals died during separate episodes and sank rapidly postmortem. Specimens are typically preserved in dorso‐ventral aspect, the most hydrodynamically stable orientation. The near‐cylindrical morphology of the body, however, allowed some carcasses to settle in or subsequently re‐orientate into, lateral orientations. Loss of skeletal elements (i.e. reduced completeness) reflects their location within the body and followed a distal to proximal trend. Two stages are identified: initial loss of a small number of phalanges, followed by loss of more proximal limb bones plus additional phalanges. Disarticulation is more complex: it occurred via several mechanisms (notably, abdominal rupture and re‐orientation of part of the body and limbs during decay) and shows no consistent pattern among specimens. The physical taphonomy of the salamanders is controlled predominantly by intrinsic biological factors, i.e. the geometry of the body and of individual skeletal elements, the orientation, inherent strength and location of specific joints and the extent to which soft tissues, particularly the skin, persist during decay. These biological factors probably control patterns of physical taphonomy of other fossil tetrapods with a similar skeletal configuration. □Articulation, completeness, Konservat‐Lagerstätten, orientation, quantitative taphonomy, salamanders.     

Systems analysis of primary Sjögren's syndrome pathogenesis in salivary glands identifies shared pathways in human and a mouse model

Bone tissue has an exceptional quality to regenerate to native tissue in response to injury. However, the fracture repair process requires mechanical stability or a viable biological microenvironment or both to ensure successful healing to native tissue. An improved understanding of the molecular and cellular events that occur during bone repair and remodeling has led to the development of biologic agents that can augment the biological microenvironment and enhance bone repair. Orthobiologics, including stem cells, osteoinductive growth factors, osteoconductive matrices, and anabolic agents, are available clinically for accelerating fracture repair and treatment of compromised bone repair situations like delayed unions and nonunions. Preclinical and clinical studies using biologic agents like recombinant bone morphogenetic proteins have demonstrated an efficacy similar or better than that of autologous bone graft in acute fracture healing. A lack of standardized outcome measures for comparison of biologic agents in clinical fracture repair trials, frequent off-label use, and a limited understanding of the biological activity of these agents at the bone repair site have limited their efficacy in clinical applications.