Interactions of herpes simplex virus type 1 with ND10 and recruitment of PML to replication compartments

Many of the events required for productive herpes simplex virus type 1 (HSV-1) infection occur within globular nuclear domains called replication compartments, whose formation appears to depend on interactions with cellular nuclear domains 10 (ND10). We have previously demonstrated that the formation of HSV-1 replication compartments involves progression through several stages, including the disruption of intact ND10 (stage I to stage II) and the formation of PML-associated prereplicative sites (stage III) and replication compartments (stage IV) (J. Burkham, D. M. Coen, and S. K. Weller, J. Virol. 72:10100-10107, 1998). In this paper, we show that some, but not all, PML isoforms are recruited to stage III foci and replication compartments. Genetic experiments showed that the recruitment of PML isoforms to stage III prereplicative sites and replication compartments requires the localization of the HSV-1 polymerase protein (UL30) to these foci but does not require polymerase catalytic activity. We also examined the stages of viral infection under conditions affecting ND10 integrity. Treatment with factors that increase the stability of ND10, arsenic trioxide and the proteasome inhibitor MG132, inhibited viral disruption of ND10, formation of replication compartments, and production of progeny virus. These results strengthen the previously described correlation between ND10 disruption and productive viral infection.     

Identification of crucial hydrogen-bonding residues for the interaction of herpes simplex virus DNA polymerase subunits via peptide display, mutational, and calorimetric approaches

The catalytic subunit, Pol, of herpes simplex virus DNA polymerase interacts via its extreme C terminus with the processivity subunit, UL42. This interaction is critical for viral replication and thus a potential target for antiviral drug action. To investigate the Pol-binding region on UL42, we engineered UL42 mutations but also used random peptide display to identify artificial ligands of the Pol C terminus. The latter approach selected ligands with homology to residues 171 to 176 of UL42. Substitution of glutamine 171 with alanine greatly impaired binding to Pol and stimulation of long-chain DNA synthesis by Pol, identifying this residue as crucial for subunit interactions. To study these interactions quantitatively, we used isothermal titration calorimetry and wild-type and mutant forms of Pol-derived peptides and UL42. Each of three peptides corresponding to either the last 36, 27, or 18 residues of Pol bound specifically to UL42 in a 1:1 complex with a dissociation constant of 1 to 2 microM. Thus, the last 18 residues suffice for most of the binding energy, which was due mainly to a change in enthalpy. Substitutions at positions corresponding to Pol residue 1228 or 1229 or at UL42 residue 171 abolished or greatly reduced binding. These residues participate in hydrogen bonds observed in the crystal structure of the C terminus of Pol bound to UL42. Thus, interruption of these few bonds is sufficient to disrupt the interaction, suggesting that small molecules targeting the relevant side chains could interfere with Pol-UL42 binding.     

Vasoconstrictor effects of insulin in skeletal muscle arterioles are mediated by ERK1/2 activation in endothelium

Insulin exerts both NO-dependent vasodilator and endothelin-dependent vasoconstrictor effects on skeletal muscle arterioles. The intracellular enzymes 1-phosphatidylinositol 3-kinase (PI3-kinase) and Akt have been shown to mediate the vasodilator effects of insulin, but the signaling molecules involved in the vasoconstrictor effects of insulin in these arterioles are unknown. Our objective was to identify intracellular mediators of acute vasoconstrictor effects of insulin on skeletal muscle arterioles. Rat cremaster first-order arterioles (n=40) were isolated, and vasoreactivity to insulin was studied using a pressure myograph. Insulin induced dose-dependent vasoconstriction of skeletal muscle arterioles (up to -22 +/- 3% of basal diameter; P <0.05) during PI3-kinase inhibition with wortmannin (50 nmol/l). Insulin-induced vasoconstriction was abolished by inhibition of extracellular signal-regulated kinase 1/2 (ERK1/2) with PD-98059 (40 micromol/l). In addition, inhibition of ERK1/2 without PI3-kinase inhibition uncovered insulin-mediated vasodilatation in skeletal muscle arterioles (up to 37 +/- 10% of baseline diameter; P <0.05). Effects of insulin on ERK1/2 activation in arterioles were then investigated by Western blot analysis. Insulin induced a transient 2.4-fold increase in ERK1/2 phosphorylation (maximal at approximately 15 min) in skeletal muscle arterioles (P <0.05). Removal of the arteriolar endothelium abolished insulin-induced vasoconstriction, which suggests that activation of ERK1/2 in endothelial cells is involved in acute insulin-mediated vasoconstriction. To investigate this, acute effects of insulin on ERK1/2 phosphorylation were studied in human microvascular endothelial cells. In support of the findings in skeletal muscle arterioles, insulin induced a 1.9-fold increase in ERK1/2 phosphorylation (maximal at approximately 15 min) in microvascular endothelial cells (P <0.05). We conclude that acute vasoconstrictor effects of insulin in skeletal muscle arterioles are mediated by activation of ERK1/2 in endothelium. This ERK1/2-mediated vasoconstrictor effect antagonizes insulin-induced, PI3-kinase-dependent vasodilatation in skeletal muscle arterioles. These findings provide a novel mechanism by which insulin may determine blood flow and glucose disposal in skeletal muscle.     

A centuries-long epidemic of scrapie in British sheep?

The apparent persistence of scrapie in British sheep for more than 250 years is difficult to explain. Susceptibility to scrapie is associated with particular alleles at a single locus, the PrP gene. As the only known effect of these alleles is to confer susceptibility to a fatal disease, natural selection is expected to reduce their frequency, as has been observed in practice during scrapie outbreaks in single sheep flocks. Susceptibility alleles, and hence scrapie itself, are therefore expected to become rare, yet the disease remains widespread. We suggest that the paradox of scrapie's persistence can be explained by the exceptionally long time-scales inherent in the epidemiology of the disease. It is proposed that scrapie should be regarded as epidemic in British sheep but, unlike more familiar epidemics, which have time-scales of months or years, the scrapie epidemic has a time-scale of centuries. This interpretation implies that scrapie should eventually disappear from the sheep population.     

The cycloidea gene can respond to a common dorsoventral prepattern in Antirrhinum

Dorsoventral asymmetry in flowers of Antirrhinum depends on expression of the cycloidea gene in dorsal regions of floral meristems. To determine how cycloidea might be regulated we analysed its expression in several contexts. We show that cycloidea is activated shortly after floral induction, and that in addition to flowers, cycloidea can be asymmetrically expressed in shoots, even though these shoots show no marked dorsoventral asymmetry. Shoots expressing cycloidea include secondary branches lying just below the inflorescence, and shoots of floricaula mutants. Asymmetric cycloidea expression may also be observed within organ primordia, such as the sepals of terminal flowers produced by centroradialis mutants. Later expression of cycloidea within flowers can be modified by mutations in organ identity genes. Taken together, the results suggest that cycloidea can respond to a common dorsoventral pre-pattern in the apex and that the specific effects of cycloidea on the flower depend on interactions with floral-specific genes.