The UL12 protein of herpes simplex virus 1 is regulated by tyrosine phosphorylation.

UNLABELLED: The herpes simplex virus 1 (HSV-1) UL12 protein (pUL12) is a nuclease that is critical for viral replication in vitro and neurovirulence in vivo. In this study, mass spectrometric analysis of pUL12 and phosphate-affinity SDS-polyacrylamide gel electrophoresis analysis identified tyrosine at pUL12 residue 371 (Tyr-371) as a pUL12 phosphorylation site: Tyr-371 is conserved in pUL12 homologs in herpesviruses in all Herpesviridae subfamilies. Replacement of Tyr-371 with phenylalanine (Y371F) in pUL12 (i) abolished its exonuclease activity in HSV-1-infected Vero, HEL, and A549 cells, (ii) reduced viral replication, cell-cell spread, and pUL12 expression in infected cells in a cell type-dependent manner, (iii) led to aberrant subcellular localization of pUL12 in infected cells in a cell type-dependent manner, and (iv) reduced HSV-1 neurovirulence in mice. The effects of the pUL12 Y371F mutation in cell cultures and mice were similar to those of a nuclease-dead double mutation in pUL12, although the Y371F mutation reduced viral replication severalfold more than the nuclease-dead double mutation in a cell type- and multiplicity-of-infection-dependent manner. Replacement of Tyr-371 with glutamic acid, which mimics constitutive phosphorylation, restored the wild-type phenotype in cell cultures and mice. These results suggested that phosphorylation of pUL12 Tyr-371 was essential for pUL12 to express its nuclease activity in HSV-1-infected cells and that this phosphorylation promoted viral replication and cell-cell spread in cell cultures and neurovirulence in mice mainly by upregulating pUL12 nuclease activity and, in part, by regulating the subcellular localization and expression of pUL12 in HSV-1-infected cells. IMPORTANCE: Herpesviruses encode a considerable number of enzymes for their replication. Like cellular enzymes, the viral enzymes need to be properly regulated in infected cells. Although the functional aspects of herpesvirus enzymes have gradually been clarified, information on how most of these enzymes are regulated in infected cells is lacking. In the present study, we report that the enzymatic activity of the herpes simplex virus 1 alkaline nuclease pUL12 was regulated by phosphorylation of pUL12 Tyr-371 in infected cells and that this phosphorylation promoted viral replication and cell-cell spread in cell cultures and neurovirulence in mice, mainly by upregulating pUL12 nuclease activity. Interestingly, pUL12 and tyrosine at pUL12 residue 371 appeared to be conserved in all herpesviruses in the family Herpesviridae, raising the possibility that the herpesvirus pUL12 homologs may also be regulated by phosphorylation of the conserved tyrosine residue.

Role of the nuclease activities encoded by herpes simplex virus 1 UL12 in viral replication and neurovirulence.

Enzyme-dead mutations in the herpes simplex virus 1 UL12 gene that abolished its endo- and exonuclease activities only slightly reduced viral replication in cell cultures. However, the UL12 null mutation significantly reduced viral replication, suggesting that a UL12 function(s) unrelated to its nuclease activities played a major role in viral replication. In contrast, the enzyme-dead mutations significantly reduced viral neurovirulence in mice, suggesting that UL12 nuclease activities were critical for viral pathogenesis in vivo.

Neurula rotation determines left-right asymmetry in ascidian tadpole larvae.

Tadpole larvae of the ascidian Halocynthia roretzi show morphological left-right asymmetry. The tail invariably bends towards the left side within the vitelline membrane. The structure of the larval brain is remarkably asymmetric. nodal, a conserved gene that shows left-sided expression, is also expressed on the left side in H. roretzi but in the epidermis unlike in vertebrates. We show that nodal signaling at the late neurula stage is required for stereotypic morphological left-right asymmetry at later stages. We uncover a novel mechanism to break embryonic symmetry, in which rotation of whole embryos provides the initial cue for left-sided expression of nodal. Two hours prior to the onset of nodal expression, the neurula embryo rotates along the anterior-posterior axis in a counterclockwise direction when seen in posterior view, and then this rotation stops when the left side of the embryo is oriented downwards. It is likely that epidermis monocilia, which appear at the neurula rotation stage, generate the driving force for the rotation. When the embryo lies on the left side, protrusion of the neural fold physically prevents it from rotating further. Experiments in which neurula rotation is perturbed by various means, including centrifugation and sandwiching between glass, indicate that contact of the left epidermis with the vitelline membrane as a consequence of neurula rotation promotes nodal expression in the left epidermis. We suggest that chemical, and not mechanical, signals from the vitelline membrane promote nodal expression. Neurula rotation is also conserved in other ascidian species.