9 research outputs found
Effects of Phosphorylation of Herpes Simplex Virus 1 Envelope Glycoprotein B by Us3 Kinase In Vivo and In Vitro â–ż
We recently reported that the herpes simplex virus 1 (HSV-1) Us3 protein kinase phosphorylates threonine at position 887 (Thr-887) in the cytoplasmic tail of envelope glycoprotein B (gB) (A. Kato, J. Arii, I. Shiratori, H. Akashi, H. Arase, and Y. Kawaguchi, J. Virol. 83:250-261, 2009; T. Wisner, C. C. Wright, A. Kato, Y. Kawaguchi, F. Mou, J. D. Baines, R. J. Roller and D. C. Johnson, J. Virol. 83:3115-3126, 2009). In the studies reported here, we examined the effect(s) of this phosphorylation on viral replication and pathogenesis in vivo and present data showing that replacement of gB Thr-887 by alanine significantly reduced viral replication in the mouse cornea and development of herpes stroma keratitis and periocular skin disease in mice. The same effects have been reported for mice infected with a recombinant HSV-1 carrying a kinase-inactive mutant of Us3. These observations suggested that Us3 phosphorylation of gB Thr-887 played a critical role in viral replication in vivo and in HSV-1 pathogenesis. In addition, we generated a monoclonal antibody that specifically reacted with phosphorylated gB Thr-887 and used this antibody to show that Us3 phosphorylation of gB Thr-887 regulated subcellular localization of gB, particularly on the cell surface of infected cells
Improved Orange and Red Ca<sup>2+</sup> Indicators and Photophysical Considerations for Optogenetic Applications
We have used protein
engineering to expand the palette of genetically encoded calcium ion
(Ca<sup>2+</sup>) indicators to include orange and improved red fluorescent
variants, and validated the latter for combined use with optogenetic
activation by channelrhodopsin-2 (ChR2). These indicators feature
intensiometric signal changes that are 1.7- to 9.7-fold improved relatively
to the progenitor Ca<sup>2+</sup> indicator, R-GECO1. In the course
of this work, we discovered a photoactivation phenomenon in red fluorescent
Ca<sup>2+</sup> indicators that, if not appreciated and accounted
for, can cause false-positive artifacts in Ca<sup>2+</sup> imaging
traces during optogenetic activation with ChR2. We demonstrate, in
both a beta cell line and slice culture of developing mouse neocortex,
that these artifacts can be avoided by using an appropriately low
intensity of blue light for ChR2 activation
Improved Orange and Red Ca<sup>2+</sup> Indicators and Photophysical Considerations for Optogenetic Applications
We have used protein
engineering to expand the palette of genetically encoded calcium ion
(Ca<sup>2+</sup>) indicators to include orange and improved red fluorescent
variants, and validated the latter for combined use with optogenetic
activation by channelrhodopsin-2 (ChR2). These indicators feature
intensiometric signal changes that are 1.7- to 9.7-fold improved relatively
to the progenitor Ca<sup>2+</sup> indicator, R-GECO1. In the course
of this work, we discovered a photoactivation phenomenon in red fluorescent
Ca<sup>2+</sup> indicators that, if not appreciated and accounted
for, can cause false-positive artifacts in Ca<sup>2+</sup> imaging
traces during optogenetic activation with ChR2. We demonstrate, in
both a beta cell line and slice culture of developing mouse neocortex,
that these artifacts can be avoided by using an appropriately low
intensity of blue light for ChR2 activation