The Us9 Gene Product of Pseudorabies Virus, an Alphaherpesvirus, Is a Phosphorylated, Tail-Anchored Type II Membrane Protein

Abstract

The Us9 gene is highly conserved among the alphaherpesviruses sequenced to date, yet its function remains unknown. In this report, we demonstrate that the pseudorabies virus (PRV) Us9 protein is present in infected cell lysates as several phosphorylated polypeptides ranging from 17 to 20 kDa. Synthesis is first detected at 6 h postinfection and is sensitive to the DNA synthesis inhibitor phosphonoacetic acid. Unlike the herpes simplex virus type 1 Us9 homolog, which was reported to be associated with nucleocapsids in the nuclei of infected cells (M. C. Frame, D. J. McGeoch, F. J. Rixon, A. C. Orr, and H. S. Marsden, Virology 150:321–332, 1986), PRV Us9 localizes to the secretory pathway (predominately to the Golgi apparatus) and not to the nucleus. By fusing the enhanced green fluorescent protein (EGFP) reporter molecule to the carboxy terminus of Us9, we demonstrated that Us9 not only is capable of targeting a Us9-EGFP fusion protein to the Golgi compartment but also is able to direct efficient incorporation of such chimeric molecules into infectious viral particles. Moreover, through protease digestion experiments with Us9-EGFP-containing viral particles, we demonstrated that the Us9 protein is inserted into the viral envelope as a type II, tail-anchored membrane protein.

An Integral Membrane Green Fluorescent Protein Marker, Us9-GFP, Is Quantitatively Retained in Cells during Propidium Iodide-Based Cell Cycle Analysis by Flow Cytometry

“…Here we report the use of a plasmid expressing an integral membrane EGFP fusion protein as a marker for transfected cells in cell cycle analysis by flow cytometry. In this construct, the EGFP gene is fused to the pseudorabies virus Us9 gene and thus is localized to the Golgi apparatus, cytoplasmic vesicles, and the plasma membrane [8]. This fusion protein has unique properties in ethanol-treated cells that make it a markedly more sensitive marker protein in comparison to the GFP fusion proteins that are currently employed for the simulta-neous identification of transfected cells and PI-based cell cycle analysis by flow cytometry [3,6].…”

A Kinesin-3 Recruitment Complex Facilitates Axonal Sorting of Enveloped Alpha Herpesvirus Capsids

“…To confirm the role of the Us7-9 proteins and further test the functionality of the transduced PRV proteins, we used AdV transduction to complement the severe anterograde spread defect of a PRV mutant lacking all three genes (PRV BaBe; ΔUs7-9) ( Figure 1 F). PRV BaBe has spread defects comparable to the parential PRV vaccine strain 'Bartha' in vivo and in vitro (22,28). AdV expressing Us7, Us8, or both in combination failed to complement the spread defect of PRV BaBe.…”

“…Three PRV proteins encoded in a gene cluster (Us7 (glycoprotein I; gI), Us8 (glycoprotein E; gE), and Us9) are required for axonal sorting, and appear to function by recruiting Kif1a to the axonal egress vesicle (17,19,20). Us7 and Us8 encode for glycosylated multifunctional PRV membrane proteins with large cytoplasmic and ecto-domains (21), non-glycosylated Us9 is a small type II membrane protein that appears to function specifically in particle sorting and transport in neurons (22)(23)(24)(25). These proteins are also conserved with HSV-1, where they play similar, but possibly non-identical, roles.…”

A hCXCR1 transgenic mouse model containing a conditional color-switching system for imaging of hCXCL8/IL-8 functions in vivo

“…1A). We chose to use the mRFP1 [29] for Marker 1, and for Marker 2, firefly luciferase fused to a membrane-localizing eGFP [30]. In Marker 2, eGFP localizes to the extracellular side of the plasma membrane and firefly luciferase to the intracellular domain.…”