Fluorescent tagging of herpes simplex virus tegument protein VP13/14 in virus infection.
The cellular site of herpesvirus tegument assembly has yet to be defined. We have previously used a recombinant herpes simplex virus type 1 expressing a green fluorescent protein (GFP)-tagged tegument protein, namely VP22, to show that VP22 is localized exclusively to the cytoplasm during infection. Here we have constructed a similar virus expressing another fluorescent tegument protein, YFP-VP13/14, and have visualized the intracellular localization of this second tegument protein in live infected cells. In contrast to VP22, VP13/14 is targeted predominantly to the nuclei of infected cells at both early and late times in infection. More specifically, YFP-13/14 localizes initially to the nuclear replication compartments and then progresses into intense punctate domains that appear at around 12 h postinfection. At even later times this intranuclear punctate fluorescence is gradually replaced by perinuclear micropunctate and membranous fluorescence. While the vast majority of YFP-13/14 seems to be targeted to the nucleus, a minor subpopulation also appears in a vesicular pattern in the cytoplasm that closely resembles the pattern previously observed for GFP-22. Moreover, at late times weak fluorescence appears at the cell periphery and in extracellular virus particles, confirming that YFP-13/14 is assembled into virions. This predominantly nuclear targeting of YFP-13/14 together with the cytoplasmic targeting of VP22 may imply that there are multiple sites of tegument protein incorporation along the virus maturation pathway. Thus, our YFP-13/14-expressing virus has revealed the complexity of the intracellular targeting of VP13/14 and provides a novel insight into the mechanism of tegument, and hence virus, assembly.
Virus Assembly Group, Marie Curie Research Institute, The Chart, Oxted, Surrey RH8 0TL, United Kingdom.
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Donnelly
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Elliott
D000818:Animals; D019556:COS Cells; D002467:Cell Nucleus / Q000378:metabolism*; D002522:Chlorocebus aethiops; D049452:Green Fluorescent Proteins; D018259:Herpesvirus 1, Human / Q000502:physiology*; D007091:Image Processing, Computer-Assisted; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism*; D018613:Microscopy, Confocal; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014709:Vero Cells; D014760:Viral Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D014771:Virion / Q000378:metabolism; D019065:Virus Assembly; D014779:Virus Replication
D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; C085822:VP13-14 protein, herpes simplex virus type 1; D014760:Viral Fusion Proteins; D049452:Green Fluorescent Proteins