Incorporation of the green fluorescent protein into the herpes simplex virus type 1 capsid.
The herpes simplex virus type 1 (HSV-1) UL35 open reading frame (ORF) encodes a 12-kDa capsid protein designated VP26. VP26 is located on the outer surface of the capsid specifically on the tips of the hexons that constitute the capsid shell. The bioluminescent jellyfish (Aequorea victoria) green fluorescent protein (GFP) was fused in frame with the UL35 ORF to generate a VP26-GFP fusion protein. This fusion protein was fluorescent and localized to distinct regions within the nuclei of transfected cells following infection with wild-type virus. The VP26-GFP marker was introduced into the HSV-1 (KOS) genome resulting in recombinant plaques that were fluorescent. A virus, designated K26GFP, was isolated and purified and was shown to grow as well as the wild-type virus in cell culture. An analysis of the intranuclear capsids formed in K26GFP-infected cells revealed that the fusion protein was incorporated into A, B, and C capsids. Furthermore, the fusion protein incorporated into the virion particle was fluorescent as judged by fluorescence-activated cell sorter (FACS) analysis of infected cells in the absence of de novo protein synthesis. Cells infected with K26GFP exhibited a punctate nuclear fluorescence at early times in the replication cycle. At later times during infection a generalized cytoplasmic and nuclear fluorescence, including fluorescence at the cell membranes, was observed, confirming visually that the fusion protein was incorporated into intranuclear capsids and mature virions.
Desai
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Virology Laboratories, Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA. pdesai@welchlink.welch.jhu.edu
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D000818:Animals; D002213:Capsid / Q000235:genetics* / Q000378:metabolism; D036022:Capsid Proteins; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular; D016679:Genome, Viral; D049452:Green Fluorescent Proteins; D018259:Herpesvirus 1, Human / Q000235:genetics*; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014709:Vero Cells; D014771:Virion
D036022:Capsid Proteins; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; C077409:capsid protein VP26, herpes simplex virus type 1; D049452:Green Fluorescent Proteins