Analysis of protein expression from within the region encoding the 2.0-kilobase latency-associated transcript of herpes simplex virus type 1.
During latent infections of sensory neurons, herpes simplex virus type 1 gene expression is restricted to the latency-associated transcripts (LATs). The association of the stable 2.0-kb LAT intron with polysomes has suggested that it might represent a novel mRNA. In this work, we investigated expression of 2.0-kb LAT open reading frames (ORFs) by inserting the gene for green fluorescent protein (GFP) within the 2.0-kb LAT sequence, both within a LAT expression plasmid and in the context of the virus. Upon transient transfection of cells of both neuronal and nonneuronal origin with LAT-GFP expression vectors, low-level GFP fluorescence was distributed over the cell cytoplasm and likely resulted from infrequent initiation at a GFP AUG codon, on either unspliced or alternately spliced LAT RNAs. A second nucleolar GFP expression pattern which resulted from fusion of GFP to a conserved ORF in exon 1 of the LAT gene was also observed. However, the abundant expression of this fusion protein was dependent upon an artificially added translation initiation codon. Expression was much reduced and restricted to a small subset of transfected cells when this initiator codon was removed. Neither the 2.0-kb LAT-GFP intron itself nor transcripts originating from the latency-associated promoter 2 (LAP2) were responsible for GFP expression. Abundant alternate splicing involving the 1.5-kb LAT splice acceptor and including splicing between the 1.5-kb LAT splice donor and acceptor, was observed in the nonneuronal Cos-1 cell line. Contrary to the results of our transfection studies, GFP expression could not be detected from a LAT-GFP virus at any stage of the infection cycle. Our results suggest that the inhibition of LAT ORF expression during viral infection occurred primarily at the level of translation.
Department of Microbiology, University of Pennsylvania, Philadelphia, Pennsylvania 19104-6076, USA.
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Lock
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Miller
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Fraser
D020121:5' Untranslated Regions / Q000378:metabolism; D017398:Alternative Splicing; D000818:Animals; D002467:Cell Nucleus / Q000378:metabolism; D002478:Cells, Cultured; D005091:Exons; D049452:Green Fluorescent Proteins; D018259:Herpesvirus 1, Human / Q000235:genetics*; D006801:Humans; D007438:Introns; D008164:Luminescent Proteins / Q000235:genetics; D051379:Mice; D008807:Mice, Inbred BALB C; D016366:Open Reading Frames; D012333:RNA, Messenger / Q000096:biosynthesis*; D012367:RNA, Viral / Q000096:biosynthesis*; D017735:Virus Latency
D020121:5' Untranslated Regions; D008164:Luminescent Proteins; D012333:RNA, Messenger; D012367:RNA, Viral; D049452:Green Fluorescent Proteins