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.
Lock
M
M
Department of Microbiology, University of Pennsylvania, Philadelphia, Pennsylvania 19104-6076, USA.
Miller
C
C
Fraser
N W
NW
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