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 ge ne expression is restricted to the latency-associated transcripts (LATs). T he 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 expres sion of 2.0-kb LAT open reading frames (ORFs) by inserting the gene for gre en 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 tran sfection of cells of both neuronal and nonneuronal origin with LAT-GFP expr ession vectors, few-level GFP fluorescence was distributed over the cell cy toplasm 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 translatio n initiation codon. Expression was much reduced and restricted to a small s ubset of transfected cells when this initator codon was removed. Neither th e 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 transfectio n 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 W T ORF expression during viral infection occurred primarily at the level of translation.