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Journal of Virology, April 2001, p. 3413-3426, Vol. 75, No. 7
0022-538X/01/$04.00+0   DOI: 10.1128/JVI.75.7.3413-3426.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Analysis of Protein Expression from within the Region Encoding the 2.0-Kilobase Latency-Associated Transcript of Herpes Simplex Virus Type 1

Martin Lock, Cathie Miller, and Nigel W. Fraser*

Department of Microbiology, University of Pennsylvania, Philadelphia, Pennsylvania 19104-6076

Received 8 August 2000/Accepted 4 January 2001

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 initator 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.

* Corresponding author. Mailing address: Department of Microbiology, University of Pennsylvania, 315 Johnson Pavilion, Philadelphia, PA 19104-6076. Phone: (215) 898-3847. Fax: (215) 898-3849. E-mail: nfraser{at}mail.med.upenn.edu.

Journal of Virology, April 2001, p. 3413-3426, Vol. 75, No. 7
0022-538X/01/$04.00+0   DOI: 10.1128/JVI.75.7.3413-3426.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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