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Mutational analysis of two herpes simplex virus type 1 late promoters.

ABSTRACT: To investigate the cis-acting sequence elements that are involved in the regulation of herpes simplex virus type 1 late gene expression, linker-scanning mutations were constructed in the promoters of the glycoprotein C and glycoprotein H genes. Each promoter mutation was inserted upstream of the Escherichia coli lacZ gene in a recombinant virus, and the relative activities of beta-galactosidase expressed from individual recombinant viruses were compared. This analysis identified three sequence elements in each promoter: a TATA element, an element that overlapped the start of transcription, and an element downstream from the start of transcription. Primer extension analysis confirmed these results and showed that mutations in either the TATA element or the initiation sequence could eliminate normal transcription initiation. Analysis of expression from hybrid promoters revealed that the TATA and the initiation elements were interchangeable, at least when correctly aligned, and that the initiation element plays a pivotal role in determining the actual site of transcription initiation.

SUBMITTER: Steffy KR

PROVIDER: S-EPMC250683 | biostudies-other | 1991 Dec

REPOSITORIES: biostudies-other

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Mutational analysis of two herpes simplex virus type 1 late promoters.

Steffy K R KR Weir J P JP

Journal of virology 19911201 12

To investigate the cis-acting sequence elements that are involved in the regulation of herpes simplex virus type 1 late gene expression, linker-scanning mutations were constructed in the promoters of the glycoprotein C and glycoprotein H genes. Each promoter mutation was inserted upstream of the Escherichia coli lacZ gene in a recombinant virus, and the relative activities of beta-galactosidase expressed from individual recombinant viruses were compared. This analysis identified three sequence e ...[more]

PMID: 1658354

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Project description:Herpes simplex virus type 1 (HSV-1) establishes latency in human sensory ganglia, during which time the viral genome is transcriptionally silent with the exception of the latency-associated transcripts (LATs). The most abundant LAT is a 2-kb RNA whose biosynthesis is poorly characterized. The 2-kb LAT may be a primary transcript, or its synthesis may involve splicing and/or other forms of processing. Two potential RNA polymerase II promoters (LAP1 and LAP2) upstream of the 2-kb LAT 5' end have been identified. To investigate the role played by LAP1 and LAP2 in the synthesis of the 2-kb LAT under lytic and latent conditions, we analyzed HSV-1 mutants which contain deletions of one or both of these promoters. During lytic infection in cell culture, the cis elements critical for the normal accumulation of the 2-kb LAT were mapped to LAP2, while LAP1 sequences were largely dispensable. The 5' ends of the major 2-kb LATs produced by the wild-type and LAP deletion viruses were examined by primer extension analysis and were all found to be identical (+/- 2 bp). The accumulation of the 2-kb LAT during latent infections of murine trigeminal ganglia was examined by Northern (RNA) blot and by reverse transcription-PCR. In contrast to the results found in lytic infections, the critical cis elements needed for 2-kb LAT accumulation during latency were mapped to LAP1. Deletion of LAP1 resulted in a 500-fold reduction in 2-kb LAT accumulation, whereas deletion of LAP2 resulted in only a 2- to 3-fold reduction. Deletion of both LAP1 and LAP2 resulted in undetectable levels of the 2-kb LAT. Our results indicate that both LAP1 and LAP2 are critical for 2-kb LAT expression but under different conditions. LAP1 is essential for LAT expression during latency, while LAP2 is primarily responsible for LAT expression in lytic infections in cell culture. LAP1 and LAP2 may prove to be functionally independent promoter elements that control 2-kb LAT expression during different stages of HSV-1 infections.

Project description:Herpes simplex virus replicates in the nucleus, where new capsids are assembled. It produces procapsids devoid of nucleic acid but containing the preVP22a scaffold protein. These thermo-unstable particles then mature into A-, B- or C-nuclear icosahedral capsids, depending on their ability to shed the proteolytically processed scaffold and incorporation of the viral genome. To study how these viral capsids differ, we performed proteomics studies of highly enriched HSV-1 A-, B- and C-nuclear capsids, relying in part on a novel and powerful flow virometry approach to purify C-capsids. We found that the viral particles contained the expected capsid components and identified several tegument proteins in the C-capsid fraction (pUL21, pUL36, pUL46, pUL48, pUL49, pUL50, pUL51 and pUS10). Moreover, numerous ribosomal, hnRNPs and other host proteins, absent from the uninfected controls, were detected on the capsids with some of them seemingly specific to C-capsids (glycogen synthase, four different keratin-related proteins, fibronectin 1 and PCBP1). A subsequent proteomics analysis was performed to rule out the presence of protein complexes that may share similar density as the viral capsids but do not otherwise interact with them. Using pUL25 or VP5 mutant viruses incapable of assembling C-nuclear or all nuclear capsids, respectively, we confirmed the bulk of our initial findings. Naturally, it will next be important to address the functional relevance of these proteins.IMPORTANCE Much is known about the biology of herpesviruses. This includes their unique ability to traverse the two nuclear envelopes by sequential budding and fusion steps. For HSV-1, this implies the pUL31/pUL34 and pUL17/pUL25 complexes that may favor C-capsid egress. However, this selection process is not clear, nor are all the differences that distinguish A-, B- and C-capsids. The present study probes what proteins compose these capsids, including host proteins. This should open up new research avenues to clarify the biology of this most interesting family of viruses. It also reiterates the use of flow virometry as an innovative tool to purify viral particles.

Dataset Information

Mutational analysis of two herpes simplex virus type 1 late promoters.

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Mutational analysis of two herpes simplex virus type 1 late promoters.

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