Summary auto-generated
This study investigates how herpes simplex virus type 1 (HSV-1) produces major latency-associated transcripts (LATs), which are the only viral genes expressed during latent neuronal infection. The researchers hypothesized that major LATs are stable introns generated by splicing from a larger precursor transcript, and that this splicing involves specific splice donor and acceptor sites flanking the major LAT region. To test this, they constructed mutant viruses with disrupted splice sites using site-directed mutagenesis while preserving the nearby IE110 gene function. During productive infection in cultured cells, disrupting the splice acceptor site completely eliminated 2 kb LAT production, and disrupting the splice donor site significantly reduced it. However, in latently infected mouse neurons, the mutations had minimal effects on LAT production. The 2 kb and 1.5 kb major LATs were produced at near-normal levels even when both splice sites were mutated. These findings demonstrate that the proposed splice sites function during productive infection but are largely dispensable for major LAT generation during latent infection, suggesting that LATs are generated through different mechanisms depending on the infection state.
Key findings
- Splice acceptor and donor sites flanking major LATs are essential for 2 kb LAT production during productive lytic infection in cultured cells
- Mutation of splice sites significantly reduces or eliminates major LAT synthesis in productively infected non-neuronal cells
- The same splice site mutations have no significant effect on major LAT production during latent infection in mouse neurons
- Suggests alternate splicing mechanisms or processing pathways are used to generate LATs during latent versus lytic infection
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Abstract
The herpes simplex virus type 1 (HSV-1) latency-associated transcripts (LATs) are the only viral gene products expressed within latently infected neurones. The most abundant (major) LATs consist of two collinear nuclear polyA- RNAs of 2 kb and 1.5 kb which it has been suggested represent stable introns derived from a less abundant primary transcript (minor LAT). Consistent with this proposition is the identification of consensus splice donor and acceptor sites flanking major LATs which are conserved between HSV types 1 and 2. Here we test the functionality of the predicted splice sites within the context of the virus genome during productive infection in vitro and latent infection in vivo. To this end viruses in which the LAT splicing signals were disrupted by site-directed mutagenesis were constructed. We report that mutation of the splice acceptor site abrogates 2 kb major LAT generation during productive infection but does not significantly influence major LAT synthesis during neuronal latency. Similarly, mutation of the splice donor site significantly reduces levels of 2 kb major LAT during productive infection but has no detectable effect on the generation of 2 kb major LAT during neuronal latency as assessed by Northern and in situ hybridization analyses of latently infected neuronal tissue. From these data it can be concluded that the proposed splice sites flanking the major LAT region are dispensable for 2 kb major LAT production in neurones latently infected with HSV-1 but constitute functional splicing signals in productively infected non-neuronal cells.