Abstract
Infection and life-long residence in the human nervous system is central to herpes simplex virus (HSV) pathogenesis. Access is gained through innervating axonal projections of sensory neurons. This distinct mode of entry separates the viral genome from tegument proteins, including the potent transactivator of viral IE genes, VP16. This, in turn, promotes a balance between lytic and latent infection which underlies the ability of the virus to invade, disseminate, and set up a large reservoir of latent infections. In the mouse ocular model, TG neurons marked as either “latent” or “lytic” at 48 h postinfection indicated that these programs were selected early and were considered distinct and mutually exclusive. More recently, a temporal analysis of viral program selection revealed a default latent-like state that begins at ~18 h postinfection and in individual neurons, precedes entry into the viral lytic cycle. Studies using refined viral mutants demonstrated that transition out of this latent program depended upon the transactivation function of VP16. Pursuit of the apparent incongruity between the established leaky-late kinetics of VP16 expression with a “preimmediate-early” function led to the discovery of an unrecognized regulatory feature of the HSV-1 VP16 promoter near/downstream of its TATA box. Among three potential sites identified was a putative Egr-1/Sp1 site. Here, we report that a refined mutation of this site, while having no impact on replication in cultured cells or cornea, resulted in ~100-fold reduction in lytic infection in TG in vivo. Notably, the HSV-2 VP16 promoter has 13 direct tandem-repeats upstream of its TATA box forming multiple potential overlapping Egr-1/Sp1 sites. Thus, despite different structures, these promoters might share function in directing the preimmediate-early VP16 protein expression. To test this, the HSV-1 VP16 promoter/5′UTR was replaced by the HSV-2 VP16 promoter/5′UTR in the HSV-1 backbone. Compared to the genomically repaired isolate, the HSV-2 VP16 promoter/5′UTR (1) accelerated the transition into the lytic cycle, and enhanced (2) virulence, and (3) entry into the lytic cycle following a reactivation stressor. These gain-of-function phenotypes support the hypothesis that the VP16 promoter regulates the latent/lytic boundary in neurons and that the HSV-1 and HSV-2 promoter/5′UTRs encode distinct thresholds for this transition.
Highlights
The US and global disease burden resulting from herpes simplex virus (HSV-1 and -2) infection includes life threatening encephalitis, blindness, devastating neonatal infection, increased risk of HIV infection, neurological disease, and a list of other disease outcomes
Simultaneous mutation of three potential transcription factor (TF) sites identified in silico greatly reduced the capacity of HSV-1 to enter lytic infection in sensory neurons in vivo, which requires de novo pre-immediate early Virion protein 16 (VP16) expression (Sawtell and Thompson, 2016a,b)
To test whether Egr-1 and/or Sp1 could bind to this site, the HSV-1 30mer sequence shown (Figure 1B) was employed in electrophoretic mobility shift assays with recombinant Egr-1 and Sp1 proteins and HeLa cell nuclear extracts as described in Section “Materials and Methods.”. Both recombinant Egr-1 and Sp1 proteins bound to the fragment with affinities similar to those found with commercial oligonucleotides for individual Egr-1 and Sp1 sites, and the HSV-1 30mer effectively competed with the canonical commercial oligos
Summary
The US and global disease burden resulting from herpes simplex virus (HSV-1 and -2) infection includes life threatening encephalitis, blindness, devastating neonatal infection, increased risk of HIV infection, neurological disease, and a list of other disease outcomes. Practical treatments to prevent transmission or eliminate the latent reservoir remain unmet goals. Greater insights into the regulatory mechanisms controlling latent and lytic program selection in the nervous system are needed to facilitate progress. Efficient infection by a single HSV-1 virion requires the VP16 transactivation function which is transported into the cell as part of the virion tegument (Campbell et al, 1984; Kristie and Roizman, 1987; Ace et al, 1988; Stern et al, 1989)
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