Two Small RNAs Encoded within the First 1.5 Kilobases of the Herpes Simplex Virus Type 1 Latency-Associated Transcript Can Inhibit Productive Infection and Cooperate To Inhibit Apoptosis

Abstract

The herpes simplex virus type 1 (HSV-1) latency-associated transcript (LAT) is abundantly expressed in latently infected trigeminal ganglionic sensory neurons. Expression of the first 1.5 kb of LAT coding sequences is sufficient for the wild-type reactivation phenotype in small animal models of infection. The ability of the first 1.5 kb of LAT coding sequences to inhibit apoptosis is important for the latency-reactivation cycle. Several studies have also concluded that LAT inhibits productive infection. To date, a functional LAT protein has not been identified, suggesting that LAT is a regulatory RNA. Two small RNAs (sRNAs) were previously identified within the first 1.5 kb of LAT coding sequences. In this study, we demonstrated that both LAT sRNAs were expressed in the trigeminal ganglia of mice latently infected with an HSV-1 strain that expresses LAT but not when mice were infected with a LAT null mutant. LAT sRNA1 and sRNA2 cooperated to inhibit cold shockinduced apoptosis in mouse neuroblastoma cells. LAT sRNA1, but not LAT sRNA2, inhibited apoptosis less efficiently than both sRNAs. When rabbit skin cells were cotransfected with plasmids that express LAT sRNA1 and HSV-1 genomic DNA, the amount of infectious virus released was reduced approximately 3 logs. Although LAT sRNA2 was less effective at inhibiting virus production, it inhibited expression of infected cell protein 4 (ICP4). Neither LAT sRNA had an obvious effect on ICP0 expression. These studies suggested that expression of two LAT sRNAs plays a role in the latency-reactivation cycle by inhibiting apoptosis and productive infection.Most adults in the United States harbor latent herpes simplex virus type 1 (HSV-1) (48, 71) in sensory neurons located in trigeminal ganglia (TG) or sacral dorsal root ganglia (34, 68). Acute infection is typically initiated in the mucocutaneous epithelium. Despite a vigorous immune response during acute infection, HSV-1 establishes latency in sensory neurons. Latent HSV-1 periodically reactivates from latency, resulting in the shedding of infectious virus and various recurrent clinical disorders (reviewed in references 34 and 35).Mice, rabbits, or humans latently infected with HSV-1 express abundant levels of the latency-associated transcript (LAT) in latently infected neurons (12,14,15,38,45,62,65,69,70). The primary LAT transcript is 8.3 kb, and splicing yields a stable 2-kb LAT and an unstable 6.3-kb LAT (14,62,73). The 2-kb LAT can be further spliced in infected neurons (43). The majority of the 2-kb LAT is not capped or polyadenylated and appears to be a stable intron (19,40). In general, HSV-1 LAT null mutants do not reactivate from latency as efficiently as LAT-expressing strains (reviewed in references 34, 35, and 68). Expression of the first 1.5 kb of LAT coding sequences (LAT nucleotides [nt] 1 to 1499) is crucial for wildtype (wt) levels of reactivation in small animal models (28,33,56).LAT reduces apoptosis in infected tissue culture cells (32) and promotes neuronal survival in the TG of infected rabbits (53...

Herpes simplex virus type 1 latency-associated transcript inhibits apoptosis and promotes neurite sprouting in neuroblastoma cells following serum starvation by maintaining protein kinase B (AKT) levels

“…Although AL3 is expressed during latency, it does not appear to inhibit apoptosis directly, because AL3 (mRNA and protein) are expressed in DC-DLAT311 cells . Two small noncoding RNAs within LAT were recently shown to inhibit cold-shock-induced apoptosis (Shen et al, 2009), suggesting that they may influence AKT protein levels and phosphorylation after serum withdrawal. AKT was important with respect to LAT inhibiting apoptosis and promoting neurite sprouting because the AKT inhibitor, AKT VIII, reduced DC-LAT6 cell survival.…”

“…Cold-shock-induced apoptosis was essentially performed as described previously (Geiser et al, 2008;Jin et al, 2004;Shen et al, 2009;Shen & Jones, 2008). In brief, cells were plated at a density of 1610 6 cells in complete growth medium in a T25 plastic flask.…”

Interactions between Herpesvirus Entry Mediator (TNFRSF14) and Latency-Associated Transcript during Herpes Simplex Virus 1 Latency

“…Construction of sncRNA1 and sncRNA2 in the plasmid vector pSilence was described previously (45). Neuro2A cells were transfected with plasmid DNA and Lipofectamine 2000 (Invitrogen, Carlsbad, CA) according to the manufacturer's protocol.…”

A comparison of herpes simplex virus type 1 and varicella-zoster virus latency and reactivation

“…Recently, a neuronal-specific miRNA (miR-138) has been shown to target sequences within HSV-1 ICP0/RF2, reducing ICP0 expression and thus expression of most lytic virus genes (Pan et al, 2014). In addition, two small (961 and 36 nt) HSV-1 RNAs reduce expression of HSV-1 ICP4, productive virus yields and cold shock-induced apoptosis (Shen et al, 2009). Recently, these two HSV-1 small noncoding RNAs were shown to increase neuronal expression of herpesvirus entry mediator (HVEM), an HSV-1 receptor protein in the tumour necrosis factor receptor superfamily that regulates cellular homeostasis (Allen et al, 2014).…”