A Herpes Simplex Virus Type 1 Mutant Expressing a Baculovirus Inhibitor of Apoptosis Gene in Place of Latency-Associated Transcript Has a Wild-Type Reactivation Phenotype in the Mouse

Abstract

The latency-associated transcript (LAT) is essential for the wild-type herpes simplex virus type 1 (HSV-1) high-reactivation phenotype since LAT ؊ mutants have a low-reactivation phenotype. We previously reported that LAT can decrease apoptosis and proposed that this activity is involved in LAT's ability to enhance the HSV-1 reactivation phenotype. The first 20% of the primary 8.3-kb LAT transcript is sufficient for enhancing the reactivation phenotype and for decreasing apoptosis, supporting this proposal. For this study, we constructed an HSV-1 LAT ؊ mutant that expresses the baculovirus antiapoptosis gene product cpIAP under control of the LAT promoter and in place of the LAT region mentioned above. Mice were ocularly infected with this mutant, designated dLAT-cpIAP, and the reactivation phenotype was determined using the trigeminal ganglion explant model. dLAT-cpIAP had a reactivation phenotype similar to that of wild-type virus and significantly higher than that of (i) the LAT Herpes simplex virus type 1 (HSV-1) establishes lifelong latent infections in host sensory neurons. This virus is widespread in the general population. When the eye is infected, the virus spreads from epithelial cells to the peripheral nerve endings and then travels to the trigeminal ganglia (TG) via retrograde axonal transport and establishes latent infection in sensory neurons of the TG. During neuronal latency, HSV-1 has no apparent impact on the infected individual. However, the latent virus can reactivate sporadically throughout the life of the individual. This occurs through a mechanism or mechanisms that are currently not fully understood. HSV-1 reactivation in the TG results in the virus returning to the eye via anterograde axonal transport. At the eye, reactivated HSV-1 can replicate, and infectious virus is shed in tears. Recurrent ocular HSV-1 infection may cause corneal disease leading to corneal scarring and loss of vision. Consequently, HSV-1 is one of the most common infectious causes of corneal blindness in the developed world.During HSV-1 neuronal latency, the latency-associated transcript (LAT) is the only abundantly transcribed viral gene (39,45). The primary LAT transcript is approximately 8.3 kb long (11, 53) and partially or completely overlaps three viral genes, those encoding AL, ICP0, and ICP34.5, in an antisense direction (33,39,45). A very stable intron, the 2-kb LAT, is spliced from the primary transcript (12) and is the major LAT RNA detected during latency (11,42,44,(50)(51)(52). LAT Ϫ mutants have impaired reactivation phenotypes in small-animal models (3,9,17,23,29,36,40,43), indicating that a LAT function enhances the HSV-1 reactivation phenotype. However, the nature of this LAT function remains unresolved.It has been proposed that LAT may affect the latency reactivation cycle by (i) antisense regulation of the important immediate-early genes for ICP0 and/or ICP4 (via an extended LAT transcript of approximately 15 kb) (6,13,39,45), (ii) association of the stable 2-kb LAT with ribosomes (26), and/or (ii...

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

“…Consequently, LAT can inhibit caspase 8-or caspase 9-induced apoptosis (Henderson et al, 2002;Jin et al, 2003;Peng et al, 2004), the two major apoptotic pathways (Krueger et al, 2001;Schmitz et al, 2000;Wang, 2001). The anti-apoptosis functions of LAT correlate with promoting spontaneous reactivation (Inman et al, 2001;Jin et al, 2003) and appear to be the crucial function of LAT because three different anti-apoptosis genes restore wt levels of spontaneous reactivation to a LAT null mutant virus (Jin et al, 2005(Jin et al, , 2008Mott et al, 2003;Perng et al, 2002b). LAT also represses productive viral gene expression in TG of mice during acute infection (Chen et al, 1997b;Garber et al, 1997).…”

Introducing point mutations into the ATGs of the putative open reading frames of the HSV-1 gene encoding the latency associated transcript (LAT) reduces its anti-apoptosis activity

“…LAT has anti-apoptosis activity [12][13][14][15][16][17][18][19]. Mutants in which LAT is replaced by an alternative anti-apoptosis gene have efficient, wild-type like reactivation phenotypes [20][21][22], indicating that LAT's anti-apoptosis activity is sufficient to account for LAT's ability to support the wild-type reactivation phenotype in experimental animal models.…”

A speculated ribozyme site in the herpes simplex virus type 1 latency-associated transcript gene is not essential for a wild-type reactivation phenotype

“…Thus there is a LAT function that resides completely within the first 1.5 kb of the primary 8.3-kb LAT transcript that is sufficient for supporting a wild-type reactivation phenotype. LAT has antiapoptosis activity (Ahmed et al, 2002;Carpenter et al, 2007;Kang et al, 2003;Perng et al, 2000) that appears to be a key factor in how LAT enhances the reactivation phenotype because (1) the region of LAT that can block apoptosis appears to co-map with the region of LAT (the first 1.5 kb of LAT) that enhances the reactivation phenotype (Inman et al, 2001); and (2) mutants in which the HSV-1 LAT gene is replaced by an alternative antiapoptosis gene have a wild-type reactivation phenotype (Jin et al, 2005Mott et al, 2003;Perng et al, 2002).…”

“…This was achieved by homologous recombination following cotransfection of RS cells with the plasmid and purified genomic dLAT2903 DNA. Viral plaques were screened with a StyI-StyI probe (LAT nts 76 to 447) for reintroduction of the LAT region into the dLAT2903 virus, as we previously described for construction of other LAT mutants (Jin et al, 2005;Perng et al, 1994). Following nine rounds of plaque purification, a viral stock was grown and subjected to Southern blot analysis to confirm that both copies of LAT had been restored to dLAT2903 by the mutated LAT region (Figure 2).…”

“…Thus there is a LAT function that resides completely within the first 1.5 kb of the primary 8.3-kb LAT transcript that is sufficient for supporting a wild-type reactivation phenotype. LAT has antiapoptosis activity (Ahmed et al, 2002;Carpenter et al, 2007;Kang et al, 2003;Perng et al, 2000) that appears to be a key factor in how LAT enhances the reactivation phenotype because (1) the region of LAT that can block apoptosis appears to co-map with the region of LAT (the first 1.5 kb of LAT) that enhances the reactivation phenotype (Inman et al, 2001); and (2) mutants in which the HSV-1 LAT gene is replaced by an alternative antiapoptosis gene have a wild-type reactivation phenotype (Jin et al, 2005Mott et al, 2003;Perng et al, 2002).Theoretically, LAT's function could be due to its genomic DNA, a LAT RNA, and/or a LAT protein. Apart from the promoter driving high levels of LAT expression, a direct DNA function is highly unlikely because plasmids or viral mutants containing just the LAT promoter or just the LAT structural region without the LAT promoter do not block apoptosis and do not have a wild-type reactivation phenotype (Hill et al, 1990;Inman et al, 2001;Jin et al, 2003;Perng et al, 1999Perng et al, , 2000.…”