Role of Immediate Early Protein ICP27 in the Differential Sensitivity of Herpes Simplex Viruses 1 and 2 to Leptomycin B

Abstract

Leptomycin B (LMB) is a highly specific inhibitor of CRM1, a cellular karyopherin-␤ that transports nuclear export signal-containing proteins from the nucleus to the cytoplasm. Previous work has shown that LMB blocks herpes simplex virus 1 (HSV-1) replication in Vero cells and that certain mutations in viral immediate early protein ICP27 can confer LMB resistance. However, little is known of the molecular mechanisms involved. Here we report that HSV-2, a close relative of HSV-1, is naturally resistant to LMB. To see whether the ICP27 gene determines this phenotypic difference, we generated an HSV-1 mutant that expresses the HSV-2 ICP27 instead of the HSV-1 protein. This recombinant was fully sensitive to LMB, indicating that one or more other viral genes must be important in determining HSV-2's LMB-resistant phenotype. In additional work, we report several findings that shed light on how HSV-1 ICP27 mutations can confer LMB resistance. First, we show that LMB treatment of HSV-1-infected cells leads to suppression of late viral protein synthesis and a block to progeny virion release. Second, we identify a novel type of ICP27 mutation that can confer LMB resistance, that being the addition of a 100-residue amino-terminal affinity purification tag. Third, by studying infections where both LMB-sensitive and LMB-resistant forms of ICP27 are present, we show that HSV-1's sensitivity to LMB is dominant to its resistance. Together, our results suggest a model in which the N-terminal portion of ICP27 mediates a nonessential activity that interferes with HSV-1 replication when CRM1 is inactive. We suggest that LMB resistance mutations weaken or abrogate this activity. Herpes simplex virus 1 (HSV-1) is a widely distributed human alphaherpesvirus that is capable of causing serious and in some cases life-threatening infections. The HSV-1 infectious cycle involves both nuclear and cytoplasmic processes, and thus, productive viral replication requires that viral and cellular protein and RNA components travel efficiently between the nucleus and cytoplasm. Such movement is largely mediated by dedicated cellular transporters, one important class of which consists of the karyopherin-␤ family of proteins (1). Among this family, CRM1 is perhaps the most well studied member. It is an essential protein that exports ribosomal subunits and various protein cargos from the nucleus to the cytoplasm (2-4). Transport by CRM1 is dependent on the presence of a specific nuclear export signal (NES) on the cargo protein. This signal consists of an ϳ15-residue consensus sequence that is rich in leucine and other hydrophobic residues. The NES binds to a hydrophobic cleft on the surface of nucleus-localized CRM1 (5, 6). Following binding, CRM1 escorts the cargo through the nuclear pore to the cytoplasm, whereupon it is released. One of the first leucine-rich NESs described was that of the human immunodeficiency virus type 1 (HIV-1) Rev protein (7). Rev uses CRM1 to facilitate the nuclear export of unspliced and partially spliced HIV-1 transcripts. S...

Functional Comparison of Herpes Simplex Virus 1 (HSV-1) and HSV-2 ICP27 Homologs Reveals a Role for ICP27 in Virion Release

“…The plasmid pT2-27, which contains the HSV-2 gene from strain HG52, was recently described (30). Additional plasmids used were the gC expression plasmid pgC⌬pro (33), ICP27 expression plasmids pBH27 (34) and pC27 (5), ICP4 expression plasmid pK1-2 (35), and ICP0 expression plasmid pSHZ (36).…”

“…HSV-2 strains HG52 and MS were obtained from Jin-Young Han, and strain G was obtained from the ATCC. The engineering and isolation of HSV-1 mutant K2F1 (and an independent isolate, K2F1) were recently described (30). HSV-1 and HSV-2 infections were carried out in 199V medium, defined as medium 199 containing 50 units/ml penicillin, 50 g/ml streptomycin, and 1% heat-inactivated newborn calf serum (NCS).…”

“…To study ICP27t2 further, we wanted to examine its activities in infected cells when expressed from its natural context in the viral genome. In a recent publication, we described the isolation of K2F1, an HSV-1 mutant that encodes ICP27t2 in place of ICP27 (30). Aside from showing that the virus is viable in Vero cells and that it is sensitive to the drug leptomycin B (similar to WT HSV-1), the phenotype of K2F1 was not further characterized.…”

“…Here, we report a detailed phenotypic analysis of K2F1. It should be mentioned that in many of the experiments described below we included a second genetically independent isolate of K2F1, designated K2F1b (30). This was done to ensure that the phenotype of the mutant was the result of the engineered ICP27 gene change, rather than the consequence of an additional unknown mutation.…”

“…For expression of ICP27t2, we used the recently described plasmid pT2-27, which contains an intact ICP27t2 gene from HSV-2 strain HG52 (30). The insert includes the gene's promoter and polyadenylation site, and thus we expected that pT2-27 would express ICP27t2 upon transfection into mammalian cells.…”