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...