Herpes simplex virus type 1 (HSV-1) is a large, enveloped, double-stranded DNA virus with a genome of approximately 150 kbp that encodes over 80 proteins, many of whose functions are still unknown (reviewed in reference 48). Productive HSV-1 infection occurs in a sequentially ordered cascade in which ␣ (immediate-early or IE) proteins precede the synthesis of ␤ (early or E) and ␥ (late or L) proteins (22,23). During the course of lytic infection, HSV-1 takes over the host cell, subjugating the cell's biomolecular synthesis machinery to serve its own reproduction. Cells react to viral infection by attempting to block viral replication, undergoing apoptosis, or by signaling neighboring cells to activate antiviral systems (reviewed in reference 31).The interplay between HSV-1 and its host involves numerous factors, and the virus employs several mechanisms to combat the many antiviral responses enacted by an infected cell. One of the most dramatic cellular responses to viral infection is the induction of apoptosis or programmed cell death. Apoptosis is characterized by cell shrinkage, membrane blebbing, redistribution of phosphatidylserine to the outer leaflet of the plasma membrane, fragmentation of nuclei (karyorrhexis), chromosomal DNA (pyknosis), and oligosomal DNA laddering (29,30,58). These structural modifications are a consequence of the activation of effector (executioner) caspases (cysteine aspartases), one of which is caspase 3, and the cleavage of poly(ADP-ribose) polymerase (PARP) and structural proteins (29,30,39,57,58). Apoptosis functions to limit virus spread by preventing viral replication, saving other cells from infection (34). HSV-1 has developed a mechanism to counteract this antiviral cell death process (3).The interaction of HSV-1 with its host cell results in the triggering of the apoptotic cell death program (2,4,33). During infection by wild-type HSV-1, caspase 3 is activated (4), the death factor DFF45 is completely cleaved (4), and phosphatidylserine is flipped from the inner to the outer membrane leaflet (25), indicating that replication-competent HSV-1 triggers the apoptosis death program and infected cells are actively in an apoptotic state. However, cells infected with wild-type HSV-1 do not show features of apoptosis, because infected cell proteins produced between 3 and 6 h postinfection (hpi), termed the apoptosis "prevention window" (4), prevent the process from killing the cells (4). Thus, human HEp-2 cells infected with either (i) viruses containing a deletion in the key viral regulators ICP4 or ICP27 or (ii) wild-type virus plus the addition of cycloheximide (CHX) at the time of infection, die by apoptosis (2,33,37). Although most recent efforts have focused on identifying viral gene products involved in apopto-* Corresponding author. Mailing address:

Left-handed DNA is shown to exist and elicit a biological response in Escherichia coli. A plasmid encoding the gene for a temperature-sensitive Eco RI methylase (MEco RI) was cotransformed with different plasmids containing inserts that had varying capacities to form left-handed helices or cruciforms with a target Eco RI site in the center or at the ends of the inserts. Inhibition of methylation in vivo was found for the stable inserts with the longest left-handed (presumably Z) helices. In vitro methylation with the purified MEco RI agreed with the results in vivo. Supercoil-induced changes in the structure of the primary helix in vitro provided confirmation that left-handed helices were responsible for this behavior. The presence in vivo of left-handed inserts elicits specific deletions and plasmid incompatibilities in certain instances.

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Full-length VP22 is necessary for efficient spread of herpes simplex virus type 1 (HSV-1) from cell to cell during the course of productive infection. VP22 is a virion phosphoprotein, and its nuclear localization initiates between 5 and 7 h postinfection (hpi) during the course of synchronized infection. The goal of this study was to determine which features of HSV-1 infection function to regulate the translocation of VP22 into the nucleus. We report the following. The molecular mechanism of herpes simplex virus type 1 (HSV-1) tegument and envelope assembly is poorly understood. Most of the major tegument proteins exhibit nuclear or perinuclear distributions late in infection (1, 8, 10-12, 26, 33, 37, 43), and there is general agreement that primary envelopment occurs as the capsid exits the nucleus (7,19,21,42,46) (for a detailed review, see reference 17).Work in our laboratory has focused on the major tegument protein VP22 and its function in virion assembly and virus replication. Full-length VP22 is necessary for efficient spread of the virus from cell to cell, inasmuch as a recombinant virus producing a truncated form of the protein exhibits a decreased plaque size in Vero cells compared to that of wild-type virus (36). In an earlier study, we reported that the nuclear localization of VP22 initiates between 5 and 7 h postinfection (hpi) during the course of synchronized infection (37). This period corresponds to the peak of viral ␤ protein production and DNA synthesis (39). Recently, computer analyses predicted two nuclear localization signals (NLS) in the primary structure of VP22 (22). Although transiently expressed VP22 can be detected in the nuclei of transfected cells (15,22), these proposed NLS have not yet been characterized in nuclear import assays. VP22 is a 301-amino-acid protein with a predicted size of 32,000 Da (28). Thus, VP22 is below the size exclusion limit (40,000 to 45,000 Da) for passive diffusion through the nuclear pore (13). That VP22 accumulates in the cytoplasm of infected cells prior to 5 hpi suggests its translocation to the nucleus is regulated during infection. One possible means of regulation could be active retention through binding of VP22 to a cytoplasmic structure.In this study, we sought to determine which features of HSV-1 replication are involved in the redistribution of VP22 during productive infection. Our investigations show the following. Regulated VP22 nuclear localization initiates after 5 hpi with HSV-1 and is independent of viral DNA and true late protein synthesis. We propose that HSV-1 induced microtubule restructuring releases VP22 from the cytoskeleton, allowing its entry into the nucleus. Stabilization of microtubules during infection or in VP22-expressing cells increases VP22 retention in the cytoplasm. Based on these results, we conclude that microtubule reorganization during herpes HSV-1 infection acts to facilitate the nuclear localization of VP22. MATERIALS AND METHODS Cells and virus.African green monkey kidney (Vero) cells were obtained from the Ameri...