Herpes simplex virus type 1 DNA replication occurs in nuclear domains termed replication compartments, which are areas of viral single-stranded DNA-binding protein (UL29) localization (M. P. Quinlan, L. B. Chen, and D. M. Knipe, Cell 36:857-868, 1984). In the presence of herpesvirus-specific polymerase inhibitors, UL29 localizes to punctate nuclear foci called prereplicative sites. Using versions of the helicase-primase complex proteins containing short peptide epitopes which can be detected in an immunofluorescence assay, we have found that the helicase-primase complex localizes to prereplicative sites and replication compartments. To determine if prereplicative site formation is dependent upon these and other essential viral replication proteins, we have studied UL29 localization in cells infected with replication-defective viruses. Cells infected with viruses that fail to express one of the three helicase-primase subunits or the origin-binding protein show a diffuse nuclear staining for UL29. However, in the presence of polymerase inhibitors, mutant-infected cells contain UL29 in prereplicative sites. Replication-defective viruses containing subtle mutations in the helicase or origin-binding proteins behaved identically to their null mutant counterparts. In contrast, cells infected with viral mutants which fail to express the polymerase protein contain prereplicative sites in the absence and presence of polymerase inhibitors. We propose that active viral polymerase prevents the formation of prereplicative sites. Models of the requirement of essential viral replication proteins in the assembly of prereplicative sites are presented.on July 10, 2020 by guest http://jvi.asm.org/ Downloaded from FIG. 3. UL29 localization in cells infected with viral mutants that fail to express UL5, UL8, UL52, or UL9. Vero cells were infected with 20 PFU of virus per cell and processed as described in Materials and Methods. (A) Cells were stained with 3-83 and fluorescein isothiocyanate-conjugated goat anti-rabbit secondary antibody. Cells were infected with the following mutants: panels A, B, and C, hr80; D, E, and F, hr99; G, H, and I, hr114. Infected cells in panels B, E, and H were treated with PAA, and those in panels C, F, and I were treated with ACG. Marker bar, 15 m. (B) hr94-infected cell doubly stained with antibodies (␣) for UL29 and BrdU. Panels A and B show the same group of hr94-infected cells doubly stained, respectively, with 3-83 (fluorescein isothiocyanate-conjugated goat anti-mouse secondary antibody) and anti-BrdU (Texas red-conjugated goat anti-mouse secondary antibody). Marker bar, 15 m.

During infection, the seven essential herpes simplex virus type 1 (HSV-1) replication proteins are found in globular nuclear structures called replication compartments. Replication compartments form adjacent to ND10, nuclear matrix-bound domains which are present in most cell types but whose function is unknown (G. G. Maul, I. M. Ishov, and R. D. Everett, Virology 217:67-75, 1996). We now demonstrate that replication compartments can be formed by cotransfecting Vero cells with constructs expressing the seven essential viral replication proteins and a plasmid containing an HSV-1 origin of DNA replication. Like replication compartments in infected cells, replication compartments formed by cotransfection contain all of the essential viral replication proteins, are sites of DNA synthesis, and are found adjacent to ND10. However, neither the viral origin-binding protein nor a plasmid containing an HSV-1 origin of DNA replication is individually required for the formation of transfection replication compartments, although the presence of each increases the efficiency of replication compartment formation. Further, we provide evidence that UL29 independently localizes adjacent to ND10 and so may play a role in directing replication compartments to these preexisting nuclear structures.

When herpes simplex virus type 1 (HSV-1) DNA replication is blocked by viral polymerase inhibitors, such as phosphonoacetic acid (PAA) or acyclovir (ACV), UL29 (ICP8) localizes to numerous punctate nuclear foci which are called prereplicative sites. Since this pattern can form in cells infected with mutants which are defective in UL5, UL8, UL9, or UL52 in the presence of polymerase inhibitors (C.

The infected cell polypeptide 0 (ICP0) protein of herpes simplex virus type 1 (HSV-1) is a promiscuous transactivator. When expressed by transfection, ICP0 forms spherical structures in the nucleus. Using a double-label immunofluorescence assay, we have found that the HSV-1 helicase/primase complex subunits accumulate within ICP0 structures in cotransfected cells. This phenomenon was also observed in cells coexpressing ICP0 and UL6, a protein thought to be involved in the cleavage and/or packaging of viral genomes. ICP0 structures were found to be proteinaceous by immunoelectron microscopy. These results suggest that ICP0 may interact nonspecifically with a variety of viral proteins.

CASE REPORTMr. A is a 34-year-old Caucasian man with a past medical history of methionine synthase deficiency (cobalamin G defect), a disorder of vitamin B 12 metabolism, who presented to the emergency department after three weeks of increasingly erratic and violent behavior toward his wife and two young sons (aged 3 and 6). His wife, who stated that "he has always had a temper, but nothing like this," had recently moved out of the house (taking the children) for a few days, only to fail to see improvement in the situation on her return. Mr. A's anger and impulsive behavior escalated to the point where he allegedly punched his wife in the stomach, kicked down a door after being locked out, and was verbally threatening toward his children. This behavior prompted Mrs. A From the Harvard Longwood Psychiatry Residency Training Program,to telephone her husband's primary care physician, who recommended that she bring him to the emergency department for a psychiatric evaluation.Mr. A, who had previously been diagnosed with a mild polyneuropathy by a neurologist at our hospital, thought that he had come to "follow up on an MRI" taken months earlier. A psychiatric evaluation in the emergency department revealed that Mr. A had ceased "keeping track" of the vitamins and medications prescribed for his underlying metabolic disorder and had not been compliant with them for several months. Mrs. A noted that she had not given her husband his daily IM injection of methylcobalamin during the days after she moved out of the house. She recalled aggressive and violent behavior in the past when he was noncompliant with his medication. She described him as otherwise being "quite laid back and relaxed"-until two months ago, when she noticed that he had become "confrontational, easily agitated, and dictatorial." On interview, Mr. A became agitated, often interrupting his wife loudly and requiring significant redirection to complete the interview. He was paranoid about his wife's motives and his visit to the hospital, stating that "my wife should be in [the hospital], not me," and that he "was tricked into coming here." His inability to control his behavior in multiple settings, coupled with the corresponding threat to his own safety and the safety of his family, warranted his admission to the psychiatric unit.Mr. A's previous psychiatric history comprised four months of couples counseling with a psychiatrist less than a year prior to this presentation. His medical history included the previously mentioned methionine synthase deficiency. This disorder regularly resulted in the accumulation of homocysteine, with a range of 65 to 202 µmol/L (normal, <14). He was not allergic to any medications and took oral 116