Herpesviruses are large, ubiquitous DNA viruses with complex host interactions, yet many of the proteins encoded by these viruses have not been functionally characterized. As a first step in functional characterization, we determined the subcellular localization of 234 epitope-tagged proteins from herpes simplex virus, cytomegalovirus, and Epstein–Barr virus. Twenty-four of the 93 proteins with nuclear localization formed subnuclear structures. Twelve of these localized to the nucleolus, and five at least partially localized with promyelocytic leukemia (PML) bodies, which are known to suppress viral lytic infection. In addition, two proteins disrupted Cajal bodies, and 19 of the nuclear proteins significantly decreased the number of PML bodies per cell, including six that were shown to be SUMO-modified. These results have provided the first functional insights into over 120 previously unstudied proteins and suggest that herpesviruses employ multiple strategies for manipulating nuclear bodies that control key cellular processes.

The baculovirus protein P143 is essential for viral DNA replication in vivo, likely as a DNA helicase. We have demonstrated that another viral protein, LEF-3, first described as a single-stranded DNA binding protein, is required for transporting P143 into the nuclei of insect cells. Both of these proteins, along with several other early viral proteins, are also essential for DNA replication in transient assays. We now describe the identification, nucleotide sequences, and transcription patterns of the Choristoneura fumiferana nucleopolyhedrovirus (CfMNPV) homologues of p143 and lef-3 and demonstrate that CfMNPV LEF-3 is also responsible for P143 localization to the nucleus. We predicted that the interaction between P143 and LEF-3 might be critical for cross-species complementation of DNA replication. Support for this hypothesis was generated by substitution of heterologous P143 and LEF-3 between two different baculovirus species, Autographa californica nucleopolyhedrovirus and CfMNPV, in transient DNA replication assays. The results suggest that the P143-LEF-3 complex is an important baculovirus replication factor.The family Baculoviridae represents a unique group of large rod-shaped enveloped viruses carrying a double-stranded circular DNA genome and replicating only in invertebrates. Many of the advances in understanding the molecular biology of baculoviruses have resulted from studies of variants of the type species Autographa californica nucleopolyhedrovirus (AcMNPV). Nucleopolyhedroviruses (NPVs) replicate in cell nuclei and are characterized by the production of two virion phenotypes, the budded virions and the occlusion-derived virions. Both forms are produced following infection of cells in culture and are characteristic of late stages of the viral replication cycle following initiation of viral DNA replication at about 8 h postinfection (37). The early events prior to this time are characterized by the expression of several viral gene products, some of which have been shown to be essential for viral DNA replication. Nine viral genes (ie-1, ie-2, p143, dnapol, lef-1, lef-2, lef-3, pe38, and p35) are involved in directing replication of plasmids carrying viral DNA inserts in transfected cells (20,31,38). These data supported earlier genetic analysis of a conditional lethal AcMNPV mutant defective in DNA replication (13), which led to the description of the p143 gene: its nucleotide sequence and the identification of the lesion in the 1,221-amino-acid open reading frame (ORF) (143 kDa) responsible for the temperature-sensitive DNA negative phenotype (29). The p143 gene is essential for viral DNA replication in vivo since no replication occurs in cells infected at the nonpermissive temperature with ts8 (29).Biochemical characterization of extracts from AcMNPV-infected cells showed that P143 copurified through hydroxylapatite and coeluted from single-stranded DNA cellulose with another viral protein called LEF-3, suggesting a possible direct interaction between P143 and LEF-3 (22, 39). LEF-3, also demonstra...

The Rev protein of HIV-1 is essential for HIV-1 proliferation due to its role in exporting viral RNA from the nucleus. We used a modified version of tandem affinity purification (TAP) tagging to identify proteins interacting with HIV-1 Rev in human cells and discovered a prominent interaction between Rev and nucleosome assembly protein 1 (Nap1). This interaction was also observed by specific retention of Nap1 from human cell lysates on a Rev affinity column. Nap1 was found to bind Rev through the Rev arginine-rich domain and altered the oligomerization state of Rev in vitro. Overexpression of Nap1 stimulated the ability of Rev to export RNA, reduced the nucleolar localization of Rev, and affected Rev nuclear import rates. The results suggest that Nap-1 may influence Rev function by increasing the availability of Rev.

We have developed a high-throughput system for generating baculoviruses and testing the expression, solubility, and affinity column purification of encoded proteins. We have used this system to generate baculoviruses for and analyze the expression of 337 proteins from three different herpesviruses (HSV-1, EBV, and CMV) and vaccinia virus. Subsets of these proteins were also tested for expression and solubility in E. coli. Comparisons of the results in the two systems are presented for each virus.