Functional order of assembly of herpes simplex virus DNA replication proteins into prereplicative site structures.

Herpes simplex virus replicates its DNA within nuclear structures called replication compartments. In contrast, in cells in which viral DNA replication is inhibited, viral replication proteins localize to punctate structures called prereplicative sites. We have utilized viruses individually mutated in each of the seven essential replication genes to assess the function of each replication protein in the assembly of these proteins into prereplicative sites. We observed that four replication proteins, UL5, UL8 UL52, and UL9, are necessary for the localization of ICP8 (UL29) to prereplicative sites natural infection conditions. Likewise, four of the seven viral DNA replication proteins, UL5, UL52, UL9, and ICP8, are necessary for the localization of the viral DNA polymerase to prereplicative sites. On the basis of these results, we present a model for prereplicative site formation in infected cells in which the helicase-primase components (UL5, UL8, and UL52), the origin-binding protein (UL9), and the viral single-stranded DNA-binding protein (ICP8) assemble together to initiate the process. This is followed by the recruitment of the viral polymerase into the structures, a step facilitated by the polymerase accessory protein, UL42. Host cell factors can apparently substitute for some of these viral proteins under certain conditions, because the viral protein requirements for prereplicative site formation are reduced in transfected cells and in infected cells treated with drugs that inhibit DNA synthesis.

The herpes simplex virus type 1 regulatory protein ICP0 enhances virus replication during acute infection and reactivation from latency.

ICP0 is a potent activator of herpes simplex virus type 1 gene expression in transient assays and in productive infection. A role for ICP0 in reactivation from latency in vivo has also been suggested on the basis of the observation that viruses with mutations in both copies of the diploid gene for ICP0 reactivate less efficiently than wild-type virus. Because the ICP0 gene is contained entirely within the coding sequences for the latency-associated transcripts (LATs), ICP0 mutants also contain mutations in LAT coding sequences. This overlap raises the question of whether mutations in ICP0 or the LATs, which have also been implicated in reactivation, are responsible for the reduced reactivation frequencies characteristic of ICP0 mutants. Two approaches were taken to examine more definitively the role of ICP0 in the establishment and reactivation of latency. First, a series of ICP0 nonsense, insertion, and deletion mutant viruses that exhibit graded levels of ICP0-specific transactivating activity were tested for parameters of the establishment and reactivation of latency in a mouse ocular model. Although these mutants are ICP0 LAT double mutants, all nonsense mutants induced the synthesis of near-wild-type levels of the 2-kb LAT, demonstrating that the nonsense linker did not disrupt the synthesis of this LAT species. All mutants replicated less efficiently than the wild-type virus in mouse eyes and ganglia during the acute phase of infection. The replication efficiencies of the mutants at these sites corresponded well with the ICP0 transactivating activities of individual mutant peptides in transient expression assays. All mutants exhibited reduced reactivation frequencies relative to those of wild-type virus, and reactivation frequencies, like replication efficiencies in eyes and ganglia, correlated well with the level of ICP0 transactivating activity exhibited by individual mutant peptides. The amount of DNA of the different mutants varied in latently infected ganglia, as demonstrated by polymerase chain reaction analysis. No correlation was evident between reactivation frequencies and the levels of viral DNA in latently infected ganglia. Thus, replication and reactivation efficiencies of ICP0 mutant viruses correlated well with the transactivating efficiency of the corresponding mutant peptides. In a second approach to examining the role of ICP0 in latency, a single copy of the wild-type gene for ICP0 was inserted into the genome of an ICP0- LAT- double mutant, 7134, which exhibits a marked impairment in its ability to replicate in the mouse eye and reactivate from latency.(ABSTRACT TRUNCATED AT 400 WORDS)

In vivo phosphorylation and membrane association of the fyn proto-oncogene product in IM-9 human lymphoblasts.

The protein product of the src-related proto-oncogene, fyn, was isolated from IM-9 cells with antibodies specific for the amino-terminal 22 residues of the fyn protein. Peptide mapping demonstrated that the fyn protein was distinct from the closely related c-src and c-fgr proteins. The fyn protein from IM-9 cells incorporated [3H]myristate in vivo and was found to be membrane associated. Phosphoamino acid analysis demonstrated that the fyn protein from IM-9 cells was phosphorylated in vivo predominantly on tyrosine and threonine with only a small amount of phosphoserine detected. the major chymotryptic phosphopeptide of the fyn protein was phosphorylated exclusively on tyrosine. This peptide was specifically precipitated by antibodies directed against a peptide modeled on the closely related carboxy termini of the c-src and fyn proteins. These results provide direct evidence for phosphorylation of tyrosine-531 in the carboxy-terminal chymotryptic peptide of the fyn protein. Phosphorylation of the corresponding site in the closely related c-src protein (tyrosine-527) represses src kinase activity and transforming ability. Loss of the phosphorylation site at tyrosine-531 may similarly contribute to the transforming abilities of carboxy-terminal deletion mutants of the fyn protein.