Using truncated forms of gD generated by recombinant DNA methods and proteolysis, epitopes III, IV, and VI were located within amino acids 1-233. The fine specificity of gC-specific CTL clones was analyzed on target cells infected with mutant viruses altered in the antigenic structure of gC. It was found that strain KOS/M established latent infections, as defined by the presence of the viral genome, in about 30% of the neurons. When either of these cosmids was replaced by a derivative comprising only ori-L forms, ori+L and ori-L progeny were obtained, and only ori-L progeny were produced when both were replaced. In striking contrast, both LATs- mutants reactivated with wild-type frequencies from lumbosacral ganglia. These results support the model of double-strand-break repair for recombination of the a sequence.
Production of an HSV-1 mutant multiply defective in the expression of IE gene products was achieved by introduction of the temperature-sensitive mutation of HSV-1tsK, which inactivates the IE transcription activator ICP4 at nonpermissive temperatures, intoin1820 to producein1820K. To determine whether HSV-1 induced CR1-like receptors or CR3-like receptors, infected cells were pretreated with EDTA, which is known to inhibit native CR3 function. Type II receptor knockout and wild-type mice had comparable viral replication and localization, with no systemic spread of HSV-1 or lethality. Selected References These references are in PubMed. K. We also found an excellent correlation between the ability of purified GST fusion proteins to stimulate pol activity in vitro and the ability of full-length nonfusion UL42 mutant genes to support DNA replication in infected cells.
The RING finger domain of ICP0 and the N terminus of VP22 were both shown to be essential but not sufficient for ICP0 packaging and complex formation. McLean, F. Halford and P. In addition, we found that UL15 colocalizes with replication compartments at early times (6 h postinfection). We also show that the virion population of bUL47 binds RNA in vitro. Infection conditions were manipulated so that approximately equal numbers of latent infections were established by the parental strains, the mutants, and their genomically restored counterparts, eliminating disparate latent pool sizes as a complicating factor.
This ICP0-mediated effect occurs at the transcriptional level and involves the ubiquitin-proteasome pathway. Despite this, transcription of lytic-phase genes could be detected within 4 h following induction of rabbits latently infected with either LAT+ or LAT- virus; this transcription diminished by 16 h following induction. In this study we have investigated the role of HAUSP in Vmw110 activity; single amino acid residues of Vmw110 required for the interaction were identified, and the effects of mutation of these residues in infected and transfected cells were then assayed. Our studies tested the ability of the 2-kb LAT to dissociate from cytoplasmic protein complexes under various salt conditions. We found that d109 was nontoxic to neural cultures and persisted in the cultures throughout their life spans. Finally, we demonstrate that the execution of apoptosis is not required for inhibition of HSV-1 replication and that the hyperphosphorylation of RPA per se is not inhibitory for HSV-1 replication, suggesting that these two processes are not directly responsible for the inhibition of HSV-1 replication by Rep.
These findings are discussed with reference to the nature, location, and potential number of HSV-1 prereplication compartments and to the dynamic aspects of HSV-1 genomes and viral products during the early stages of lytic infection. The magnitude of bioluminescence from firefly luciferase measured in vivo correlated directly with input titers of recombinant virus used for infection. Three different classes of lysosomotropic agents, which raise endosomal pH, blocked HSV entry into primary and transformed human keratinocytes, but not into human neurons or neuroblastoma lines. We found that the inhibition of HSV-1 replication required Rep DNA-binding and ATPase/helicase activities but not endonuclease activity. Two strains of HSV1 were used for the inoculation: SC16, a wild type strain considered as highly neuroinvasive, and KOS, previously described as poorly neurovirulent. The viral strain and the combination of mutant viral genes that ultimately may serve as a safe and optimal backbone for such products are still being explored.
The gE promoter is a late viral promoter that has some activity in the absence of viral DNA replication and is representative of the γ1 class of HSV-1 promoters.