We have previously demonstrated that efficient replication of mutant herpes simplex virus which fails to synthesize the polypeptide ICP34.5 is cell type and cell state dependent. ICP34.5 negative viruses do not grow in stationary state mouse embryo fibroblast 3T6 cells whereas the growth kinetics in BHK cells are indistinguishable from those of wild-type. We now demonstrate that this defect is not due to an inability of mutant virus to adsorb to 3T6 cells but rather to an inability to spread from the initially infected cells. Electron microscopic studies with wild-type HSV in both BHK and 3T6 cells revealed virus particles equally distributed between nucleus and cytoplasm, and additionally in the extracellular matrix. In BHK cells infected with the ICP34.5 negative mutant 1716, virus is likewise distributed between nucleus and cytoplasm but in 50 % of the infected cells there is marked delamination and swelling of the nuclear membrane. In addition there is evidence of a significant number of particles trapped between the nuclear lamellae. When 1716 is used to infect 3T6 cells, over 90% of the virus particles are confined to the nuclei and the number of infected cells remains constant between 24 and 48 h with no increase in the proportion of extracellular virus. Failure to express ICP34.5 appears therefore to result in a defect in virus maturation and egress from the nuclei of infected cells. Egress of HSV from the nuclei to the extracellular space is thought to occur via two pathways. We postulate that lack of expression of ICP34.5 results in one of these pathways being blocked. In BHK cells this leads to overloading of the alternative pathway with a buildup of particles in the nuclear lamellae and associated endoplasmic reticulum. In stationary state 3T6 cells, it appears that there is no functional alternative pathway. We conclude that ICP34.5 exerts an effect on HSV maturation by controlling the passage of virus through infected cells.
The DNA sequence of herpes simplex virus type 1 (HSV-1) strain 17 + in the region coding for the polypeptide ICP34.5 predicts a protein of 248 amino acids with a proposed M r of 26158. The entire RL1 open reading frame was cloned into the expression vector pET8c to enable over-expression of ICP34.5 in Escherichia coli. The expressed protein was partially purified and used as an immunogen to produce a polyclonal antiserum in rabbits. Construction of an ICP34.5 null mutant (1771), demonstrated that the predicted open reading frame for ICP34.5 in strain 17 + is correct and confirmed that HSV-1 strain 17 + ICP34.5 specifically determines neurovirulence. The specificity of the antiserum directed against the E. coli-expressed ICP34.5 was defined by Western blotting of wild-type and RL1-negative infected cell extracts.
In peripheral sensory ganglia latently infected with herpes simplex virus type 1 (HSV-1) transcription is restricted. A set of viral latency-associated transcripts, the LATs, have been characterized by Northern blotting and in situ hybridization. These transcripts have previously been mapped to a 3 kb region of the viral genome within the repeat long region. However, transcription from adjacent regions of the genome can be detected by in situ hybridization, which cannot be detected by Northern blotting. These RNAs are termed minor LATs or m-LAT. In this study we show that in ganglia latently infected with the HSV-1 variant 1704, which is deleted in one complete copy of the LAT gene and in the promoter and 5' portion of the other copy, m-LATs are not detected by in situ hybridization. Furthermore, the levels of DNA in nervous system tissue latently infected with the parental and the 1704 variant virus are similar. Thus we propose that the sequence elements necessary for initiating transcription or stabilizing m-LATs are within the region deleted in variant 1704 that codes for the promoter and the 5' end of the LATs.
The viral and host factors involved in herpes simplex virus (HSV) recombination are little understood. To identify features of the process, recombination in HSV-1 and HSV-2 has been studied by analysing the segregation of unselected markers in the form of restriction endonuclease (RE) sites. By confining parental interactions to only one strain of virus of each serotype, restrictions imposed by non-homology are overcome and differential growth phenotypes can be discounted. The analysis of unselected and selected recombinants using RE sites in conjunction with temperature-sensitive mutations is consistent with (i) HSV being highly recombinogenic, (ii) parental and progeny molecules taking part in the process, (iii) the four genomic isomers participating in recombination, (iv) genome alignment being part of the recombination process and (v) cellular factors in conjunction with genome homology influencing the efficiency of recombination.
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