Isolation and characterization of deletion mutants of herpes simplex virus type 1 in the gene encoding immediate-early regulatory protein ICP4

Abstract

Using Vero cells tranformed with the wild-type gene for ICP4 as the permissive host cell, we isolated herpes simplex virus type 1 (HSV-1) mutants containing deletions in both copies of the ICP4 gene. The mutants, d120 and d202, contained deletions of 4.1 and 0.5 kilobases, respectively, in each copy of ICP4. ICP4 mRNA synthesized in d202-infected Vero cells was 0.5 kilobases smaller than that synthesized in cells infected with the wild-type virus. No ICP4 mRNA was detected in d120-infected Vero cells. d120 and d202 specified polypeptides that reacted with ICP4 antiserum and were smaller than the wild-type ICP4 polypeptide by 130 and 30 kilodaltons, respectively. The only other HSV-1 gene products detectable on infection of Vero cells with d120 and d202 were ICP6 (of the early kinetic class of HSV-1 polypeptides), ICPO (immediate early), ICP22 (immediate early), and ICP27 (immediate early). Immediate-early polypeptides ICPO and ICP27 were expressed to a higher level in Vero cells infected with an ICP4 temperature-sensitive (ts) mutant (tsB32) at 39°C, indicating immediate-early stimulatory activity associated with the ts ICP4 polypeptide. In addition, the patterns of complementation of d120, d202, and tsB32 in ICP4-transformed cells also demonstrated inhibitory activity associated with the ts form of the ICP4 polypeptide.

Herpes Simplex Virus ICP27 Activation of Stress Kinases JNK and p38

“…79,2005 HSV ACTIVATION OF SAPKs 8351 on April 30, 2020 by guest http://jvi.asm.org/ frame (27); and (iv) d27-1, which is deleted for the entire ICP27 gene (74). The n12 mutant was reported to express only IE proteins and the DE protein ICP6 (18,19). Following infection with wild-type and mutant viruses at an MOI of 10, whole-cell lysates were prepared at 8 h postinfection, separated by SDS-PAGE, and Western blotted for phospho-specific forms of p38 and JNK.…”

“…The mutant tsB7 (9,47), the parental HFEM strain of HSV-1, and the KOS ICP27 deletion mutant d27-1 (74) were provided by David Knipe (Harvard University). The ICP4 mutants vi13, n12, and d120 (18,19,85) were provided by Neal DeLuca (University of Pittsburgh). The IE multiple mutants d100 (0 Ϫ /4 Ϫ ), d103 (4 Ϫ /22 Ϫ /47 Ϫ ), d106 (4 Ϫ / 22 Ϫ /27 Ϫ /47 Ϫ ), d107 (4 Ϫ /27 Ϫ ), and d109 (0 Ϫ /4 Ϫ /22 Ϫ /27 Ϫ /47 Ϫ ) were also provided by Neal DeLuca (80).…”

Protein Kinase B/Akt Is Present in Activated Form throughout the Entire Replicative Cycle of ΔUS3 Mutant Virus but Only at Early Times after Infection with Wild-Type Herpes Simplex Virus 1

“…The recombinant virus R7041, lacking the U S 3 gene (⌬U S 3), is described in reference 37. The HSV-1(KOS) d120 mutant, a kind gift from N. DeLuca (University of Pittsburgh, Pittsburgh, Pa.), lacks both copies of the ␣4 gene (⌬ICP4) and was grown in a Vero-derived cell line (E5) expressing the ␣4 gene (12). In all experiments in which cells were infected with virus, cells grown in 25-cm 2 flasks were either mock infected or exposed to approximately 10 PFU of virus per cell for 1 h and 30 min at 37°C in medium 199V (mixture 199 [Sigma] supplemented with 1% calf serum) and then maintained in DMEM supplemented with the proper amount of serum as described above.…”

Protective effect of HSV‐mediated gene transfer of nerve growth factor in pyridoxine neuropathy demonstrates functional activity of trkA receptors in large sensory neurons of adult animals

“…The construction of the NGF-expressing replication-incompetent HSV vector SHN has been described previously (Goins et al, 1999). Briefly, a plasmid containing the coding sequence for murine NGF flanked by the human cytomegalovirus immediate-early promoter and SV40 polyadenylation sequence was inserted into the thymidine kinase (TK) locus of an ICP4-deleted HSV 1 recombinant (DeLuca et al, 1985). The control vector SHZ was created in a similar manner with the Escherichia coli lacZ cDNA in place of NGF (Goins et al, 1999).…”