Herpes simplex virus type 1 ICP0 plays a critical role in the de novo synthesis of infectious virus following transfection of viral DNA

Abstract

As a first step in identifying the functions and intramolecular functional domains of herpes simplex virus type 1 infected cell protein 0 (ICP0) in productive infection and latency, a series of mutant plasmids specifying varying amounts of the ICP0 primary amino acid sequence were constructed. In transient expression assays with mutant and wild-type plasmids, the N-terminal half of the ICP0 molecule was found to be sufficient to transactivate a variety of viral promoters. Although promoters representing the immediate-early, early, and late kinetic classes were transactivated by wild-type ICP0, individual promoters responded to mutant forms of ICP0 in a manner consistent with the possibility that ICP0 transactivates different promoters by different mechanisms. Unlike infection with virus particles, which contain the 65-kilodalton transcriptional transactiovator, the initiation of viral replication after transfection of cells with purified viral DNA requires de novo protein synthesis. In order to assess the role of ICP0 in the de novo synthesis of infectious virus, Vero cells were transfected with purified DNA of wild-type virus or an ICP0 null mutant and the production of infectious virus was monitored. In cells transfected with mutant DNA, virus production was delayed by 2 days and the level of virus was reduced by several orders of magnitude relative to Vero cells transfected with wild-type viral DNA, suggesting an important role for ICP0 in the de novo synthesis of infectious particles. In cotransfection experiments with infectious DNA of the ICP0 null mutant and a plasmid specifying wild-type ICP0 titers of infectious virus were significantly enhanced relative to transfection with mutant DNA alone, confirming the role of ICP0 in de novo synthesis. These findings are consistent with the proposed role of ICP0 in reactivation of herpes simplex virus from latency (D. A. Leib, D. M. Coen, C. L. Bogard, K. A. Hicks, D. R. Yager, D. M. Knipe, K. L. Tyler, and P. A. Schaffer, J. Virol. 63:759-768, 1989), a process also thought to require de novo protein synthesis. The complementing activities of ICP0 mutant plasmids for ICP0 null mutant DNA in cotransfection assays correlated well with their transactivating activities for viral promoters in transient assays, indicating that the transactivating function of ICP0 is a critical factor in the de novo synthesis of infectious particles.(ABSTRACT TRUNCATED AT 400 WORDS)

Varicella-zoster virus open reading frame 61 protein is functionally homologous to herpes simplex virus type 1 ICP0

“…(i) Efficiency of plaque formation. ICPO is not essential for productive infection in cell culture but plays a critical role in viral growth, as indicated by the poor efficiency of plaque formation and the impaired growth of a null mutant (HSV-1 7134 (1]). Transfection of cells with a plasmid expressing ICPO and HSV-1 7134 viral DNA results in complementation of the 7134 virus (1).…”

“…Each of these cysteine-rich sequences resembles a zinc-finger motif common to cellular proteins that may interact with DNA (17). The cysteine-rich region of HSV-1 ICPO is thought to be important for trans activation (in the absence of ICP4), for virus reactivation from latency, and for normal growth of HSV-1 in cell culture (1,5,(12)(13)(14). Thus, the homologous cysteine-rich region of ORF61 may play similar critical roles in the biology of VZV.…”

Native RNA sequencing on nanopore arrays redefines the transcriptional complexity of a viral pathogen

“…NHDFs were cultured in DMEM supplemented with 5% FBS, ARPE-19s in DMEM/F12 supplemented with 10% FBS and neuronal cultures as previously described 36 . For HSV-1 infections, we utilized multiple viruses corresponding to four different HSV-1 strains including GFP-Us11 Patton 23, 36 , Kos 37 , 17syn + , F 38 , KOS N12 (ΔICP4) 39 , and strain F R2621 (Δvhs) 40 , always at a multiplicity of infection (MOI) of three for either 6 or 18 h prior to collecting total RNA or protein. Note that FBS concentrations were halved during the infection and post-infection periods.…”

Recruitment of activated IRF-3 and CBP/p300 to herpes simplex virus ICP0 nuclear foci: Potential role in blocking IFN-β induction

“…The HSV-1 wild type (wt) KOS virus strain was propagated and titrated as described previously (Knipe and Spang, 1982). The 7134 (ICP0 null) virus (Cai and Schaffer, 1989) and the ICP0 nonsense mutants (n212, n428, n525, n680, n720, and n770) were described in the indicated references. The HSV-1 d106 virus has been described elsewhere (Samaniego, Neiderhiser, and DeLuca, 1998).…”