Structure–Function Analysis of the Herpes Simplex Virus Type 1 UL12 Gene: Correlation of Deoxyribonuclease Activityin Vitrowith Replication Function

Abstract

Although the product of the UL12 gene of herpes simplex virus type 1 (HSV-1) has been shown to possess both exonuclease and endonuclease activities in vitro, and deletion of most of the gene within the viral genome results in inefficient production and maturation of infectious virions, the function of the deoxyribonuclease (DNase) activity per se in virus replication remains unclear. In order to correlate the in vitro and in vivo activities of the protein encoded by UL12, mutant proteins were tested for nuclease activity in vitro by a novel hypersensitivity cleavage assay and for their ability to complement the replication of a DNase null mutant, AN-1. Rabbit reticulocyte lysates programmed with wild-type UL12 RNA cleaved at the same sites cleaved by purified HSV-1 DNase, but distinct from those cleaved by DNase 1 or micrococcal nuclease. All mutants which lacked DNase activity in vitro also failed to complement the replication of AN-1 in nonpermissive cells. Likewise, all mutants which contained HSV-1 DNase activity, as detected by the hypersensitivity cleavage assay, were capable of complementing the replication of the DNase null mutant, though to varying extents. Of particular note was the d1-126 mutant protein, which, despite having the same specific activity as the wild-type enzyme in vitro, complemented the replication of AN-1 significantly less than the wild-type protein. The results suggest that DNase activity per se is required for efficient replication of HSV-1 in vivo. However, residues, including the N-terminal 126 amino acids, which are dispensable for enzymatic activity in vitro may facilitate the accessibility or activity of the protein in vivo.

Mitochondrial Nucleases ENDOG and EXOG Participate in Mitochondrial DNA Depletion Initiated by Herpes Simplex Virus 1 UL12.5

“…Intriguingly, a previous study had shown that two mutations in the N-terminal regions of UL12 and UL12.5 that are absent from UL12 M185 inactivate detectable nuclease activity in vitro (23). Taken together, these data raised the possibility that UL12 M185 lacks enzymatic activity and, by extension, that the nuclease activity of UL12.5 is not required for mtDNA depletion.…”

“…The L150K mutation was introduced using a modified site-directed mutagenesis protocol (25) with primers F4 and R3. The ⌬N mutation (deletes residues W128 to R148 [23]) and the ⌬MLS mutation (deletes residues R188 to R212 [10]) were both introduced using a QuikChange XL site-directed mutagenesis kit (Stratagene) with primers F5 and R4 and primers F6 and R5, respectively. The ⌬C mutation (deletes residues P578 to R626 [23]) was created by PCR using primers F2 and R6.…”

“…The smaller mRNA encodes an inframe, N-terminally truncated version of UL12 called UL12.5 which initiates at UL12 codon M127 (22). While UL12.5 displays enzymatic properties similar to those of UL12, it cannot functionally replace UL12 during viral replication (17,22,23). This lack of redundancy has been attributed to two factors: UL12 is much more abundant than UL12.5 (22), and UL12 and UL12.5 have distinct subcellular locations (17).…”

Replication, recombination and packaging of amplicon DNA in cells infected with the herpes simplex virus type 1 alkaline nuclease null mutant ambUL12

“…In addition, analysis of replicating concatemeric DNA suggests that in the absence of AN the replicative intermediates have a more complex structure with an increased frequency of branches (Martinez et al, 1996a), whilst structural abnormalities have also been detected in the genomes of progeny virions (Porter & Stow, 2004). Taken together, these observations suggest a possible role for AN in the resolution of recombination intermediates prior to DNA packaging, and this model is supported by the observation that the nuclease function, per se, of the UL12 protein is necessary for efficient replication (Goldstein & Weller, 1998b;Henderson et al, 1998). It can be envisaged that failure to correctly resolve branched structures could have an indirect impact upon DNA synthesis and the assembly of the infectious virus particle.…”

Identification of a DNA-binding domain and an active-site residue of pseudorabies virus DNase

“…Several studies also indicated that similar modes of action are present in herpesviral DNases purified from different sources [14,15]. A structure-function analysis of the herpesviral DNase showed that mutations at the N-terminus of gammaherpesvirus (Epstein-Barr virus ; EBV) eliminate its exonuclease activity [16] ; however, other studies indicated that the N-terminus of herpes simplex virus type 1 (HSV-1) DNase might not have a role in its enzymic activity [17]. Goldstein and Weller [18] showed also that Abbreviations used : DEPC, diethyl pyrocarbonate ; EBV, Epstein-Barr virus ; HSV-1, herpes simplex virus type 1 ; PRV, pseudorabies virus ; UL12, alkaline nuclease.…”