An Intertypic Herpes Simplex Virus Helicase-Primase Complex Associated with a Defect in Neurovirulence Has Reduced Primase Activity

Abstract

R13-1 is an intertypic recombinant virus in which the left-hand 18% of the herpes simplex virus type 1 (HSV-1) genome is replaced by homologous sequences from HSV-2. R13-1 is nonneurovirulent and defective in DNA replication in neurons. The defect was localized to the UL5 open reading frame by using marker rescue analysis (D. C. Bloom and J. G. Stevens, J. Virol. 68:3761-3772, 1994). To provide conclusive evidence that UL5 is the only HSV-2 gene involved in the restricted replication phenotype of R13-1, we have characterized the phenotype of a recombinant virus (IB1) in which only the UL5 gene of HSV-1 was replaced by HSV-2 UL5. Data from 50% lethal dose determinations and the in vivo yields of virus suggested that IB1 has the same phenotypic characteristics as R13-1. UL5 is the helicase component of a complex with helicase and primase activities. All three subunits of this complex (UL5, UL8, and UL52) are required for viral DNA replication in all cell types. The intertypic complex HSV-2 UL5-HSV-1 UL8-HSV-1 UL52 was purified and biochemically characterized. The primase activity of the intertypic complex was 10-fold lower than that of HSV-1 UL5-HSV-1 UL8-HSV-1 UL52. The ATPase activity was comparable to that of the HSV-1 enzyme complex, and although the helicase activity was threefold lower, this did not interfere with the synthesis of leading strands by the HSV polymerase. One explanation for these findings is that the interactions between the subunits of the helicase-primase intertypic complex that are important for the full function of each subunit are inappropriate or weak.

Key Role of Template Sequence for Primer Synthesis by the Herpes Simplex Virus 1 Helicase−Primase

“…As mentioned in the introduction, the discrete sites where HSV-1 helicase-primase preferentially initiates primer synthesis on the pBS ssDNA [ 3′ ...GTCCTTCCG... 5′ (25)] and the φX174 ssDNA [ 3′ ...GCCCTCCCATC... 5′ (1), Table 2] have been identified. Comparing the sequence of these two reported template initiation sites (underlined above) revealed three similarities: (1) in both cases, primer initiation began opposite a template pyrimidine; (2) the template sequence just after the initiation site is pyrimidine rich, and (3) a noncoding template deoxyguanylate is found just prior to the base that codes for the 5′-terminal nucleotide of the primer.…”

“…Interestingly, primer synthesis occurred only at discrete sites on these long ssDNAs, therefore indicating that HSV-1 helicase−primase has specific template requirements for primer synthesis. The principal sites of HSV-1 primer synthesis have been identified for both the pBS and φX174 templates ( , ). Furthermore, sequence variations of the φX174 site significantly affected HSV-1 primer synthesis ().…”