DNA Binding Activity of the Herpes Simplex Virus Type 1 Origin Binding Protein, UL9, Can Be Modulated by Sequences in the N Terminus: Correlation between Transdominance and DNA Binding

Abstract

UL9, the origin binding protein of herpes simplex virus type 1, is a member of the SF2 family of helicases. Cotransfection of cells with infectious viral DNA and plasmids expressing either full-length UL9 or the C-terminal DNA binding domain alone results in the drastic inhibition of plaque formation which can be partially relieved by an insertion mutant lacking DNA binding activity. In this work, C-terminally truncated mutants which terminate at or near residue 359 were shown to potentiate plaque formation, while other C-terminal truncations were inhibitory. Thus, residues in the N-terminal region appear to regulate the inhibitory properties of UL9. To identify which residues were involved in this regulation, a series of N-terminally truncated mutants were constructed which contain the DNA binding domain and various N-terminal extensions. Mutants whose N terminus is either at residue 494 or 535 were able to bind the origin efficiently and were inhibitory to plaque formation, whereas constructs whose N terminus is at residue 304 or 394 were defective in origin binding activity and were able to relieve inhibition. Since UL9 is required for viral infection at early but not late times and is inhibitory to infection when overexpressed, we propose that the DNA binding activities of UL9 are regulated during infection. For infection to proceed, UL9 may need to switch from a DNA binding to a non-DNA binding mode, and we suggest that sequences residing in the N terminus play a role in this switch.Replication of the 152-kb double-stranded genome of herpes simplex virus type 1 (HSV-1) in cell culture requires at least seven essential virus-encoded proteins: a highly processive heterodimeric DNA polymerase (UL30/UL42), a heterotrimeric helicase-primase complex (UL5/UL8/UL52), a singlestranded DNA binding protein (UL29), and an origin binding protein (UL9) (reviewed in references 5 and 20). UL9 is a multifunctional 94-kDa protein exhibiting the following activities: DNA-stimulated nucleotide triphosphatase, 3Ј-to-5Ј DNA helicase on partially double-stranded substrates, ability to form dimers in solution, and cooperative origin-specific DNA binding (reviewed in reference 20). The N-terminal 534 residues define a domain containing seven conserved motifs, which are characteristic of the superfamily II helicases. Mutations within five of the seven conserved motifs inactivated the function of UL9 in an in vivo complementation assay (21) and also inactivated the helicase activity (19). These results suggest that the helicase domain is essential for biological activity.The HSV-1 genome contains three origins of replication, a single copy of Ori L and two copies of Ori S , and each contains high-affinity binding sites for UL9 (29,30,(32)(33)(34). The 75-bp minimal Ori S contains three closely related 11-bp palindromic sequences, two of which (box I and box II) have been shown to be essential for efficient DNA replication (33). UL9 binds specifically to box I and box II in a highly cooperative manner which may be important for distor...

Cathepsin B Mediates Cleavage of Herpes Simplex Virus Type 1 Origin Binding Protein (OBP) To Yield OBPC-1, and Cleavage Is Dependent upon Viral DNA Replication

“…It has recently been demonstrated, however, that C-terminal forms of OBP with various amounts of N-terminal sequence behave differently during infection, with some acting as inhibitors of viral replication and others actually increasing viral replication (9). Indeed, C-terminal fragments of OBP of similar size to OBPC-1 were not able to bind origin sequences in this study (9). Therefore, it is unclear if OBPC-1 is able to bind to origins.…”

“…Based on these considerations, we hypothesized that OBPC-1 may play a role in the negative regulation of OBP activity by inhibiting its binding to origin sequences and thereby facilitating the switch from origin-dependent DNA replication to origin-independent DNA replication (2). It has recently been demonstrated, however, that C-terminal forms of OBP with various amounts of N-terminal sequence behave differently during infection, with some acting as inhibitors of viral replication and others actually increasing viral replication (9). Indeed, C-terminal fragments of OBP of similar size to OBPC-1 were not able to bind origin sequences in this study (9).…”

“…Based on these observations, it has been hypothesized that OBP functions and/or levels must be regulated during infection (8,9). Specific posttranslational modifications of OBP may be used for this purpose.…”

Direct Interaction between the N- and C-Terminal Portions of the Herpes Simplex Virus Type 1 Origin Binding Protein UL9 Implies the Formation of a Head-to-Tail Dimer

“…We speculate that if excess UL9 is present and bound to viral DNA, origin clearance may be delayed or inhibited, resulting in lower overall viral yields. In support of this model, we recently reported that residues in the N-terminal region of UL9 can regulate the inhibitory properties of UL9 by modulating the DNA binding ability of the C terminus (9). The ability of residues within the N terminus of UL9 to modulate the DNA binding domain in the C terminus may be a result of physical interaction between these two apparently independent functional domains of UL9.…”

“…Plasmids pCDNA3-UL9-WT, UL9-1-450, UL9-1-354, UL9-1-321, and UL9-C were reported previously (9,27). The UL9 mutant plasmids used in this study and the PCR primers used for generating these plasmids are listed in Tables 1 and 2, respectively.…”