The origin-binding domain of the herpes simplex virus type 1 UL9 protein is not required for DNA helicase activity

2003

J Virol

UL9 is a multifunctional protein required for herpes simplex virus type 1 (HSV-1) replication in vivo. UL9 is a member of the superfamily II helicases and exhibits helicase and origin-binding activities. We have previously shown that mutations in the conserved helicase motifs of UL9 can have either a transdominant or potentiating effect on the plaque-forming ability of infectious DNA from wild-type virus (A. J. Malik and S. K. Weller, J. Virol. 70:7859-7866, 1996). In this paper, the mechanisms of transdominance and potentiation are explored. We show that the motif V mutant protein containing a G to A substitution at residue 354 is unstable when expressed by transfection and is either processed to a 38-kDa N-terminal fragment or degraded completely. The overexpression of the MV mutant protein is able to influence the steady-state protein levels of wild-type UL9 and to override the inhibitory effects of wild-type UL9. Potentiation correlates with the ability of the UL9 variants containing the G354A mutation to be processed or degraded to the 38-kDa form. We propose that the MV mutant protein is able to interact with full-length UL9 and that this interaction results in a decrease in the steady-state levels of UL9, which in turn leads to enhanced viral infection. Furthermore, we demonstrate that inhibition of HSV-1 infection can be obtained by overexpression of full-length UL9, the C-terminal third of the protein containing the origin-binding domain, or the N-terminal two-thirds of UL9 containing the conserved helicase motifs and the putative dimerization domain. Our results suggest that transdominance can be mediated by overexpression, origin-binding activity, and dimerization, whereas potentiation is most likely caused by the ability of the UL9 MV mutant to influence the steady-state levels of wild-type UL9. Taken together, the results presented in this paper suggest that the regulation of steady-state levels of UL9 may play an important role in controlling viral infection.The UL9 gene is required for herpes simplex virus type 1 (HSV-1) replication in vivo (6, 9). The UL9 protein is a dimer in solution and exhibits helicase, ATPase, and origin-binding activities (8, 13). UL9 is believed to play a key role in the initiation of HSV-1 replication by binding the HSV-1 origin of replication via its C-terminal domain and unwinding it in the presence of ATP and ICP8, the HSV-1 single-stranded DNA binding protein. It is likely that UL9 plays an important role in the assembly of the viral replisome (10, 20, 26, 41) through its interactions with other viral replication proteins (7,28,29).UL9 is a member of the superfamily II helicases (14). The conserved helicase motifs that are characteristic of this superfamily are positioned within the N-terminal domain of the protein (14). Genetic studies have previously shown that conserved residues within the helicase motifs are essential for HSV-1 replication in vivo; most engineered motif mutants fail to complement the growth of hr94, a UL9 null virus (24,27). Furthermore, biochemi...

Section: Resultssupporting

confidence: 83%

mentioning

confidence: 99%

Existence of Transdominant and Potentiating Mutants of UL9, the Herpes Simplex Virus Type 1 Origin-Binding Protein, Suggests that Levels of UL9 Protein May Be Regulated during Infection

2003

J Virol

“…It is interesting that although the engineered motif Ia mutants exhibited defects in ssDNA-binding activity, their ability to bind the double-stranded HSV-1 origin of replication was similar to that of the wild type. This finding is consistent with the notion that the N-terminal domain of UL9 harbors all functions attributable to a helicase (including ssDNA binding), whereas the C-terminal domain harbors the dsDNA originbinding function (1,3). proteins to bind OriS was tested by a double-membrane filter-binding assay, performed as described in Materials and Methods.…”

Section: Vol 77 2003 Structure-function Analysis Of Ul9 Helicase Mosupporting

confidence: 70%

“…On the other hand, they resemble the wild-type protein in their abilities to dimerize and bind the HSV-1 origin of replication (30). The C terminus of UL9 (amino acids 535 to 851) forms a domain responsible for the recognition of the HSV origins of replication (1,3), and it is believed that this domain is responsible for targeting UL9 to the origins during the initiation of DNA replication. Once bound at the origin, however, UL9 is expected to unwind the origin and translocate along ssDNA, an activity that requires the ability to bind ssDNA (37).…”

mentioning

confidence: 99%

Helicase Motif Ia Is Involved in Single-Strand DNA-Binding and Helicase Activities of the Herpes Simplex Virus Type 1 Origin-Binding Protein, UL9

2003

J Virol

On the basis of sequence conservation between several UL9 homologs within the Herpesviridae family and distant homology with helicases whose structures have been solved, we designed specific mutations in motif Ia and analyzed them genetically and biochemically. Mutant proteins with residues predicted to be involved in ssDNA binding (R112A and R113A/F115A) exhibited wild-type levels of intrinsic ATPase activity and moderate to severe defects in ssDNA-stimulated ATPase activity and ssDNA binding. The S110T mutation targets a residue not predicted to contact ssDNA directly. The mutant protein with this mutation exhibited wild-type levels of intrinsic ATPase activity and near wild-type levels of ssDNA-stimulated ATPase activity and ssDNA binding. All mutant proteins lack helicase activity but were able to dimerize and bind the HSV-1 origin of replication as well as wild-type UL9. Our results indicate that residues from motif Ia contribute to the ssDNA-binding and helicase activities of UL9 and are essential for viral growth. This work represents the successful application of an approach based on a combination of bioinformatics and structural information from related proteins to deduce valuable information about a protein of interest.The UL9 gene is essential for herpes simplex virus type 1 (HSV-1) replication in vivo (5, 9). UL9 is a multifunctional protein, exhibiting intrinsic and single-stranded DNA (ssDNA)-stimulated ATPase activity, 3Ј to 5Ј helicase activity, ssDNAbinding and origin-binding activities (8,15). It is thought that UL9 binds the HSV-1 origin of replication and unwinds it in the presence of ATP and ICP8, the HSV-1 ssDNA-binding protein. In addition, UL9 interacts with several viral proteins that are essential for HSV-1 replication: ICP8 (6); UL42, the processivity subunit of the viral polymerase (34) and UL8, a subunit of the heteromeric helicase/primase complex (33).UL9 is a superfamily II (SFII) helicase and possesses seven conserved helicase motifs, positioned within the N-terminal domain of the protein (16). Using site-directed mutagenesis, we have previously shown that conserved residues within helicase motifs I to VI of UL9 are essential for HSV-1 replication in vivo; most of the engineered motif mutants fail to complement the growth of UL9 null virus (32). Consistent with the genetic data, the UL9 helicase motif mutant proteins exhibit defects in ATPase and helicase activity in vitro (30). On the other hand, they resemble the wild-type protein in their abilities to dimerize and bind the HSV-1 origin of replication (30). The C terminus of UL9 (amino acids 535 to 851) forms a domain responsible for the recognition of the HSV origins of replication (1, 3), and it is believed that this domain is responsible for targeting UL9 to the origins during the initiation of DNA replication. Once bound at the origin, however, UL9 is expected to unwind the origin and translocate along ssDNA, an activity that requires the ability to bind ssDNA (37). The UL9 residues responsible for ssDNA binding have not bee...

“…1) as follows: a C-terminal origin-binding domain (residues 535-851) and an N-terminal helicase domain (residues 1-535) that contains the SFII-conserved helicase motifs (3). Each domain expressed separately exhibits the expected biochemical activities (26,27). UL9 has been hypothesized to bind and unwind the HSV-1 origin of replication, creating a replication bubble and promoting the assembly of the viral replication machinery (28 -30); however, direct confirmation of this hypothesis has been lacking.…”

mentioning

confidence: 99%

Residues within the Conserved Helicase Motifs of UL9, the Origin-binding Protein of Herpes Simplex Virus-1, Are Essential for Helicase Activity but Not for Dimerization or Origin Binding Activity

Journal of Biological Chemistry

2001

Here we present the characterization of the biochemical properties of the UL9 helicase motif mutants. We report that mutations in motifs I-IV and VI affect the ATPase activity, and all but the motif III mutation completely abolish the helicase activity. In addition, mutations in these motifs do not interfere with UL9 dimerization or the ability of UL9 to bind the HSV-1 origin of replication. Based on the similarity of the helicase motif sequences between UL9 and UvrB, another superfamily II member with helicase-like activity, we were able to map the UL9 mutations on the structure of the UvrB protein and provide an explanation for the observed phenotypes. Our results indicate that the helicase function of UL9 is indispensable for viral replication, supporting the hypothesis that UL9 is essential for unwinding the HSV-1 origin of replication in vivo. Furthermore, the data presented provide insights into the mechanism of transdominance of the UL9 helicase motif mutants.