TY - JOUR
T1 - Herpes simplex virus 1 ICP8 mutant lacking annealing activity is deficient for viral DNA replication
AU - Weerasooriya, Savithri
AU - DiScipio, Katherine A.
AU - Darwish, Anthar S.
AU - Bai, Ping
AU - Weller, Sandra K.
N1 - Funding Information:
ACKNOWLEDGMENTS. We thank members of the S.K.W. laboratory and Dr. Lee Wright, Dr. Lynn Thomason, and Dr. James Sawitzke for discussion and careful reading of the manuscript. Additionally, we thank Maya Yankova and the EM Core facility for assistance with EM, Bing Hao and Kazuya Machida for assistance with fluorescence polarization, and Mitali Adlakha for assistance with confocal microscopy. We thank the US Department of Health and Human Services, and the NIH provided funding (to S.K.W.) under Grants AI069136 and AI021747.
Publisher Copyright:
© 2019 National Academy of Sciences. All Rights Reserved.
PY - 2019/1/15
Y1 - 2019/1/15
N2 - Most DNA viruses that use recombination-dependent mechanisms to replicate their DNA encode a single-strand annealing protein (SSAP). The herpes simplex virus (HSV) single-strand DNA binding protein (SSB), ICP8, is the central player in all stages of DNA replication. ICP8 is a classical replicative SSB and interacts physically and/or functionally with the other viral replication proteins. Additionally, ICP8 can promote efficient annealing of complementary ssDNA and is thus considered to be a member of the SSAP family. The role of annealing during HSV infection has been difficult to assess in part, because it has not been possible to distinguish between the role of ICP8 as an SSAP from its role as a replicative SSB during viral replication. In this paper, we have characterized an ICP8 mutant, Q706A/F707A (QF), that lacks annealing activity but retains many other functions characteristic of replicative SSBs. Like WT ICP8, the QF mutant protein forms filaments in vitro, binds ssDNA cooperatively, and stimulates the activities of other replication proteins including the viral polymerase, helicase–primase complex, and the origin binding protein. Interestingly, the QF mutant does not complement an ICP8-null virus for viral growth, replication compartment formation, or DNA replication. Thus, we have been able to separate the activities of ICP8 as a replicative SSB from its annealing activity. Taken together, our data indicate that the annealing activity of ICP8 is essential for viral DNA replication in the context of infection and support the notion that HSV-1 uses recombination-dependent mechanisms during DNA replication.
AB - Most DNA viruses that use recombination-dependent mechanisms to replicate their DNA encode a single-strand annealing protein (SSAP). The herpes simplex virus (HSV) single-strand DNA binding protein (SSB), ICP8, is the central player in all stages of DNA replication. ICP8 is a classical replicative SSB and interacts physically and/or functionally with the other viral replication proteins. Additionally, ICP8 can promote efficient annealing of complementary ssDNA and is thus considered to be a member of the SSAP family. The role of annealing during HSV infection has been difficult to assess in part, because it has not been possible to distinguish between the role of ICP8 as an SSAP from its role as a replicative SSB during viral replication. In this paper, we have characterized an ICP8 mutant, Q706A/F707A (QF), that lacks annealing activity but retains many other functions characteristic of replicative SSBs. Like WT ICP8, the QF mutant protein forms filaments in vitro, binds ssDNA cooperatively, and stimulates the activities of other replication proteins including the viral polymerase, helicase–primase complex, and the origin binding protein. Interestingly, the QF mutant does not complement an ICP8-null virus for viral growth, replication compartment formation, or DNA replication. Thus, we have been able to separate the activities of ICP8 as a replicative SSB from its annealing activity. Taken together, our data indicate that the annealing activity of ICP8 is essential for viral DNA replication in the context of infection and support the notion that HSV-1 uses recombination-dependent mechanisms during DNA replication.
KW - Annealing
KW - DNA replication
KW - HSV-1
KW - ICP8
KW - Recombination
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U2 - 10.1073/pnas.1817642116
DO - 10.1073/pnas.1817642116
M3 - Article
C2 - 30598436
AN - SCOPUS:85060060045
SN - 0027-8424
VL - 116
SP - 1033
EP - 1042
JO - Proceedings of the National Academy of Sciences of the United States of America
JF - Proceedings of the National Academy of Sciences of the United States of America
IS - 3
ER -