Identification of Conserved Amino Acids in the Herpes Simplex Virus Type 1 UL8 Protein Required for DNA Synthesis and UL52 Primase Interaction in the Virus Replisome

Abstract

Background:The herpes simplex virus replisome is composed of seven proteins encoded by the virus. Results: Analyses of hybrid replisomes revealed species-specific interactions, and mutagenesis of UL8 identified amino acids required for DNA synthesis and UL52 primase interaction. Conclusion:The herpesvirus replisome is a conserved molecular machine with species-specific interactions. Significance: This work provides functional and mechanistic insights into herpesvirus replication.

UL52 Primase Interactions in the Herpes Simplex Virus 1 Helicase-Primase Are Affected by Antiviral Compounds and Mutations Causing Drug Resistance

“…Similarly, HA-tagged UL5 was created by direct insertion of the HA-coding sequence right upstream the ul5 gene in the PE5 vector ( 24 ). The FLAG- and emerald GFP (emGFP)-tagged UL8 constructs were as described previously ( 12 ). All HA-UL5 and eGFP-UL52 mutants were made using the kit referred to above, and all DNA oligonucleotides were produced by Eurofins Genomics.…”

“…The remaining viral replisome components UL30-UL42 DNA polymerase and the trimeric helicase-primase complex are then assumed to be recruited to the activated origins of replication. The replisome can be operationally defined as a molecular machine inasmuch as none of its components can be exchanged for a functional homolog from a closely related virus ( 12 ). HSV-1 helicase-primase was first discovered as a DNA-dependent ATPase and subsequently shown to consist of the UL5 helicase, the UL8 accessory protein, and the UL52 primase subunits ( 13 , 14 ).…”

“…To perform these tasks, helicase-primase will most likely undergo a series of conformational changes triggered by interactions with the DNA template and/or by protein interactions between the three subunits and with the other enzymes involved in DNA synthesis. Little is known about these interactions, but it has been shown in yeast two-hybrid experiments that the UL8 protein interacts with the C-terminal 2/3 of the UL52 primase and that a conserved sequence motif in the C-terminal part of UL8 is required for the UL8-UL52 interaction ( 12 , 23 ). Other protein-protein interactions within the helicase-primase complex as well as between helicase-primase and remaining replisome components remain to be elucidated.…”

Production and characterisation of Epstein–Barr virus helicase–primase complex and its accessory protein BBLF2/3

“…Using sequence alignments with Clustal omega, we showed that the accessory proteins of a-, b-and c-herpesvirus families share sequence homology over the full length of their sequence. For HSV UL8 some important residues for the function in DNA replication and the interaction with the primase UL52 have been identified [46]. The residue required for DNA replication (mutant A23, [46]) maps into the conserved motif 1 and the one important for interaction with UL52 (mutant A49, [46]) to motif 4.…”

“…For HSV UL8 some important residues for the function in DNA replication and the interaction with the primase UL52 have been identified [46]. The residue required for DNA replication (mutant A23, [46]) maps into the conserved motif 1 and the one important for interaction with UL52 (mutant A49, [46]) to motif 4. As it has been proposed very recently that aherpesvirus accessory proteins might be related to family B polymerases [21], this might also be true for BBLF2/3 and herpesvirus accessory proteins in general.…”