The Exonuclease Activity of Herpes Simplex Virus 1 UL12 Is Required for Production of Viral DNA That Can Be Packaged To Produce Infectious Virus.

The herpes simplex virus (HSV) type I alkaline nuclease, UL12, has 5'-to-3' exonuclease activity and shares homology with nucleases from other members of the Herpesviridae family. We previously reported that a UL12-null virus exhibits a severe defect in viral growth. To determine whether the growth defect was a result of loss of nuclease activity or another function of UL12, we introduced an exonuclease-inactivating mutation into the viral genome. The recombinant virus, UL12 D340E (the D340E mutant), behaved identically to the null virus (AN-1) in virus yield experiments, exhibiting a 4-log decrease in the production of infectious virus. Furthermore, both viruses were severely defective in cell-to-cell spread and produced fewer DNA-containing capsids and more empty capsids than wild-type virus. In addition, DNA packaged by the viral mutants was aberrant, as determined by infectivity assays and pulsed-field gel electrophoresis. We conclude that UL12 exonuclease activity is essential for the production of viral DNA that can be packaged to produce infectious virus. This conclusion was bolstered by experiments showing that a series of natural and synthetic α-hydroxytropolones recently reported to inhibit HSV replication also inhibit the nuclease activity of UL12. Taken together, our results demonstrate that the exonuclease activity of UL12 is essential for the production of infectious virus and may be considered a target for development of antiviral agents.IMPORTANCE Herpes simplex virus is a major pathogen, and although nucleoside analogs such as acyclovir are highly effective in controlling HSV-1 or -2 infections in immunocompetent individuals, their use in immunocompromised patients is complicated by the development of resistance. Identification of additional proteins essential for viral replication is necessary to develop improved therapies. In this communication, we confirm that the exonuclease activity of UL12 is essential for viral replication through the analysis of a nuclease-deficient viral mutant. We demonstrate that the exonuclease activity of UL12 is essential for the production of viral progeny and thus provides an attractive, druggable enzymatic target.

ICP8 Filament Formation Is Essential for Replication Compartment Formation during Herpes Simplex Virus Infection.

UNLABELLED: Herpes simplex virus (HSV) dramatically reorganizes the infected-cell nucleus, leading to the formation of prereplicative sites and replication compartments. This process is driven by the essential viral single-stranded DNA (ssDNA) binding protein ICP8, which can form double-helical filaments in the absence of DNA. In this paper, we show that two conserved motifs, FNF (F1142, N1143, and F1144) and FW (F843 and W844), are essential for ICP8 self-interactions, and we propose that the FNF motif docks into the FW region during filament formation. Mammalian expression plasmids bearing mutations in these motifs (FNF and FW) were unable to complement an ICP8-null mutant for growth and replication compartment formation. Furthermore, FNF and FW mutants were able to inhibit wild-type (WT) virus plaque formation and filament formation, whereas a double mutant (FNF-FW) was not. These results suggest that single mutant proteins are incorporated into nonproductive ICP8 filaments, while the double mutant is unable to interact with WT ICP8 and does not interfere with WT growth. Cells transfected with WT ICP8 and the helicase-primase (H/P) complex exhibited punctate nuclear structures that resemble prereplicative sites; however, the FNF and FW mutants failed to do so. Taken together, these results suggest that the FNF and FW motifs are required for ICP8 self-interactions and that these interactions may be important for the formation of prereplicative sites and replication compartments. We propose that filaments or other higher-order structures of ICP8 may provide a scaffold onto which other proteins can be recruited to form prereplicative sites and replication compartments. IMPORTANCE: For nuclear viruses such as HSV, efficient DNA replication requires the formation of discrete compartments within the infected-cell nucleus in which replication proteins are concentrated and assembled into the HSV replisome. In this paper, we characterize the role of filament formation by the single-stranded DNA binding protein ICP8 in the formation of prereplicative sites and replication compartments. We propose that ICP8 protein filaments generate a protein scaffold for other cellular and viral proteins, resulting in a structure that concentrates both viral DNA and replication proteins. Replication compartments may be similar to other types of cellular membraneless compartments thought to be formed by phase separations caused by low-affinity, multivalent interactions involving proteins and nucleic acids within cells. ICP8 scaffolds could facilitate the formation of replication compartments by mediating interactions with other components of the replication machinery.

HSV-1 protein expression using recombinant baculoviruses.

The baculovirus expression system is an invaluable method for the expression of Herpes Simplex Virus 1 (HSV-1) proteins. The use of insect cells provides a eukaryotic system for the robust expression of heterologous proteins under control of the baculovirus polyhedrin gene promoter that naturally drives the high expression of the polyhedrin protein. Additionally, insect cells often initiate the necessary posttranslational modifications and/or disulfide-bond formation important for the proper folding of the protein. We and others have successfully expressed and purified several HSV-1 proteins including the polymerase, helicase-primase, single-strand DNA binding protein, and alkaline nuclease. The following protocol is based on 15 years of experience from our laboratory.

The variable subdomain of Escherichia coli SecA functions to regulate SecA ATPase activity and ADP release.

Bacterial SecA proteins can be categorized by the presence or absence of a variable subdomain (VAR) located within nucleotide-binding domain II of the SecA DEAD motor. Here we show that VAR is dispensable for SecA function, since the VAR deletion mutant secAΔ519-547 displayed a wild-type rate of cellular growth and protein export. Loss or gain of VAR is extremely rare in the history of bacterial evolution, indicating that it appears to contribute to secA function within the relevant species in their natural environments. VAR removal also results in additional secA phenotypes: azide resistance (Azi(r)) and suppression of signal sequence defects (PrlD). The SecAΔ(519-547) protein was found to be modestly hyperactive for SecA ATPase activities and displayed an accelerated rate of ADP release, consistent with the biochemical basis of azide resistance. Based on our findings, we discuss models whereby VAR allosterically regulates SecA DEAD motor function at SecYEG.

Characterization of the Escherichia coli SecA signal peptide-binding site.

SecA signal peptide interaction is critical for initiating protein translocation in the bacterial Sec-dependent pathway. Here, we have utilized the recent nuclear magnetic resonance (NMR) and Förster resonance energy transfer studies that mapped the location of the SecA signal peptide-binding site to design and isolate signal peptide-binding-defective secA mutants. Biochemical characterization of the mutant SecA proteins showed that Ser226, Val310, Ile789, Glu806, and Phe808 are important for signal peptide binding. A genetic system utilizing alkaline phosphatase secretion driven by different signal peptides was employed to demonstrate that both the PhoA and LamB signal peptides appear to recognize a common set of residues at the SecA signal peptide-binding site. A similar system containing either SecA-dependent or signal recognition particle (SRP)-dependent signal peptides along with the prlA suppressor mutation that is defective in signal peptide proofreading activity were employed to distinguish between SecA residues that are utilized more exclusively for signal peptide recognition or those that also participate in the proofreading and translocation functions of SecA. Collectively, our data allowed us to propose a model for the location of the SecA signal peptide-binding site that is more consistent with recent structural insights into this protein translocation system.