Comparison of exonucleolytic activities of herpes simplex virus type‐1 DNA polymerase and DNase

Replication of the human herpesvirus Epstein-Barr virus drastically impairs cellular protein synthesis. This shutoff phenotype results from mRNA degradation upon expression of the early lytic-phase protein BGLF5. Interestingly, BGLF5 is the viral DNase, or alkaline exonuclease, homologues of which are present throughout the herpesvirus family. During productive infection, this DNase is essential for processing and packaging of the viral genome. In contrast to this widely conserved DNase activity, shutoff is only mediated by the alkaline exonucleases of the subfamily of gammaherpesviruses. Here, we show that BGLF5 can degrade mRNAs of both cellular and viral origin, irrespective of polyadenylation. Furthermore, shutoff by BGLF5 induces nuclear relocalization of the cytosolic poly(A) binding protein. Guided by the recently resolved BGLF5 structure, mutants were generated and analyzed for functional consequences on DNase and shutoff activities. On the one hand, a point mutation destroying DNase activity also blocks RNase function, implying that both activities share a catalytic site. On the other hand, other mutations are more selective, having a more pronounced effect on either DNA degradation or shutoff. The latter results are indicative of an oligonucleotide-binding site that is partially shared by DNA and RNA. For this, the flexible “bridge” that crosses the active-site canyon of BGLF5 appears to contribute to the interaction with RNA substrates. These findings extend our understanding of the molecular basis for the shutoff function of BGLF5 that is conserved in gammaherpesviruses but not in alpha- and betaherpesviruses.

Section: Discussionmentioning

confidence: 99%

The “Bridge” in the Epstein-Barr Virus Alkaline Exonuclease Protein BGLF5 Contributes to Shutoff Activity during Productive Infection

Horst

Burmeister

Boer

et al. 2012

“…Herpes simplex virus type 1 (HSV-1) encodes a 5Ј-to-3Ј exonuclease (17,23,32,52) termed alkaline nuclease, the product of the UL12 open reading frame (29). Recently, computer database searches have revealed that the HSV-1 UL12 gene shares homology with bacteriophage lambda Red␣, commonly known as exonuclease (1,36).…”

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confidence: 99%

“…The UL12 gene product, like Red␣ and RecE, is a 5Ј33Ј exonuclease (17,23,32,52). Analogous to the interaction of lambda Red␣ and the ssDNA binding protein (SSB) lambda Red␤, UL12 interacts with the SSB of HSV-1, ICP8 (53,55).…”

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confidence: 99%

The Herpes Simplex Virus Type 1 Alkaline Nuclease and Single-Stranded DNA Binding Protein Mediate Strand Exchange In Vitro

Reuven

Staire

Myers

et al. 2003

108

126

The replication of herpes simplex virus type 1 (HSV-1) DNA is associated with a high degree of homologous recombination. While cellular enzymes may take part in mediating this recombination, we present evidence for an HSV-1-encoded recombinase activity. HSV-1 alkaline nuclease, encoded by the UL12 gene, is a 533 exonuclease that shares homology with Red␣, commonly known as exonuclease, an exonuclease required for homologous recombination by bacteriophage lambda. The HSV-1 single-stranded DNA binding protein ICP8 is an essential protein for HSV DNA replication and possesses single-stranded DNA annealing activities like the Red␤ synaptase component of the phage lambda recombinase. Here we show that UL12 and ICP8 work together to effect strand exchange much like the Red system of lambda. Purified UL12 protein and ICP8 mediated the complete exchange between a 7.25-kb M13mp18 linear double-stranded DNA molecule and circular single-stranded M13 DNA, forming a gapped circle and a displaced strand as final products. The optimal conditions for strand exchange were 1 mM MgCl 2 , 40 mM NaCl, and pH 7.5. Stoichiometric amounts of ICP8 were required, and strand exchange did not depend on the nature of the double-stranded end. Nuclease-defective UL12 could not support this reaction. These data suggest that diverse DNA viruses appear to utilize an evolutionarily conserved recombination mechanism.Herpes simplex virus type 1 (HSV-1) is a double-stranded DNA (dsDNA) virus with a 152-kb linear genome. Replication of HSV-1 DNA takes place in the host nucleus. The first step of viral replication involves the circularization of the genome (13, 43). Shortly thereafter, replication intermediates appear as longer-than-unit-length head-to-tail concatemers (19) that have undergone genomic inversion (2, 27, 47, 58). The genome concatemers are not linear but rather consist of a mixture of complex structures such as Y-and X-shaped branches, replication bubbles, and tangled masses (20,28,48,49). The presence of these structures and the inversion of the L and S genome segments suggest that recombination plays a role in the replication of HSV-1 DNA. In fact, high levels of recombination are known to accompany HSV infection (3,10,11,46,54). While cellular recombinases may be involved in mediating some of these processes (57), the possibility exists that HSV-1 encodes recombinases that can also participate.Herpes simplex virus type 1 (HSV-1) encodes a 5Ј-to-3Ј exonuclease (17, 23, 32, 52) termed alkaline nuclease, the product of the UL12 open reading frame (29). Recently, computer database searches have revealed that the HSV-1 UL12 gene shares homology with bacteriophage lambda Red␣, commonly known as exonuclease (1, 36). The Red␣ protein is a 5Ј33Ј exonuclease which is part of the Red recombinase previously shown to be required for recombination by bacteriophage lambda in a RecA Ϫ host (8,9,50). Red␣ operates in conjunction with a single-stranded DNA (ssDNA) binding protein, lambda Red␤, which promotes ssDNA annealing (34). The lambda Red recomb...

“…HSV isolates, whether from patients or cell culture, are heterogeneous populations and thus contain preexisting drugresistant TK variants (six to eight mutants per 10 4 plaqueforming viruses) (12,31,36). The infidelity of the HSV DNA replication process is directly responsible for this naturally occurring variation (17,24,25,28), with errors in the viral DNA introduced spontaneously during DNA replication and not requiring the presence of drug. However, exposure to a nucleoside analog may provide selective pressure leading to the enrichment of such preexisting drug-resistant viruses.…”

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confidence: 99%

Characterization of Herpes Simplex Viruses Selected in Culture for Resistance to Penciclovir or Acyclovir

Sarisky¹,

Quail²,

Clark³

et al. 2001

Penciclovir (PCV), an antiherpesvirus agent in the same class as acyclovir (ACV), is phosphorylated in herpes simplex virus (HSV)-infected cells by the viral thymidine kinase (TK).Resistance to ACV has been mapped to mutations within either the TK or the DNA polymerase gene. An identical activation pathway, the similarity in mode of action, and the invariant cross-resistance of TK-negative mutants argue that the mechanisms of resistance to PCV and ACV are likely to be analogous. A total of 48 HSV type 1 (HSV-1) and HSV-2 isolates were selected after passage in the presence of increasing concentrations of PCV or ACV in MRC-5 cells. Phenotypic analysis suggested these isolates were deficient in TK activity. Moreover, sequencing of the TK genes from ACV-selected mutants identified two homopolymeric G-C nucleotide stretches as putative hot spots, thereby confirming previous reports examining Acv r clinical isolates. Surprisingly, mutations identified in PCV-selected mutants were generally not in these regions but distributed throughout the TK gene and at similar frequencies of occurrence within A-T or G-C nucleotides, regardless of virus type. Furthermore, HSV-1 isolates selected in the presence of ACV commonly included frameshift mutations, while PCV-selected HSV-1 mutants contained mostly nonconservative amino acid changes. Data from this panel of laboratory isolates show that Pcv r mutants share cross-resistance and only limited sequence similarity with HSV mutants identified following ACV selection. Subtle differences between PCV and ACV in the interaction with viral TK or polymerase may account for the different spectra of genotypes observed for the two sets of mutants.The introduction of penciclovir [PCV;9-(4-hydroxy-3-hydroxymethylbut-1-yl)guanine] and its prodrug, famciclovir, (FCV), resulted in the use of antivirals alternative to acyclovir (ACV) for treatment of herpes simplex virus (HSV) infections. Biochemical studies have indicated that PCV, like ACV, is phosphorylated by the viral thymidine kinase (TK) to a monophosphate and subsequently converted by cellular enzymes to a triphosphate, which inhibits the HSV DNA polymerase (Pol) (44). Although PCV and ACV have identical activation pathways and similar modes of action (14, 44), and the frequencies with which resistance in HSV arises to PCV and ACV in cell culture are identical (36), the affinities and therefore the fine molecular interactions of PCV, ACV, and their triphosphates with TK and Pol differ (14). The last point raises the possibility that drug-resistant mutants selected by these antiviral agents may differ.Resistance to acyclovir typically arises by a single mutation in either the TK or Pol gene (11,23,29). The viral TK, unlike DNA polymerase, is not essential for virus replication in cell culture (13), although in vivo analyses implicate it in HSV virulence, pathogenicity, and reactivation from latency (9,15,20,41). Mutations in HSV TK are the most common causes of clinical resistance to ACV (7,34), and the majority of mutants completely lac...