The essential 65-kilodalton DNA-binding protein of herpes simplex virus stimulates the virus-encoded DNA polymerase

Gallo, Maria F; Dorsky, David I.; Crumpacker, Clyde S.; Parris, Deborah S.

doi:10.1128/jvi.63.12.5023-5029.1989

Cited by 87 publications

(59 citation statements)

References 25 publications

(29 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Polymerase processivity factors have been identified for DNA viruses such as the gp45 protein of bacteriophage T4 (64), the UL42 protein of herpes simplex virus 1 (65,66), or E. coli thioredoxin for bacteriophage T7 (67,68). To our knowledge, coronaviruses now are the first RNA viruses known to use an RdRp processivity factor, to expedite replication of their ∼30-kb RNA genome.…”

Section: Discussionmentioning

confidence: 99%

One severe acute respiratory syndrome coronavirus protein complex integrates processive RNA polymerase and exonuclease activities

Subissi

Posthuma

Collet

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

547

709

View full text Add to dashboard Cite

In addition to members causing milder human infections, the Coronaviridae family includes potentially lethal zoonotic agents causing severe acute respiratory syndrome (SARS) and the recently emerged Middle East respiratory syndrome. The ∼30-kb positivestranded RNA genome of coronaviruses encodes a replication/ transcription machinery that is unusually complex and composed of 16 nonstructural proteins (nsps). SARS-CoV nsp12, the canonical RNA-dependent RNA polymerase (RdRp), exhibits poorly processive RNA synthesis in vitro, at odds with the efficient replication of a very large RNA genome in vivo. Here, we report that SARSCoV nsp7 and nsp8 activate and confer processivity to the RNAsynthesizing activity of nsp12. Using biochemical assays and reverse genetics, the importance of conserved nsp7 and nsp8 residues was probed. Whereas several nsp7 mutations affected virus replication to a limited extent, the replacement of two nsp8 residues (P183 and R190) essential for interaction with nsp12 and a third (K58) critical for the interaction of the polymerase complex with RNA were all lethal to the virus. Without a loss of processivity, the nsp7/nsp8/nsp12 complex can associate with nsp14, a bifunctional enzyme bearing 3′-5′ exoribonuclease and RNA cap N7-guanine methyltransferase activities involved in replication fidelity and 5′-RNA capping, respectively. The identification of this tripartite polymerase complex that in turn associates with the nsp14 proofreading enzyme sheds light on how coronaviruses assemble an RNA-synthesizing machinery to replicate the largest known RNA genomes. This protein complex is a fascinating example of the functional integration of RNA polymerase, capping, and proofreading activities.replicative complex reconstitution | processivity factor A virus-encoded RNA-dependent RNA polymerase (RdRp) is the central enzyme in the replicative cycle of RNA viruses (1). In the case of mammalian positive-strand RNA (+RNA) viruses, the enzyme is generated by the translation of the incoming viral genome, which yields a polyprotein precursor from which the RdRp-containing subunit is released by proteolytic cleavage. Subsequently, the RdRp is integrated into a membraneassociated viral enzyme complex that drives the production of negative-strand RNA (−RNA), new genome molecules, and in many virus groups also subgenomic (sg) messenger RNAs (mRNAs) (2-4). Compared with the replication of either viral or cellular DNA sequences, RNA virus genomes are copied with relatively low fidelity, because the products of replication are believed to be neither proofread nor edited (5). This property is a major factor in the evolution, adaptation, and epidemiology of RNA viruses.Among +RNA viruses, coronaviruses (CoVs) (order Nidovirales) stand out for having the largest single-stranded RNA genomes known to date (6,7). Research into the molecular and structural biology of CoVs was boosted significantly by the emergence, in 2003, of a previously undiscovered CoV that caused the severe acute respiratory syndrome (SARS) epidemic ...

show abstract

Section: Discussionmentioning

confidence: 99%

One severe acute respiratory syndrome coronavirus protein complex integrates processive RNA polymerase and exonuclease activities

Subissi

Posthuma

Collet

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

547

709

View full text Add to dashboard Cite

show abstract

“…This plasmid contains the PRV pap ORF as well as 15 bp upstream from the putative translation initiation codon and 53 bp downstream from the consensus polyadenylation signal. Plasmids pLBN 19A, encoding full-length HSV-1 UL42 (22), and pT7-7.1, encoding HSV-1 Pol lacking the first 67 amino acids (17), have been described elsewhere.…”

Section: Cells and Virusesmentioning

confidence: 99%

“…Pol assay. Pol activity was measured as incorporation of [ 3 H]dTTP (42 Ci/ mmol; Amersham) into trichloroacetic acid-insoluble radioactivity as described previously, using activated calf thymus DNA as the template (17,18,22), except that KCl concentration was varied as indicated in the figure legends. In vitro translation products from the indicated pol and pap genes (25 l) were added and incubated in a final volume of 100 l for 30 min at 37ЊC.…”

Section: Cells and Virusesmentioning

confidence: 99%

“…It has been shown to be tightly associated with a 65-kDa double-stranded DNA-binding protein encoded by the UL42 gene of HSV-1 (9,23,29,35,55,68), a gene that is essential for DNA synthesis and virus replication (38,47). The product of the UL42 gene stimulates the basal activity and processivity of Pol (22,29,35), properties which appear to be essential for HSV-1 replication (13,14,59,63).…”

mentioning

confidence: 99%

See 1 more Smart Citation

Cloning, sequencing, and functional characterization of the two subunits of the pseudorabies virus DNA polymerase holoenzyme: evidence for specificity of interaction

Berthomme

Monahan

Parris

et al. 1995

J Virol

Self Cite

View full text Add to dashboard Cite

The pseudorabies virus (PRV) genes encoding the two subunits of the DNA polymerase were located on the genome by hybridization to their herpes simplex virus type 1 (HSV-1) homologs, pol and UL42, and subsequently were sequenced. Like the HSV-1 homologs, in vitro translation products of the PRV gene encoding the catalytic subunit (pol) possessed activity in the absence of the Pol accessory protein (PAP). However, the PRV PAP stimulated the activity of Pol fourfold in the presence of 150 mM KCl, using an activated calf thymus DNA template. The stimulation of Pol activity by PAP under high-salt conditions and the inhibition of Pol activity by PAP when assayed in low salt (0 mM KCl) together were used to determine the specificity with which PAP interacted with Pol. Despite functional similarity, HSV-1 UL42 and PRV PAP could neither stimulate the noncognate Pols at high salt nor inhibit them at low salt. Furthermore, a PRV Pol mutant lacking the 30 C-terminal amino acids retained basal Pol activity but could be neither stimulated nor inhibited by the PRV PAP. Sequence comparisons of the Pol proteins of the alphaherpesviruses reveal a conserved domain in the C terminus which terminates immediately before the last 41 residues of both PRV and HSV-1 proteins. These results indicate that the ability and specificity for interaction of the PRV Pol with PAP most likely resides predominantly in the extreme Pol C terminus.

show abstract

“…(b) Peptides corresponding to the C-terminal region of the catalytic subunit or small molecules mimicking the side-chain of residues crucial for subunit interaction are capable of disrupting the DNA polymerase complex, thus inhibiting the processivity of the virus enzyme. processivity of the enzyme [19,20] without increasing the rate of catalysis per primer-binding event [21]. Mutations of UL30 and UL42 which disrupt the interaction between the two enzyme subunits specifically were shown to inhibit virus replication, thus supporting the essential role of the UL30 ⁄ UL42 association [22][23][24][25][26].…”

Section: Herpes Simplex Virus Dna Polymerasementioning

confidence: 91%

Disruption of the interactions between the subunits of herpesvirus DNA polymerases as a novel antiviral strategy

Loregian

Palù

2005

Clinical Microbiology and Infection

View full text Add to dashboard Cite

Most biological processes depend on the co-ordinated formation of protein-protein interactions. Besides their importance for virus replication, several interactions between virus proteins have been proposed as attractive targets for antiviral drug discovery, as the exquisite specificity of such cognate interactions affords the possibility of interfering with them in a highly specific and effective manner. There is a considerable need for new drugs active against herpesviruses, since available agents, most of which target the polymerisation activity of the virus DNA polymerase, are limited by pharmacokinetic issues, toxicity and antiviral resistance. A potential novel target for anti-herpesvirus drugs is the interaction between the two subunits of the virus DNA polymerase. This review focuses on recent developments using peptides and small molecules to inhibit protein-protein interactions between herpesvirus DNA polymerase subunits.

show abstract

The essential 65-kilodalton DNA-binding protein of herpes simplex virus stimulates the virus-encoded DNA polymerase

Cited by 87 publications

References 25 publications

One severe acute respiratory syndrome coronavirus protein complex integrates processive RNA polymerase and exonuclease activities

One severe acute respiratory syndrome coronavirus protein complex integrates processive RNA polymerase and exonuclease activities

Cloning, sequencing, and functional characterization of the two subunits of the pseudorabies virus DNA polymerase holoenzyme: evidence for specificity of interaction

Disruption of the interactions between the subunits of herpesvirus DNA polymerases as a novel antiviral strategy

Contact Info

Product

Resources

About