Two regions of the herpes simplex virus type 1 UL42 protein are required for its functional interaction with the viral DNA polymerase

Monahan, Steven J.; Barlam, Tamar F.; Crumpacker, Clyde S.; Parris, Deborah S.

doi:10.1128/jvi.67.10.5922-5931.1993

Cited by 28 publications

(15 citation statements)

References 56 publications

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“…Although these three domains correspond to only 57% of the PRV polypeptide, they include all of the identical amino acids and 94% of the similar residues. Although the relationship between the structure and the function of the HSV-1 UL42 protein is not completely understood, most of the results from mutagenesis studies support the importance of the conserved domains in the amino-terminal portion of the HSV-1 UL42 (14,53,59). The strongest divergence, which occurs downstream of PRV residue 317, results not only from the longer HSV-1 C-terminal protein but also from poorly conserved homology, even in pairwise alignments of this region or in threeway alignments without the HSV-1 protein (not shown).…”

Section: Comparison Of Prv Pol Sequences With Sequences Of Herpesvirumentioning

confidence: 99%

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

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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.

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Section: Comparison Of Prv Pol Sequences With Sequences Of Herpesvirumentioning

confidence: 99%

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

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“…I-160 bound dsDNA, failed to bind UL30, and lacked UL30-stimulatory activity (4). I-140, in contrast, failed to stimulate UL30 but could coprecipitate UL30 (20). Because UL42 ⌬129-163 bound to UL30 (20), it appears unlikely that the I-160 mutant identified a UL30-binding region of UL42.…”

Section: Discussionmentioning

confidence: 95%

“…The presence of neutralizing epitopes on a region of BMRF1 dispensable for in vitro function suggests that the carboxy-terminal 102 residues are situated in the native structure such that they stabilize an essential region of BMRF1 or such that the binding of the antibody interferes with BMRF1 function through steric hindrance. MAb 6898 against HSV UL42, in contrast, recognized a carboxy-terminal epitope in UL42 residues 363 to 369 (which are not required for UL30 stimulation or binding) (21) and precipitated a UL42-UL30 complex efficiently (20). Because of the requirement of both BALF5 and BMRF1 components for processive EBVpol activity, it seemed likely that a physical complex between them could be detected by immunoprecipitation, as has been described for the HSVpol UL30-UL42 complex (3)(4)(5)20).…”

Section: Discussionmentioning

confidence: 99%

“…MAb 6898 against HSV UL42, in contrast, recognized a carboxy-terminal epitope in UL42 residues 363 to 369 (which are not required for UL30 stimulation or binding) (21) and precipitated a UL42-UL30 complex efficiently (20). Because of the requirement of both BALF5 and BMRF1 components for processive EBVpol activity, it seemed likely that a physical complex between them could be detected by immunoprecipitation, as has been described for the HSVpol UL30-UL42 complex (3)(4)(5)20). However, attempts to detect coimmunoprecipitation of BALF5 by anti-BMRF1 antibodies failed to identify such a complex with MAbs R3 and 90E2 (6).…”

Section: Discussionmentioning

confidence: 99%

“…Mutational analysis of UL42 indicated that the carboxy-terminal 172 residues are dispensable for dsDNA binding and UL30-stimulatory activity (4,32), while the largest internal deletions described for HSV UL42 which preserved dsDNA binding were ⌬9-20 and ⌬242-250 (4). Two regions of UL42 required for functional interaction with the catalytic subunit were identified by deletion mutations ⌬129-163 and ⌬202-337, and the upstream mutant retained its ability to coprecipitate the catalytic subunit, while the downstream did not, suggesting that residues 202 to 337 were important for direct interaction with UL30 (20). Another mutational analysis of UL42 mapped dsDNA binding to within the carboxy-terminal 340 residues but showed that ⌬C282 still partially bound UL30, whereas ⌬250-308 lacked both dsDNA-and UL30-binding activities (4); loss of functional activity (stimulation of UL30 processivity) correlated with loss of either dsDNA binding or UL30 binding.…”

Section: Discussionmentioning

confidence: 99%

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Bipartite DNA-binding region of the Epstein-Barr virus BMRF1 product essential for DNA polymerase accessory function

Kiehl

Dorsky

1995

J Virol

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The Epstein-Barr virus (EBV) BMRF1 gene product is necessary for DNA polymerase catalytic subunit (BALF5) activity in 100 mM ammonium sulfate. To map regions of BMRF1 necessary for polymerase accessory function, linker insertion and deletion mutant BMRF1 polypeptides were expressed by in vitro transcriptiontranslation and assayed for DNA polymerase elongation activity and binding to double-stranded DNA (dsDNA)-cellulose. Amino-terminal deletions up to residue 303 were defective for stimulation of elongation. Deletions between residues 44 and 194 and residues 238 and 303 abolished binding to dsDNA-cellulose. The region from residues 194 to 238, therefore, is necessary for stimulation of BALF5 elongation but dispensable for dsDNA-cellulose binding. Deletion analysis also localized reactive epitopes of two neutralizing monoclonal antibodies to BMRF1 to a carboxy-terminal region which is dispensable for activity. These data suggest that a bipartite DNA-binding region is an essential component of the DNA polymerase accessory function and that the two noncontiguous regions are separated by a region (residues 194 to 217) which is essential for stimulation; therefore, it may interact with the BALF5 catalytic subunit of EBV DNA polymerase. Both EBV BMRF1 and herpes simplex virus UL42 gene products are DNA polymerase accessory proteins which bind dsDNA and increase the processivity of their corresponding catalytic components. Outstanding similarities between their primary amino acid sequences are not evident. However, it appears that their structural organizations are similar.

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A Major DNA Binding Protein Encoded by BALF2 Open Reading Frame of Epstein–Barr Virus (EBV) Forms a Complex with Other EBV DNA-Binding Proteins: DNAase, EA-D, and DNA Polymerase

Zeng

Middeldorp²,

Madjar

et al. 1997

Virology

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A major 135-kDa DNA binding protein (mDBP) encoded by the BALF2 open reading frame of Epstein-Barr Virus (EBV) is known to be an essential protein for the induction of the lytic cycle. The present investigation was carried out to know whether this protein forms a complex in vivo with other viral DNA binding proteins (DBP) involved in DNA replication: DNA polymerase, EA-D (diffused early antigen), and DNAase. Immunoprecipitation assays followed by mono- and two-dimensional electrophoresis showed that mDBP forms a complex with these three DBP. Other complexes were also found such as EA-D/DNAase, DNA polymerase/DNAase, and DNA polymerase/EA-D. The complexed forms already exist in the early stage of EBV cycle before DNA synthesis is induced in the EBV producer P3HR-1 cell line. The exonuclease activity encoded by DNAase was found to be inhibited when this enzyme complexed with mDBP, while the EBV DNA polymerase retained its activity in the complexed form with mDBP. Our results suggest that these complexes already present before DNA synthesis are necessary for EBV DNA synthesis.

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Two regions of the herpes simplex virus type 1 UL42 protein are required for its functional interaction with the viral DNA polymerase

Cited by 28 publications

References 56 publications

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

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

Bipartite DNA-binding region of the Epstein-Barr virus BMRF1 product essential for DNA polymerase accessory function

A Major DNA Binding Protein Encoded by BALF2 Open Reading Frame of Epstein–Barr Virus (EBV) Forms a Complex with Other EBV DNA-Binding Proteins: DNAase, EA-D, and DNA Polymerase

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