The Positively Charged Surface of Herpes Simplex Virus UL42 Mediates DNA Binding

Abstract

Herpes simplex virus DNA polymerase is a heterodimer composed of UL30, a catalytic subunit, and UL42, a processivity subunit. Mutations that decrease DNA binding by UL42 decrease long chain DNA synthesis by the polymerase. The crystal structure of UL42 bound to the C terminus of UL30 revealed an extensive positively charged surface ("back face"). We tested two hypotheses, 1) the C terminus of UL30 affects DNA binding and 2) the positively charged back face mediates DNA binding. Addressing the first hypothesis, we found that the presence of a peptide corresponding to the UL30 C terminus did not result in altered binding of UL42 to DNA. Addressing the second hypothesis, previous work showed that substitution of four conserved arginine residues on the basic face with alanines resulted in decreased DNA affinity. We tested the affinities for DNA and the stimulation of long chain DNA synthesis of mutants in which the four conserved arginine residues were substituted individually or together with lysines and also a mutant in which a conserved glutamine residue was substituted with an arginine to increase positive charge on the back face. We also engineered cysteines onto this surface to permit disulfide cross-linking studies. Last, we assayed the effects of ionic strength on DNA binding by UL42 to estimate the number of ions released upon binding. Our results taken together strongly suggest that the basic back face of UL42 contacts DNA and that positive charge on this surface is important for this interaction.Most replicative DNA polymerases depend on accessory proteins known as processivity factors to achieve prolonged association with DNA. In this way polymerases are able to synthesize long stretches of DNA before dissociating from their templates. The most studied processivity factors include proliferating nuclear antigen (PCNA) 3 from eukaryotes and archaebacteria (1, 2), the ␤-subunit from Escherichia coli (3), and gp45 from T4 and RB69 bacteriophage (4, 5). These ring shaped proteins, also known as "sliding clamps," cannot bind DNA on their own and require protein complexes called clamp loaders to be loaded onto DNA as toroidal homomultimers in an ATP-dependent manner (6). They are then able to physically tether the catalytic subunit of polymerase, thus ensuring its processivity (7-9).The DNA polymerase encoded by herpes simplex virus (HSV) is a heterodimer composed of a catalytic subunit, UL30, and a processivity subunit, UL42 (10 -12). UL42 interacts with the C terminus of UL30, increases affinity of the polymerase for primer/template DNA, and stimulates long chain DNA synthesis (13-18). In contrast to sliding clamps, UL42 binds DNA directly as a monomer with nanomolar affinity and does not require ATP hydrolysis or accessory proteins for binding (10,14,18,19). Even though UL42 binds DNA tightly, it is able to diffuse on DNA and does not impede UL30 elongation (18,20).The crystal structure of UL42 has been solved in complex with the C-terminal 36 residues of UL30 (21). The structural fold of UL42 is remarkabl...

Nuclear Import of HSV-1 DNA Polymerase Processivity Factor UL42 Is Mediated by a C-Terminally Located Bipartite Nuclear Localization Signal

“…This finding is consistent with the recent identification of functional NLSs at the C-terminus of PAPs from other herpesviruses (16)(17)(18)(19). Our data clearly show that UL42 binds to different IMPR isoforms and localizes exclusively to the cell nucleus when transiently expressed in the absence of other viral proteins, as detected by coimmunoprecipitation assays and fluorescent microscopic analysis of living cells (see Figures 1,3,7,8,and 9). UL42 is transported to the nucleus through a mechanism that is inhibited by RanQ69L, similar to its HCMV (see above) and HHV-7 homologues (19).…”

Nuclear Import of HSV-1 DNA Polymerase Processivity Factor UL42 Is Mediated by a C-Terminally Located Bipartite Nuclear Localization Signal

“…This finding is consistent with the recent identification of functional NLSs at the C-terminus of PAPs from other herpesviruses (16)(17)(18)(19). Our data clearly show that UL42 binds to different IMPR isoforms and localizes exclusively to the cell nucleus when transiently expressed in the absence of other viral proteins, as detected by coimmunoprecipitation assays and fluorescent microscopic analysis of living cells (see Figures 1,3,7,8,and 9). UL42 is transported to the nucleus through a mechanism that is inhibited by RanQ69L, similar to its HCMV (see above) and HHV-7 homologues (19).…”

Mutations That Increase DNA Binding by the Processivity Factor of Herpes Simplex Virus Affect Virus Production and DNA Replication Fidelity

“…One possible explanation for decreased infectivity is an increase in the number of lethal mutations or mutations that otherwise decrease virion infectivity and spread resulting from the UL42 mutations. However, we have previously observed viruses that exhibit mutation frequencies similar to those observed here with greater defects in virus production and mutants with higher mutation frequencies than those observed here with similar decreases in virus production (22). Another possibility is that the mutations affect some function unrelated to DNA synthesis and fidelity.…”

“…Mutations increasing binding of UL42 to DNA. Previous studies demonstrated that the substitution Q282R results in binding of UL42 to DNA with increased binding affinity without an effect on the interaction with Pol peptide (22). Here we found that a UL42 mutant with fewer negatively charged residues due to the D270A/D271A mutations also showed increased DNA binding to the same extent observed for the Q282R mutant.…”

Viral DNA polymerase structures reveal mechanisms of antiviral drug resistance

“…UL42 interactions with DNA are essentially identical in the closed, open, and editing conformations, and UL42 moves as a rigid module with Pol in concert during its conformational transitions. As predicted from the original UL42 structure and supported by mutational analysis and cross-linking data 5 , 12 , 45 , a cluster of UL42 basic residues bind the backbone of newly synthesized DNA ( Figure 6B ). These contacts include several arginines that are conserved among α-herpesviruses for which substitutions have been shown to reduce UL42 binding to DNA, processive DNA synthesis, replication fidelity, and viral replication (e.g., R113 UL42 , R279 UL42 , and R280 UL42 ) ( Figure 6B ) 12 , 45 , 46 .…”

“…As predicted from the original UL42 structure and supported by mutational analysis and cross-linking data 5 , 12 , 45 , a cluster of UL42 basic residues bind the backbone of newly synthesized DNA ( Figure 6B ). These contacts include several arginines that are conserved among α-herpesviruses for which substitutions have been shown to reduce UL42 binding to DNA, processive DNA synthesis, replication fidelity, and viral replication (e.g., R113 UL42 , R279 UL42 , and R280 UL42 ) ( Figure 6B ) 12 , 45 , 46 . Comparison of the trajectories of DNA bound to the HSV polymerase holoenzyme with the human DNA polymerase δ/PCNA complex 14 reveals marked differences in the angle with which DNA traverses the back face of the respective processivity factors ( Figures 6C and 6D ).…”

Nuclear Import of HSV-1 DNA Polymerase Processivity Factor UL42 Is Mediated by a C-Terminally Located Bipartite Nuclear Localization Signal

“…This finding is consistent with the recent identification of functional NLSs at the C-terminus of PAPs from other herpesviruses (16)(17)(18)(19). Our data clearly show that UL42 binds to different IMPR isoforms and localizes exclusively to the cell nucleus when transiently expressed in the absence of other viral proteins, as detected by coimmunoprecipitation assays and fluorescent microscopic analysis of living cells (see Figures 1,3,7,8,and 9). UL42 is transported to the nucleus through a mechanism that is inhibited by RanQ69L, similar to its HCMV (see above) and HHV-7 homologues (19).…”

Mutations That Increase DNA Binding by the Processivity Factor of Herpes Simplex Virus Affect Virus Production and DNA Replication Fidelity

“…One possible explanation for decreased infectivity is an increase in the number of lethal mutations or mutations that otherwise decrease virion infectivity and spread resulting from the UL42 mutations. However, we have previously observed viruses that exhibit mutation frequencies similar to those observed here with greater defects in virus production and mutants with higher mutation frequencies than those observed here with similar decreases in virus production (22). Another possibility is that the mutations affect some function unrelated to DNA synthesis and fidelity.…”

“…Mutations increasing binding of UL42 to DNA. Previous studies demonstrated that the substitution Q282R results in binding of UL42 to DNA with increased binding affinity without an effect on the interaction with Pol peptide (22). Here we found that a UL42 mutant with fewer negatively charged residues due to the D270A/D271A mutations also showed increased DNA binding to the same extent observed for the Q282R mutant.…”

Viral DNA polymerase structures reveal mechanisms of antiviral drug resistance

“…UL42 interactions with DNA are essentially identical in the closed, open, and editing conformations, and UL42 moves as a rigid module with Pol in concert during its conformational transitions. As predicted from the original UL42 structure and supported by mutational analysis and cross-linking data 5 , 12 , 45 , a cluster of UL42 basic residues bind the backbone of newly synthesized DNA ( Figure 6B ). These contacts include several arginines that are conserved among α-herpesviruses for which substitutions have been shown to reduce UL42 binding to DNA, processive DNA synthesis, replication fidelity, and viral replication (e.g., R113 UL42 , R279 UL42 , and R280 UL42 ) ( Figure 6B ) 12 , 45 , 46 .…”

“…As predicted from the original UL42 structure and supported by mutational analysis and cross-linking data 5 , 12 , 45 , a cluster of UL42 basic residues bind the backbone of newly synthesized DNA ( Figure 6B ). These contacts include several arginines that are conserved among α-herpesviruses for which substitutions have been shown to reduce UL42 binding to DNA, processive DNA synthesis, replication fidelity, and viral replication (e.g., R113 UL42 , R279 UL42 , and R280 UL42 ) ( Figure 6B ) 12 , 45 , 46 . Comparison of the trajectories of DNA bound to the HSV polymerase holoenzyme with the human DNA polymerase δ/PCNA complex 14 reveals marked differences in the angle with which DNA traverses the back face of the respective processivity factors ( Figures 6C and 6D ).…”

Nuclear Import of HSV-1 DNA Polymerase Processivity Factor UL42 Is Mediated by a C-Terminally Located Bipartite Nuclear Localization Signal

“…This finding is consistent with the recent identification of functional NLSs at the C-terminus of PAPs from other herpesviruses (16)(17)(18)(19). Our data clearly show that UL42 binds to different IMPR isoforms and localizes exclusively to the cell nucleus when transiently expressed in the absence of other viral proteins, as detected by coimmunoprecipitation assays and fluorescent microscopic analysis of living cells (see Figures 1,3,7,8,and 9). UL42 is transported to the nucleus through a mechanism that is inhibited by RanQ69L, similar to its HCMV (see above) and HHV-7 homologues (19).…”

Mutations That Increase DNA Binding by the Processivity Factor of Herpes Simplex Virus Affect Virus Production and DNA Replication Fidelity

“…One possible explanation for decreased infectivity is an increase in the number of lethal mutations or mutations that otherwise decrease virion infectivity and spread resulting from the UL42 mutations. However, we have previously observed viruses that exhibit mutation frequencies similar to those observed here with greater defects in virus production and mutants with higher mutation frequencies than those observed here with similar decreases in virus production (22). Another possibility is that the mutations affect some function unrelated to DNA synthesis and fidelity.…”

“…Mutations increasing binding of UL42 to DNA. Previous studies demonstrated that the substitution Q282R results in binding of UL42 to DNA with increased binding affinity without an effect on the interaction with Pol peptide (22). Here we found that a UL42 mutant with fewer negatively charged residues due to the D270A/D271A mutations also showed increased DNA binding to the same extent observed for the Q282R mutant.…”

Viral DNA polymerase structures reveal mechanisms of antiviral drug resistance

“…UL42 interactions with DNA are essentially identical in the closed, open, and editing conformations, and UL42 moves as a rigid module with Pol in concert during its conformational transitions. As predicted from the original UL42 structure and supported by mutational analysis and cross-linking data 5 , 12 , 45 , a cluster of UL42 basic residues bind the backbone of newly synthesized DNA ( Figure 6B ). These contacts include several arginines that are conserved among α-herpesviruses for which substitutions have been shown to reduce UL42 binding to DNA, processive DNA synthesis, replication fidelity, and viral replication (e.g., R113 UL42 , R279 UL42 , and R280 UL42 ) ( Figure 6B ) 12 , 45 , 46 .…”

“…As predicted from the original UL42 structure and supported by mutational analysis and cross-linking data 5 , 12 , 45 , a cluster of UL42 basic residues bind the backbone of newly synthesized DNA ( Figure 6B ). These contacts include several arginines that are conserved among α-herpesviruses for which substitutions have been shown to reduce UL42 binding to DNA, processive DNA synthesis, replication fidelity, and viral replication (e.g., R113 UL42 , R279 UL42 , and R280 UL42 ) ( Figure 6B ) 12 , 45 , 46 . Comparison of the trajectories of DNA bound to the HSV polymerase holoenzyme with the human DNA polymerase δ/PCNA complex 14 reveals marked differences in the angle with which DNA traverses the back face of the respective processivity factors ( Figures 6C and 6D ).…”