HSV-1 DNA polymerase 3'-5' exonuclease-deficient mutant D368A exhibits severely reduced viral DNA synthesis and polymerase expression

Abstract

Herpesviruses, including herpes simplex virus-1, encode and express a DNA polymerase that is required for replication of their dsDNA genomes. The catalytic subunit of this enzyme contains a 3'-5' exonuclease that is involved in proofreading during replication. Although certain mutations that severely impair exonuclease activity are not lethal to the virus, it was reported that virus containing the substitution of alanine for aspartate 368 (D368A), which ablates exonuclease activity, could not be recovered, raising the possibility that this activity is essential for viral replication. To investigate this issue, we produced virus containing this mutation (D368A Pol) using a complementing cell line. D368A Pol virus was unable to form plaques on non-complementing cells. Viral DNA synthesis and polymerase activity were severely inhibited in D368A-infected cells, as was expression of the enzyme, suggesting that effects on polymerase expression rather than on exonuclease activity per se largely explain the lethal phenotype of this mutation.

Keywords: 3’-5’ exonuclease; DNA polymerase; DNA replication; Herpes smplex virus-1.

Fig. 1.

D368A Pol virus replication is severely inhibited in non-complementing cells, and is also affected in complementing cells. (a) Assay for plaque formation of the denoted viruses titrated side-by-side on Vero and complementing PolB3 cells. The lower limit of the vertical axis denotes the limit of detection for plaque formation in this assay. (b) Twenty times magnification images of wells from plaque assays fixed at 72 h p.i. and stained with crystal violet. (c) Plaque diameters for each denoted virus infection on PolB3 cells were analysed using one-way analysis of variance (ANOVA) with Šídák correction for multiple comparisons. *, P-value <0.0001, ns, not significant. (d) Single-cycle replication kinetics for the denoted viruses collected from either Vero or PolB3 cells, titrated on both Vero and PolB3 cells. Vero cells (left column) or PolB3 cells (right column) were infected with the indicated viruses [multiplicity of infection (m.o.i.) of 10], the cells were washed at 1 h p.i. and supernatants were collected at time points ranging from 4 to 24 h p.i. The supernatants were then titrated on either PolB3 cells (top row) or Vero cells (bottom row), and the resulting titres of the supernatants are indicated on the y-axis of each graph.

Fig. 2.

D368A Pol virus is deficient in viral DNA synthesis and for polymerase activity. (a) qPCR analysis of viral genome copy numbers at 8 and 12 h p.i. compared to those present following adsorption [1 h p.i.; analysed using one-way analysis of variance (ANOVA) with Šídák correction for multiple comparisons]. *, P-value <0.002, ns, not significant. (b) ³²P dTTP incorporated into activated salmon sperm DNA in the presence of high ammonium sulfate concentrations compared to the WT incorporation after 20 min in the presence or absence of HSV-1 Pol inhibitor PAA. The data from three assays were combined and plotted as a percentage of incorporated radioactivity compared to WT Pol, as marked with the dotted line, with the standard deviations denoted by the error bars.

Fig. 3.

D368A Pol expression is substantially decreased in infected cells compared to WT. (a) Western blots of cell lysates collected at 8 h p.i. and detected using antibodies specific for the proteins listed on the left. Whereas the antibody for Pol detects protein expressed both by the virus and the PolB3 cells, the FLAG antibody only detects Pol expression from the virus and does not detect expression of the WT pol gene in PolB3 cells. (b) Lysates of WT-infected Vero cells or HP66-infected PolB3 cells were serially diluted as indicated at the top, and then probed with the antibodies against the proteins indicated on the left.