Direct evidence that HSV DNA damaged by ultraviolet (UV) irradiation can be repaired in a cell type-dependent manner

Abstract

Infection of permissive cells, in tissue culture, with herpes simplex virus (HSV) has been reported to induce host DNA damage repair responses that are necessary for efficient viral replication. However, direct repair of the damaged viral DNA has not, to our knowledge, been shown. In this report, we detect and determine the amount of damaged HSV-1 DNA, following introduction of experimentally damaged HSV genomes into tissue cultures of permissive Vero, NGF differentiated PC12 cells and primary rat neurons, using a method of detection introduced here. The results show that HSV-1 strain 17 DNA containing UV-induced DNA damage is efficiently repaired, in Vero, but not NGF differentiated PC12 cells. The primary rat neuronal cultures were capable of repairing the damaged viral DNA, but with much less efficiency than did the permissive Vero cells. Moreover, by conducting the experiments with either an inhibitor of the HSV polymerase (phosphonoacetic acid [PAA]) or with a replication defective DNA polymerase mutant virus, HP66, the results suggest that repair can occur in the absence of a functional viral polymerase, although polymerase function seems to enhance the efficiency of the repair, in a replication independent manner. The possible significance of varying cell type mediated repair of viral DNA to viral pathogenesis is discussed.

Figure 1

PCR-based DNA damage assay was performed by determining the sensitivity of a DNA preparation to long amplicon PCR. DNA to be tested was quantified by quantitative real time PCR using an HSV-1 TK standard sized small amplicon primer pair whose product is approximately 70 bp in length. The amount of DNA damage in these studies was not so extensive as to impair the PCR reaction with these small amplicons. Based on the qPCR, samples were diluted to equal HSV DNA concentration and used in subsequent endpoint PCR reactions in which a second small (~70 bp) and a long amplicon (2 kb) PCR were used. The small amplicon endpoint PCR was used as a verification of the qPCR and should remain equal in all samples. The 2 kb amplicon spanned the two small amplicon sites. PCR reactions containing serial dilutions of an HSV-1 DNA standard were run with the experimental samples to ensure that differences in PCR product reflected changes in the amount of template. Endpoint PCR reactions were run on 2% agarose gels to monitor changes in the fractional PCR efficiencies of long and short amplicons.

Figure 2

Analysis of ultraviolet irradiation induced DNA damage using the T4EndoV digests and a PCR processivity assay. (A) Purifed HSV-1 DNA was irradiated as described in the materials and methods for 0, 1, 10 and 100 sec. 10 pg of each virion DNA sample was subjected to PCR using TK primer pair TK1.70 (70 bp amplicon) and TK1.2500 (2.5 kb amplicon). “nd” represents “not determined”. (B) Irradiated DNA samples (100 ng) were digested with T4EndoV nuclease and subsequently run on a denaturing alkaline agarose gel.

Figure 3

UV damaged HSV-1 infections in Vero cells show conversion of damaged DNA to undamaged DNA by 24 hr post infection. (A) Vero cells were infected with unirradiated or irradiated HSV-1 strain 17+ (MOI = 5) for the indicated time points after which DNA was prepared and quantified for HSV-1 DNA content by SYBR green real time PCR using standard primers (TK1.70). Data from 3 experiments were averaged to show standard deviation at the 0, 2 and 24 hour time points. (B) DNA was diluted as necessary to equalize HSV-1 amounts and a long range (2 kb amplicon) and short range (70 bp amplicon) end point PCR was performed using TK1.2000 primer and TK2.70 primers, respectively. PCR products were separated on 2% agarose gels and stained with ethidium bromide. PCR results for 24 hour infections containing phosphono acetic acid (PAA) are labeled 24+PAA.

Figure 4

Repair of UV damaged virus DNA can occur in the absence of viral polymerase. (A) Vero cells were infected with unirradiated or irradiated HSV-1 strain HP66 (MOI = 5) for the indicated time points after which total DNA was prepared and quantified for HSV-1 DNA content by SYBR green real time PCR using standard primers (TK1.70). Error bars represent the standard deviation of 4 data points derived from two independent experiments (B) DNA was diluted as necessary to equalize HSV-1 DNA amounts and a long range (2 kb amplicon) and short range (70 bp amplicon) end point PCR was performed using TK1.2000 primer and TK2.70 primers, respectively. PCR products were separated on 2% agarose gels and stained with ethidium bromide. (C) Total DNA preparations at each time point were quantified by A260 spectrophotometry and equal amounts (2 µg) were spotted onto nitrocellulose membranes in triplicate. Membranes were probed for cyclobutane pyrimidine dimers (CPDs) by antibodies directed against CPDs. (D) CPD dot blots were quantified. Data points represent the average of three spots with standard deviation.

Figure 5

HSV-1 infection of NGF differentiated PC12 cells shows no evidence of repair of UV damaged HSV. (A) NGF differentiated PC12 cells were infected with unirradiated (UV-0), 10 sec (UV-10) irradiated, or 100 sec (UV-100) irradiated HSV-1 strain 17+ (MOI = 20) for the indicated time points after which DNA was prepared and quantified for HSV-1 DNA content by SYBR green real time PCR using standard primers (TK1.70) Error bars represent the standard deviation of three independent experiments. (B) DNA was diluted as necessary to equalize HSV-1 amounts and a long range (2 kb amplicon) and short range (70 bp amplicon) end point PCR was performed using TK1.2000 primer and TK2.70 primers, respectively. PCR products were separated on 2% agarose gels and stained with ethidium bromide.

Figure 6

HSV-1 infection of embryonic rat hippocampal neurons shows evidence of repair of UV damaged HSV-1. (A) Primary rat hippocampal neurons (4×10⁴ cells per infection) were infected with unirradiated (UV-0) or irradiated (UV-100) HSV-1 at an MOI of 20. Samples of culture supernatants were titered for infectious HSV-1 at the indicated times post infection. Data represents the average of three infected cultures with standard deviation. (B) At 0, 1 and 3 days post infection, the neuron cultures were collected and DNA was prepared and tested for HSV-1 TK DNA by SYBR green real time PCR using standard primers (TK1.70). The data shows viral TK DNA normalized to cellular GAPDH DNA. (C) DNA was diluted as necessary to equalize HSV-1 DNA amounts and a long range (2 kb amplicon) and short range (70 bp amplicon) end point PCR was performed using TK1.2000 primer and TK2.70 primers, respectively. PCR products were separated on 2% agarose gels and stained with ethidium bromide.