Chromatin assembly on herpes simplex virus 1 DNA early during a lytic infection is Asf1a dependent

Abstract

Herpes simplex virus (HSV) is a large DNA virus which is characterized by its ability to form latent infections in neurons of the peripheral nervous system. Although histones are found in the capsids of small DNA viruses (papovaviruses), none are found in the capsids of large HSV. However, after entry into the infected cell nucleus, the HSV genome begins to associate with nucleosomes during the earliest stages of infection. In contrast, late during infection, newly replicated viral DNA does not appear to associate with nucleosomes, suggesting that histones are deposited specifically on input viral DNA. The mechanisms of deposition and removing histones from the viral genome are unclear. Recently, histone chaperones, involved in the assembly and disassembly of nucleosomes, have been identified. Human antisilencing factor 1 (Asf1) is one such factor which is involved in both the assembly and disassembly of nucleosomes in cellular systems. In this study, we have examined the effect of small interfering RNA (siRNA) knockdown of Asf1a on HSV infections in HeLa cells. Both viral replication and growth were found to be decreased. Also, viral DNA was significantly less protected from micrococcal nuclease (MNase) digestion up to 6 h postinfection (hpi). However, transcription of the immediate early (IE) genes ICP0 and ICP4 was significantly upregulated at 3 h postinfection. Also, these genes were found to be less protected from MNase digestion and, therefore, less associated with nucleosomes. These results suggest that Asf1a plays a role in regulating IE genes by assembling chromatin onto histone-free viral DNA by 3 h postinfection.

Fig 1

Effect of Asf1a knockdown on HSV-1 growth. HeLa cells were transfected with Asf1a siRNA for 2 days, infected with HSV, and harvested at the indicated time p.i. (A) Asf1a siRNA knocks down Asf1a protein levels. Cells were lysed and immunoblotted with rabbit anti-Asf1a or β-tubulin or CAF-1 antibody. The reduction in the Asf1a protein is 93%. Interestingly, HSV infection induces Asf1a expression. (B) Asf1a siRNA specifically knocks down Asf1a mRNA, not Asf1b. Total mRNA was isolated and reverse transcribed. Asf1a and Asf1b mRNA levels (relative to a mock-infected sample) were determined at various times p.i. by qPCR and normalized to 18S rRNA levels. (C) Asf1a siRNA delayed viral DNA replication. Accumulated viral DNA was quantified by qPCR with TK promoter primers and normalized to GAPDH (glyceraldehyde-3-phosphate dehydrogenase) levels. The relative viral DNA amount was calculated based on a 3-hpi sample in negative (neg) siRNA-treated cells. (D) Asf1a siRNA delayed virus replication. Viral yield from Asf1a knockdown cells was significantly reduced at 8 hpi (18-fold), 10 hpi (7-fold), and 16 hpi (4-fold). Viral yields were determined by a plaque assay as described in Materials and Methods. All data are the averages of three independent experiments, and error bars represent the standard deviation. Student t test values are displayed with asterisks (*, P < 0.01; **, P < 0.001; ***, P < 0.0001).

Fig 2

The HSV genome is less protected from MNase digestion in Asf1a siRNA-treated cells at early times of infection. (A) Asf1a siRNA decreased the protection of viral DNA from MNase digestion. Nuclei were isolated and digested with MNase. DNA was purified, electrophoresed, transferred into membrane, and hybridized with ³²P-labeled nick-translated probes prepared from 5 cosmids covering the entire HSV-1 genome. These data represent one of three independent experiments. (B) The amount of viral genome protected from MNase digestion was significantly decreased even after correction for the delayed accumulation of viral DNA in the Asf1a siRNA-treated cells. The 150-bp viral DNA fragment was measured by a phosphor imager and normalized to the total viral DNA at each time course. The relative amount of protected viral DNA was calculated based on the 3-hpi sample in Asf1a siRNA-treated cells. (C) The 150-bp fragment in total viral DNA at 3 hpi was lower than at other times (1%). The signal from 150-bp fragments and total viral DNA from Fig. 2A Southern data were measured by a phosphor imager, and the value of the 150-bp fragments was divided by the value of the total viral DNA in the lane to give the percentage of HSV DNA in nucleosome-like particles. All data are the averages of three independent experiments, and error bars represent the standard deviation.

Fig 3

Asf1a knockdown increased IE viral transcription. HeLa cells were transfected with Asf1a siRNA for 2 days, infected, and harvested at the indicated time. Asf1a siRNA treatment dramatically increased mRNA levels of ICP0 (A) and ICP4 (B), while it did not affect mRNA levels of TK (C) and VP16 (D). Total mRNA was isolated and reverse transcribed. Relative mRNA levels for ICP0, ICP4, TK, and VP16 were determined by qPCR and normalized to 18S rRNA levels. The relative mRNA amount was calculated based on a 3-hpi sample in negative siRNA-treated cells. Three independent experiments were performed, and the error bars represent the standard deviation.

Fig 4

Asf1a knockdown only increased ICP0 protein expression. Total lysates from siRNA-treated and infected cells at various times postinfection were Western blotted and probed with ICP0, ICP4, and VP16 antibodies. β-Tubulin was used as a loading control.

Fig 5

Asf1a knockdown lowers the deposition of histone H3 on viral promoter regions at 3 hpi. The levels of histone H3 on promoter and transcribed regions of the ICP0 (A), ICP4 (B), TK (C), and VP16 (D) genes were investigated by ChIP. Values are calculated as percentages of input. Student t test values are displayed with asterisks (*, P < 0.01; **, P < 0.001; ***, P < 0.0001). All data are the averages of three independent experiments, and error bars represent the standard deviation.

Fig 6

Asf1a knockdown lowers the protection of the viral genome from MNase digestion at 3 hpi. The levels of histone H3 on promoter and transcribed regions of the ICP0 (A), ICP4 (B), TK (C), and VP16 (D) genes were examined by qPCR with 150-bp fragments purified after MNase digestion. Values are calculated as percentages of input. Student t test values are displayed with asterisks (*, P < 0.01; **, P < 0.001; ***, P < 0.0001). All data are the averages of three independent experiments, and error bars represent the standard deviation.