Herpes simplex virus-1 (HSV-1) is a double-stranded DNA virus that causes various high-mortality central nervous system (CNS) diseases, including herpes simplex virus encephalitis (HSE) and meningitis (Baringer J R, 2008; Kennedy P G, et al., 2002). HSV-1 has a complex gene structure, and its genetic replication is regulated by host cellular genes. HSV-1 can latently infect the CNS, thereby escaping attacks from the host immune system. It is important to understand the molecular biological processes involved in CNS infection in order to further understand the replication process and pathological mechanisms of HSV-1.
As one of the major cells involved in the immune functions of the CNS, astrocytes play an important role in the pathological processes of HSV-1-induced encephalitis. Astrocytes are involved in host antiviral responses via pattern recognition receptors (PRRs), such as Toll-like receptors (TLRs), and HSV-1 infection also affects pathological processes. However, the exact role of astrocytes in pathological infection remains unclear; in particular, little is known about molecular HSV-1 replication in astrocytes.
Our previous studies used siRNA library screening to preliminarily confirm that phosphatase affects HSV-1 replication in astrocytes and bifunctional polynucleotide phosphatase/kinase (PNKP) is a key gene that affects HSV-1 replication. PNKP is a dual-specificity nucleotide phosphatase(Zolner A E, et al., 2011)involved in base excision repair, single-and double-strand break repair, and nonhomologous end joining. (Weinfeld M, et al., 2011). However, the function of PNKP in HSV-1 replication remains unknown. To confirm the mechanisms of PNKP, this study investigates the effects of PNKP on HSV-1 replication. This study also confirms that PNKP inhibits the formation of the circular double-stranded HSV-1 genome, thus affecting viral replication in host cells.
HSV-1 replication in the CNS is a major event in HSE pathology; however, the mechanisms responsible for proliferative infection in neuronal cells remain unclear. Although our previous study found that PNKP might affect the proliferation of HSV-1 in astrocytes, its mechanism also remains unknown. In this study, we used Western blot analysis to examine the role of siRNA in the downregulation of PNKP (Fig. 1). Further analysis of cell proliferation confirmed that siRNA has no significant effect on the proliferation of astrocytes (Fig. 2). These results not only confirm the efficiency and safety of siRNA but also provide fundamental information for future studies on the effects of PNKP on HSV-1 proliferation.
Figure 1. RNAi-mediated knockdown of PNKP. When the cultured primary astrocytes formed a monolayer, the cells were transfected with the nonfunctional siRNA (NC-siRNA) or PNKP-specific siRNA (PNKP-siRNA) (50 nM). Seventy-two hours after transfection, cells were harvested and lysed to collect the total protein. Western blot analysis was performed to detect changes in the PNKP protein levels. β-actin was used as the experimental control.
Figure 2. Proliferation of astrocytes was not influenced by siRNA transfection. The negative control siRNA (NC-siRNA) and the PNKP-specific siRNA (PNKP-siRNA) were transfected into the monolayer of primary cultured Rhesus astrocytes. Cells were collected at different time points (0, 4, 8, 12, 24, 36, 48, 60, or 72 hours), and the WST-1 reagent was used to detect cell proliferation. Normal and non-siRNA-transfected cells were used as the experimental controls.
To further confirm the effects of PNKP on HSV-1 proliferation and observe the infection characteristics of HSV-1 in PNKP-downregulated astrocytes, this study used siRNA to downregulate PNKP. This was followed by HSV-1 infection (MOI = 0.01) in order to observe any changes in the viral growth curves. PNKP downregulation significantly inhibited viral proliferation (Fig. 3).
Figure 3. Changes in viral proliferation curves following PNKP downregulation. siRNA (50 nM) was transfected after the primary astrocytes formed a monolayer. Forty-eight hours after transfection, cells were infected with HSV-1 (MOI = 0.01), and the viral solution was collected at different time points (0, 12, 24, 36, 48, 60, 72, 84, or 96 hours). The plaque assay was used to determine the viral titers of each sample, and growth curves were plotted. Cells transfected with nonfunctional siRNA (NC-siRNA) were used as the experimental control.
The synthesis of viral proteins was inhibited when HSV-1 proliferation was affected. To determine the influence of viral DNA replication, viral gene transcription—ICP22 (immediate early), TK (early), and gB (late)—was investigated during different stages. PNKP downregulation significantly inhibited the genetic transcription of these viral genes at different stages (Fig. 4).
Figure 4. Effect of PNKP downregulation on viral gene transcription following HSV-1 infection. siRNA (50 nmol/L) was transfected after the primary astrocytes formed a monolayer. Forty-eight hours after transfection, cells were infected with HSV-1 (MOI = 8) and the viral solution was collected at different time points (2, 4, 6, 8, 12, 16, or 20 hours). Changes in viral gene transcription during different stages are shown. The results at each time point were used to perform 2-ΔΔCt relative quantitation analysis (β-actin was used as the reference gene; n = 4). The negative control group consisted of cells that were transfected with nonfunctional siRNA (NC-siRNA), and the experimental groups consisted of cells transfected with PNKP-siRNA. *p < 0.05.
PNKP downregulation reduced HSV-1 genetic transcription in different stages. We believe that this inhibition occurs after the virus enters the cell and is the initial stage of HSV-1 genome replication (Lehman I R, et al., 1999). In accordance with viral proliferation curve analyses (Fig. 3), we hypothesize that PNKP affects viral genome replication. Here, we used quantitative polymerase chain reaction (qPCR) to detect any changes in the copy number of the viral genome during approximately 1 replication cycle.
Astrocytes were transfected with specific siRNAs that downregulated PNKP, then infected with HSV-1 (MOI = 1) 48 hours later. Within 1 replication cycle, changes in the HSV-1 copy number were detected using qPCR and ICP0, TK, and GC were assessed. When PNKP was not inhibited, the viral copy number peaked 8 hours after infection in comparison with the control group (Fig. 5). When PNKP was downregulated, the viral copy number did not increase and was always at a relatively low level. These results indicate that PNKP downregulation affects the replication of the viral genome in host cells; thus, the viral copy number remained relatively low.
Figure 5. Effect of PNKP downregulation on viral genome copy number after HSV-1 infection. siRNA (50 nM) was transfected after the primary astrocytes formed a monolayer. Forty-eight hours after transfection, the cells were infected with HSV-1 (MOI = 8), and the viral solution was collected at different time points (1, 2, 3, 4, 5, 6, 8, 10, 12, or 14 hours). Changes in the viral copy number at different time points are shown (the copy numbers of the ICP0, TK, and GC genes are used to represent the viral copy numbers). The results at 1 hour and each time point were used to perform 2-ΔΔCt relative quantitation analysis (β-actin was used as the reference gene; n = 4). The negative control group consisted of cells that were transfected with nonfunctional siRNA (NC-siRNA), and the experimental groups consisted of cells that were transfected with PNKP-siRNA.
Several models describe HSV-1 replication. The classic model is the θ model: following infection, viral genome cyclization immediately starts (the double-stranded linear genome links head-to-end to form the circular double-stranded genome). θ replication then occurs, and the concatemer structure of the progeny DNA is synthesized by rolling circle replication (Muylaert I, et al., 2011). Using the results of string analysis (http://string-db.org/), we predicted five functional partners (Table 1): LOC712291 (XRCC4), XRCC1, LIG4, C9orf114 and LOC716387. XRCC4, XRCC1 and LIG4 may have relationship with DNA repair according to the descriptions. Interestingly, PNKP, XRCC4, and LigaseIV repair the ends of double-stranded DNA (Weinfeld M, et al., 2011). XRCC4 and LigaseIV are associated with the circular replication of the HSV-1 genome (Muylaert I, et al., 2007). Based on these results, we hypothesize that PNKP plays an important role in the circular replication of the HSV-1 genome. We designed primers that target the two ends of the HSV-1 genome and specifically amplified the junction fragment after genome cyclization as a representation of the copy number of the circular viral genome. We also used the copy number of the TK gene to represent the copy number of the total viral genome. We transfected siRNA into astrocytes to downregulate PNKP and used RT-PCR to detect differences in the ratios of the circular genomes during the early stage of HSV-1 infection.
Name Description Score LOC712291 Similar to X-ray repair cross complementing protein 4 isoform 1 0.894 XRCC1 X-ray repair complementing defective repair in Chinese hamster cells 1 0.815 LIG4 Ligase IV, DNA, ATP-dependent 0.660 C9orf114 Uncharacterized protein C9orf114 0.591 LOC716387 Synovial sarcoma translocation gene on chromosome 18-like 2 0.591
Table 1. Predicted functional partners
Compared with the copy number of the control group, the circular HSV-1 genome demonstrated a significant decrease 1 hour after HSV-1 infection (Fig. 6). This result confirms that PNKP plays an important role in host genome cyclization following HSV-1 infection. PNKP downregulation could affect viral genome cyclization and replication, possibly indicating genome cyclization during virus replication.
Figure 6. Effect of PNKP on HSV-1 genome cyclization. The copy number of the circular fragment of the experimental group relative to the control group and the copy number of the TK gene were calculated using 2-ΔΔCt relative quantitation analysis (β-actin was used as the reference genes; n = 3). The histogram at each time point shows the ratio of the copy number of the circular fragment to the copy number of the TK gene, which represents the ratio of the copy number of the circular virus genome to the total copy number of the genome. *p < 0.05.