The Modulation of Phosphatase Expression Impacts the Proliferation Efficiency of HSV-1 in Infected Astrocytes

<div><p>Herpes Simplex Virus 1 (HSV-1) is a major pathogen that causes human neurological diseases, including herpes simplex encephalitis (HSE). Previous studies have shown that astrocytes are involved in HSV-1 systemic pathogenesis in the central nervous system (CNS), although the mechanism remains unclear. In this study, a high-throughput RNAi library screening method was used to analyze the effect of host phosphatase gene regulation on HSV-1 replication using <i>Macaca mulatta</i> primary astrocytes in an <i>in vitro</i> culture system. The results showed that the downregulation of five phosphatase genes (PNKP, SNAP23, PTPRU, LOC714621 and PPM1M) significantly inhibited HSV-1 infection, suggesting that these phosphatases were needed in HSV-1 replication in rhesus astrocytes. Although statistically significant, the effect of downregulation of these phosphatases on HSV-1 replication in a human astrocytoma cell line appears to be more limited. Our results suggest that the phosphatase genes in astrocytes may regulate the immunological and pathological reactions caused by HSV-1 CNS infection through the regulation of HSV-1 replication or of multiple signal transduction pathways.</p></div

Replication of HSV-1 in <i>Macaca mulatta</i> astrocytes.

<p>(a) HSV-1 infection of primary astrocytes. Cellular morphology of astrocyte, U-87MG and HEK293 before and after HSV-1 infection under the light microscope; Upper panels: negative control; Lower panels: 12 hours post HSV-1 infection (MOI = 1). The magnification is 200 X. (b) Virus morphology in astrocytes infected with HSV-1. The monolayer of astrocytes was infected with HSV-1 (MOI  = 2). The infected astrocytes were observed at 24 h post infection using Transmission Electron Microscope (TEM), and the typical structure was observed for HSV-1 virus capsids. The magnification is 6000 X. (c) Viral replication curve in rhesus primary astrocytes, U-87MG and HEK293 cells infected with HSV-1. The primary astrocytes, U-87MG and HEK293 cells were infected with HSV-1 (MOI  = 0.01). Samples were collected at 0, 12, 24, 36, 48, 60, 72, 84 and 96 hr post infection. A plaque assay was used to determine the virus titer in all of the samples to generate the viral replication curve. Error bars represent the standard deviation from triplicate samples.</p

Identification of Phosphatase Genes Associated with HSV-1 Replication.

<p>Identification of Phosphatase Genes Associated with HSV-1 Replication.</p

PNKP, SNAP23, PTPRU, LOC714621 and PPM1M genes in astrocytes were identified as key genes affecting HSV-1 replication.

<p>Three siRNAs per gene (PNKP, SNAP23, PTPRU, LOC714621 and PPM1M) were transfected in astrocytes (a), U-87MG (b) and HEK293 (c) followed by infection with HSV-1 (at an MOI of 0.01) in three independent experiments. Virus solutions were harvested at 48 h post infection, and the virus titer was determined by the CPE method. * indicates P<0.05.</p

siRNA transfection effectively downregulated corresponding phosphatase RNA expression in astrocytes.

<p>(a) Determination of the silencing effect of six genes: APP6, ALPL, DUSP11, PPP1CC, PTPN11 and PNKP. The siRNAs of the six genes (ACP6, ALPL, DUSP11, PPP1CC, PTPN11 and PNKP), were used to transfect astrocytes (50 nM). Total RNA was extracted at 48 h post transfection; a relative quantification method was used to calculate the target gene transcription. Data were analyzed using 2^−ΔΔCt. β-actin was used as the internal control. Error bars represent the standard deviation from triplicate samples. * indicates P<0.05. (b,c) Relationship between transfection time and the silencing effect of the ALPL and PNKP genes. The siRNAs of the ALPL and PNKP genes were used to transfect astrocytes (50 nM), and the total RNA was extracted at 24, 48 and 72 hr post transfection; a relative quantification method was used to calculate the target gene transcription. Data were analyzed using 2^−ΔΔCt. β-actin was used as the internal control. Error bars represent the standard deviation from triplicate samples. * indicates P<0.05.</p

siRNA knockdown of different phosphatases led to different responses to HSV-1 infections.

<p>Three siRNAs per gene were transfected in astrocytes followed by infection with HSV-1 (at an MOI of 0.01) in three independent experiments. Virus solutions were harvested at 48 h post infection, and the virus titer was determined by the CPE method and analyzed by calculating the normalized fold of CCID50/mL. The color scale reflects the fold of reduction or increase level of viral replication. These values (0.003-80) represent ratios of CCID50/mL (target/NC). NC refers to the negative control (value considered as 1).</p