The nuclear localization signal-mediated nuclear targeting of herpes simplex virus 1 early protein UL2 is important for efficient viral production

Abstract

Herpes simplex virus 1 (HSV-1) is a representative alphaherpesvirus that can provoke a series of severe diseases to human being, but its exact pathogenesis is not perfectly understood. UL2, a uracil-DNA glycosylase involved in the process of HSV-1 DNA replication, has been shown to be predominantly targeted to the nuclei in our previous study, yet little is established regarding the subcellular localization signal or its related function of UL2 during HSV-1 propagation. Here, by creating a number of UL2 variants merged with enhanced yellow fluorescent protein, an authentic nuclear localization signal (NLS) of UL2 was, for the first time, identified and profiled to amino acids (aa) 1 to 17 (MKRACSRSPSPRRRPSS), and ¹²RRR¹⁴ was indispensable for its nuclear accumulation. Besides, the predicted nuclear export signal (aa 225 to 240) of UL2 was determined to be nonfunctional. Based on the HSV-1 bacterial artificial chromosome and homologous recombination technique, three recombinant viruses with mutations of the identified NLS, deletion and revertant of UL2 were constructed to assess the effect of UL2 nuclear targeting on HSV-1 replication. Compared to the wild type HSV-1, UL2 deletion remarkably restrained viral production, and mutation of NLS targeting UL2 to cytoplasm (pan-cellular distribution) in recombinant virus-infected cells showed a certain degree of deficiency in HSV-1 proliferation. Moreover, recombinant virus with UL2 deletion exhibited serious damages of viral DNA synthesis and mRNA expression, and these processes were partially disrupted in the recombinant virus with UL2 NLS mutation. Collectively, we had established a functional NLS in UL2 and showed that the NLS-mediated nuclear translocation of UL2 was important for efficient production of HSV-1. These data were of significance for further clarifying the biological function of UL2 during HSV-1 infection.

Keywords: HSV-1 UL2; nuclear localization signal; recombinant virus.

Figure 1

Preliminary determination of the NLS and NES regions of UL2. (A) Schematic diagram of full-length UL2(1-334), UL2(1-224) and UL2(225-334) fused with an EYFP monomer at its N terminus. (B) Subcellular localization of UL2(1-334)-EYFP, UL2(1-224)-EYFP, UL2(225-334)-EYFP and EYFP in live COS-7 cells. (C) Bioinformatics analysis of potential NLS and NES of UL2. Blue color labelled residues represent potential NLS1 and NLS2. Red color labelled residues represent potential NES. All scale bars indicate 10 μm.

Figure 2

The predicted NLS2 of UL2 is nonfunctional. (A) Schematic representation of WT UL2 and its deletion mutants UL2(1-75), UL2(69-224), UL2(61-224), UL2(69-75) and UL2(61-75) fused with EYFP. (B) Subcellular localization of these UL2 deletion mutants (shown in A) in plasmid-transfected live COS-7 cells. All scale bars indicate 10 μm.

Figure 3

Characterization of the functional NLS and its key aa of UL2. (A) Schematic representation of WT UL2 and its deletion mutants UL2(1-68), UL2(1-31), UL2(18-31), UL2(1-17), UL2(1-8), UL2(9-17) and UL2(12-14)mut fused with EYFP. (B–D) Subcellular localization of these UL2 deletion mutants (B and D, shown in A), EYFP-ECRF4, or UL2(NLS)-EYFP-ECRF4 (C) in plasmid-transfected live COS-7 cells. All scale bars indicate 10 μm.

Figure 4

Determination the presμmed NES function of UL2. (A) Schematic representation of WT UL2 and its deletion mutants UL2(225-277), UL2(278-334) and UL2(225-240) fused with EYFP. (B) Subcellular localization of these UL2 deletion mutants (shown in A) in plasmid-transfected live COS-7 cells. All scale bars indicate 10 μm.

Figure 5

BACs construction of UL2-related recombinant HSV-1. (A) PCR analysis of the constructed recombinant BACs. The UL2, UL31 and Kan^r genes were amplified from WT pBAC (lane 1), pBAC/UL2Mu (lane 2), pBAC/UL2Del (lane 3) and pBAC/UL2Rev (lane 4), respectively. (B) Gel electrophoresis (0.8%) of WT pBAC (lane 1) and recombinant BACs pBAC/UL2Mu (lane 2), pBAC/UL2Del (lane 3) and pBAC/UL2Rev (lane 4) analyzed by BamHI and HindIII restriction digestion, respectively. The red asterisk indicates the specific band that was disappeared only in pBAC/UL2Del genome when all the BACs were treated with BamHI digestion. Marker sizes in kb are indicated on the right side of the gels.

Figure 6

Protein expression and subcellular localization of UL2 in different recombinant viruses-infected cells. (A) Monolayer HEK293T cells were infected with different reconstitute virus vUL2, vUL2Del, vUL2Mu or vUL2Rev at an MOI of 1 for 24 h, and cells were harvested when CPE reached 90-95%. Cell lysates were then subjected to WB analysis using the prepared anti-UL2 pAb and AP-conjugated goat anti-rat IgG. (B) Vero cells infected with different reconstitute virus vUL2, vUL2Del, vUL2Mu or vUL2Rev at an MOI of 1 for 24 h, then cells were subjected to IFA using anti-UL2 pAb and Dylight 649 conjugated goat anti-rat IgG, to show the subcellular localization of UL2. Cells were finally counterstained with Hoechst to visualize the nuclei. GFP was also captured to show the cells were successfully infected by HSV-1. All scale bars indicate 10 μm.

Figure 7

Nuclear targeting of UL2 is important for efficient HSV-1 production. (A) Plaque analysis of WT HSV-1 (vUL2) and its derived recombinant viruses (vUL2Del, vUL2Mu and vUL2Rev) by live cells fluorescence microscope. Confluent Vero cells were infected with the indicated viruses at an MOI of 1. After adsorption at 37°C for 2 h, virus was washed away and the plate was covered with DMEM-2% FBS, then the fluorescences (GFP) derived from these viruses were analyze by fluorescence microscope after infection for 24 h. (B) Growth curve analysis of WT HSV-1 and its derived recombinant viruses. Vero cells were infected with the indicated viruses at an MOI of 1 for 6, 12, 24 and 36 h, then virus was harvested, and their titers were determined on the Vero monolayer by plaque method (with crystal violet staining). The data shown was the average results from three independent experiments. (C) Luciferase activity was used to determine the viral replication of WT HSV-1 and its derived recombinant viruses in HEK293T cells. HEK293T cells were infected with the indicated viruses at an MOI of 1 for 24 h, then luciferase activity was detected by harvesting the lysates of the virus-infected HEK293T cells. Data were expressed as means ± SD from three independent experiments. Statistical analysis was performed using student’s t test, and *** indicates P < 0.001. All scale bars indicate 30 μm.

Figure 8

Viral DNA replication and mRNA expression analysis of WT HSV-1 and its derived recombinant viruses. (A) DNA replication analysis of WT HSV-1 and its derived recombinant viruses. HEK293T cells were mock-infected or infected with WT HSV-1 (vUL2) and its derived recombinant viruses (vUL2Del, vUL2Mu and vUL2Rev) at an MOI of 1 for 24 h. Then, total cellular DNA was purified and PCR was performed with the primers specific for UL54 (IE gene), UL42 (E gene) and UL3 (L gene) to quantify DNA levels. To ensure that an equal amount of DNA was used from each sample, the DNA of each sample was normalized with GAPDH. (B) mRNA expression analysis of WT HSV-1 and its derived recombinant viruses. HEK293T cells were mock-infected or infected with WT HSV-1 (vUL2) and its derived recombinant viruses (vUL2Del, vUL2Mu and vUL2Rev) at an MOI of 1 for 24 h. Then, total RNA was isolated, and the mRNA expression levels of UL54, UL42, UL3 and GAPDH were assessed by RT-PCR. GAPDH was served as an internal control. Densitometry of UL54, UL42 and UL3 bands were normalized to the control GAPDH. Data were expressed as means ± SD from three independent experiments. Statistical analysis was performed using student’s t test, and * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001.