Research Paper Volume 12, Issue 3 pp 2921—2938

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

Meili Li 1, *, , Xingmei Zou 1, *, , Yuanfang Wang 1, *, , Zuo Xu 1, , Xiaowen Ou 1, , Yiwen Li 1, , Delong Liu 1, , Yingjie Guo 1, , Yangxi Deng 1, , Si Jiang 1, , Tong Li 1, , Shaoxuan Shi 1, , Yilong Bao 1, , Tao Peng 2, 3, , Mingsheng Cai 1, ,

  • 1 Guangdong Provincial Key Laboratory of Allergy and Clinical Immunology, Second Affiliated Hospital of Guangzhou Medical University, The Sixth Affiliated Hospital of Guangzhou Medical University, Qingyuan People’s Hospital, Sino-French Hoffmann Institute, School of Basic Medical Science, Guangzhou Medical University, Guangzhou 510260, Guangdong, China
  • 2 State Key Laboratory of Respiratory Diseases, Sino-French Hoffmann Institute, School of Basic Medical Science, Guangzhou Medical University, Panyu, Guangzhou 511436, Guangdong, China
  • 3 South China Vaccine Corporation Limited, Guangzhou Science Park, Guangzhou 510663, Guangdong, China
* Equal contribution and co-first authors

received: September 27, 2019 ; accepted: January 19, 2020 ; published: February 7, 2020 ;

https://doi.org/10.18632/aging.102786
How to Cite

Copyright © 2020 Li et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY 3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

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.

Abbreviations

Aa: Amino acids; AP: Alkaline phosphatase; BAC: Bacterial artificial chromosome; BCIP: 5-bromo-4-chloro-3-indolylphosphate; DMEM: Dulbecco’s modified MEM; EBV: Epstein-Barr virus; EYFP: Enhanced yellow fluorescent protein; FBS: Fetal bovine serum; HCMV: Human cytomegalovirus; HEK: Human embryonic kidney; HSV-1: Herpes simplex virus 1; IFA: Indirect immunofluorescence analysis; MOI: Multiplicity of infection; NBT: Nitroblue tetrazolium; NES: Nuclear export signal; NLS: Nuclear localization signal; pAb: Polyclonal antibody; PRV: Pseudorabies virus; RT-PCR: Reverse transcription PCR; SD: Standard deviations; SDS: Sodium dodecyl sulfate; UDG: Uracil-DNA glycosylase; WB: Western blot; WT: Wild type.