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.
Highlights
Herpes simplex virus 1 (HSV-1), a epidemic human pathogen with a high ratio of infection in the population, can cause a number of diseases that is extremely adverse to public health
To figure out the nuclear localization signal (NLS) region of UL2, amino acids 1 to 334 of full-length UL2 was firstly cut into two sections aa 1 to 224 and aa 225 to 334, since the former section is rich in basic aa, whereas the latter is rich in hydrophobic aa
These two segments were fused to the N-terminus of enhanced yellow fluorescent protein (EYFP) (Figure 1A), and the related constructs were transfected into COS-7 cells for the detection of their subcellular localizations
Summary
Herpes simplex virus 1 (HSV-1), a epidemic human pathogen with a high ratio of infection in the population, can cause a number of diseases that is extremely adverse to public health. It’s shown that one of the homologues of HSV-1 UL2, human cytomegalovirus (HCMV) UL114 protein, is required for viral DNA replication [8], which functions in cooperation with UL44 (HCMV processivity factor) and UL54 (HCMV DNA polymerase) [9, 10]. Our previous study demonstrated that in live cells, HSV-1 UL2 is almost absolutely targeted to the nucleus without the presence of other viral components [14, 15], yet little is established about its functional localization motif(s). This is unquestionably of interest and impel us to investigate its subcellular localization signals, as well as their functions in the course of HSV1 infection
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