Meili Li

Characterization of the nuclear import and export signals of pseudorabies virus UL31

The pseudorabies virus (PRV) UL31 protein (pUL31) is a homologue of the herpes simplex virus 1 pUL31, which is a multifunctional protein that is important for HSV-1 infection. However, little is known concerning the subcellular localization signal of PRV UL31. Here, by transfection with a series of PRV UL31 deletion mutants fused to an enhanced yellow fluorescent protein (EYFP) gene, a bipartite nuclear localization signal (NLS) and a PY motif NLS of UL31 were identified and mapped to amino acids (aa) 4 to 20 (RRRLLRRKSSAARRKTL) and aa 21 to 34 (TRAARDRYAPYFAY), respectively. Additionally, the predicted nuclear export signal (NES) was shown to be nonfunctional. Taken together, this information opens up new avenues for investigating the biological functions of UL31 during PRV infection.

Characterization of Synonymous Codon Usage Bias in the Pseudorabies Virus US1 Gene

In the present study, we examined the codon usage bias between pseudorabies virus (PRV) US1 gene and the US1-like genes of 20 reference alphaherpesviruses. Comparative analysis showed noticeable disparities of the synonymous codon usage bias in the 21 alphaherpesviruses, indicated by codon adaptation index, effective number of codons (ENc) and GC3s value. The codon usage pattern of PRV US1 gene was phylogenetically conserved and similar to that of the US1-like genes of the genus Varicellovirus of alphaherpesvirus, with a strong bias towards the codons with C and G at the third codon position. Cluster analysis of codon usage pattern of PRV US1 gene with its reference alphaherpesviruses demonstrated that the codon usage bias of US1-like genes of 21 alphaherpesviruses had a very close relation with their gene functions. ENc-plot revealed that the genetic heterogeneity in PRV US1 gene and the 20 reference alphaherpesviruses was constrained by G+C content, as well as the gene length. In addition, comparison of codon preferences in the US1 gene of PRV with those of E. coli, yeast and human revealed that there were 50 codons showing distinct usage differences between PRV and yeast, 49 between PRV and human, but 48 between PRV and E. coli. Although there were slightly fewer differences in codon usages between E.coli and PRV, the difference is unlikely to be statistically significant, and experimental studies are necessary to establish the most suitable expression system for PRV US1. In conclusion, these results may improve our understanding of the evolution, pathogenesis and functional studies of PRV, as well as contributing to the area of herpesvirus research or even studies with other viruses.

Characterization of the nuclear import signal of herpes simplex virus 1 UL31

The herpes simplex virus 1 (HSV-1) UL31 protein is a multifunctional nucleoprotein that is important for viral infection; however, little is known concerning its subcellular localization signal. Here, by transfection with a series of HSV-1 UL31 deletion mutants fused to enhanced yellow fluorescent protein (EYFP), a bipartite nuclear localization signal (NLS) was identified and mapped to amino acids (aa) 1 to 27 (MYDTDPHRRGSRPGPYHGKERRRSRSS). Additionally, fluorescence results showed that the predicted nuclear export signal (NES) might be nonfunctional, and the functional NES of UL31 might require a specific conformation. Taken together, these results would provide significant information for the study of the biological function of UL31 during HSV-1 infection.

Cloning, expression, purification, antiserum preparation and its characteristics of the truncated UL6 protein of herpes simplex virus 1

The herpes simplex virus 1 (HSV-1) portal protein UL6 is important for HSV-1 replication, however, its precise functions in the virus life cycle are poorly understood. As we known, a relatively important tool for disclosing these functions is the antiserum specifically detecting UL6 in the HSV-1-infected cell. To this end, a recombinant protein consisting of C-terminal 297–676 amino acids of UL6 fused to His-tag was expressed in E. coli and purified from inclusion body by the Ni2+-NTA affinity chromatography under denaturing conditions, which was then refolded and used for the preparation of antiserum in rabbit. As results, western blot and immunofluorescence assay showed that this antiserum could specifically detect the purified truncated UL6 as well as native UL6 in the HSV-1 infected cells, indicating that the prepared antiserum could serve as a valuable tool for further exploring the functions of UL6.

Probing the nuclear import signal and nuclear transport molecular determinants of PRV ICP22

BackgroundHerpes simplex virus 1 (HSV-1) ICP22 is a multifunctional protein and important for HSV-1 replication. Pseudorabies virus (PRV) ICP22 (P-ICP22) is a homologue of HSV-1 ICP22 and is reported to be able to selectively modify the transcription of different kinetic classes of PRV genes, however, the subcellular localization, localization signal and molecular determinants for its transport to execute this function is less well understood.ResultsIn this study, by utilizing live cells fluorescent microscopy, P-ICP22 fused to enhanced yellow fluorescent protein (EYFP) gene was transient expressed in live cells and shown to exhibit a predominantly nucleus localization in the absence of other viral proteins. By transfection of a series of P-ICP22 deletion mutants fused to EYFP, a bona fide nuclear localization signal (NLS) and its key amino acids (aa) of P-ICP22 was, for the first time, determined and mapped to aa 41–60 (PASTPTPPKRGRYVVEHPEY) and aa 49–50 (KR), respectively. Besides, the P-ICP22 was demonstrated to be targeted to the nucleus via Ran-, importin α1-, and α7-mediated pathway.ConclusionsOur findings reported herein disclose the NLS and molecular mechanism for nuclear transport of P-ICP22, these results will uncover new avenues for depicting the biological roles of P-ICP22 during PRV infection.

Characterization of the nuclear import mechanisms of HSV-1 UL31.

Abstract As an important protein, UL31 has been demonstrated to play multiple roles in herpes simplex virus 1 (HSV-1) replication. Previous studies showed that UL31 predominantly locates in the nucleus in chemical fixed cells and live cells, however, the determining mechanisms for its nuclear translocation is not clear. In the present study, by utilizing live cells fluorescent microscopy and co-immunoprecipitation assays, the nuclear import of UL31 was characterized to be dependent on Ran-, importin α1- and transportin-1-mediated pathway. Therefore, these results will promote the understanding of UL31-mediated biological functions in HSV-1 infection cycle.

Identification of molecular determinants for the nuclear import of pseudorabies virus UL31

Herpes simplex virus 1 (HSV-1) UL31 is a multifunctional protein and important for HSV-1 infection. Pseudorabies virus (PRV) UL31 is a late protein homologous to HSV-1 UL31. Previous studies showed that PRV UL31 is predominantly localized to nucleus, however, the molecular determinants for its nuclear import were unclear to date. Here, by utilizing live cells fluorescent microscopy, UL31 fused with enhanced yellow fluorescent protein was transiently expressed in live cells and confirmed to exclusively target to the nucleus in the absence of other viral proteins. Furthermore, the nuclear import of UL31 was found to be dependent on the Ran-, importin α1-, α3-, α5-, α7-, β1-and transportin-1-mediated pathway. Therefore, these results would open up new avenues for depicting the biological functions of UL31 during PRV infection.

Evaluation of the immune response in Shitou geese (Anser anser domesticus) following immunization with GPV-VP1 DNA-based and live attenuated vaccines

Background: Goose parvovirus (GPV) is a highly contagious and deadly disease for goslings and Muscovy ducklings. Objectives: To compare the differences in immune response of geese immunized with GPV-VP1 DNA-based and live attenuated vaccines. Animals and methods: Shitou geese were immunized once with either 20 μg pcDNA-GPV-VP1 DNA gene vaccine by gene gun bombardment via intramuscular injection, or 300 μg by im injection, or 300 μL live attenuated vaccine by im injection, whereas 300 μg pcDNA3.1 (+) im or 300 μL saline im were used as positive and negative controls, respectively. Each group comprised 28 animals. Peripheral blood samples were collected from 2–210 days after immunization and the proliferation of T lymphocytes, the number of CD4+ and CD8+ T cells and the level of IgG assessed. Statistical analysis was performed using a one-way analysis of variance with group multiple comparisons via Tukeys test. Results: The pcDNA-GPV-VP1 DNA and attenuated vaccine induced cellular and humoral responses, and there were no differences between the 20 and 300 μg group in the responses of proliferation of T lymphocyte and the CD8+ T-cell. However, as to CD4+ T-cell response and humoral immunity, the 20 μg group performed better than the 300 μg group, which induced better cellular and humoral immunity than live attenuated vaccine. Conclusions: This study showed that it is possible to induce both cellular and humoral response using DNA-based vaccines and that the pcDNA-GPV-VP1 DNA gene vaccine induced better cellular and humoral immunity than live attenuated vaccine.

Characterization of the subcellular localization of Epstein-Barr virus encoded proteins in live cells

Epstein-Barr virus (EBV) is the pathogenic factor of numerous human tumors, yet certain of its encoded proteins have not been studied. As a first step for functional identification, we presented the construction of a library of expression constructs for most of the EBV encoded proteins and an explicit subcellular localization map of 81 proteins encoded by EBV in mammalian cells. Viral open reading frames were fused with enhanced yellow fluorescent protein (EYFP) tag in eukaryotic expression plasmid then expressed in COS-7 live cells, and protein localizations were observed by fluorescence microscopy. As results, 34.57% (28 proteins) of all proteins showed pan-nuclear or subnuclear localization, 39.51% (32 proteins) exhibitted pan-cytoplasmic or subcytoplasmic localization, and 25.93% (21 proteins) were found in both the nucleus and cytoplasm. Interestingly, most envelope proteins presented pan-cytoplasmic or membranous localization, and most capsid proteins displayed enriched or complete localization in the nucleus, indicating that the subcellular localization of specific proteins are associated with their roles during viral replication. Taken together, the subcellular localization map of EBV proteins in live cells may lay the foundation for further illustrating the functions of EBV-encoded genes in human diseases especially in its relevant tumors.

Characterization of the subcellular localization and nuclear import molecular mechanisms of herpes simplex virus 1 UL2.

Abstract As a crucial protein, the herpes simplex virus 1 (HSV-1) UL2 protein has been shown to take part in various stages of viral infection, nonetheless, its exact subcellular localization and transport molecular determinants are not well known thus far. In the present study, by using live cells fluorescent microscopy assay, UL2 tagged with enhanced yellow fluorescent protein was transiently expressed in live cells and showed a completely nuclear accumulation without the presence of other HSV-1 proteins. Moreover, the nuclear transport of UL2 was characterized to be assisted by multiple transport pathways through Ran-, importin α1-, α5-, α7-, β1- and transportin-1 cellular transport receptors. Consequently, these results will improve understanding of UL2-mediated biological functions in HSV-1 infection cycles.