Molecular Cloning of Alternative Splicing Variants of the Porcine PML Gene and Its Expression Patterns During Japanese Encephalitis Virus Infection

Abstract

Promyelocytic leukemia (PML) protein is a crucial component of PML-nuclear bodies (PML-NBs). PML and PML-NBs are involved in the regulation of various cellular functions, including the antiviral immune response. The human PML gene can generate several different isoforms through alternative splicing. However, little is known about the porcine PML alternative splicing isoforms and their expression profiles during Japanese encephalitis virus (JEV) infection. In the present study, we cloned seven mature transcripts of porcine PML, all of which contained the same N-terminal sequence but differed in the C-terminal sequences due to alternative splicing. These seven transcripts encoded five proteins all of which had the RBCC motif and sumoylation sites. Amino acid sequence homology analysis showed that porcine PML-1 had relatively high levels of identity with human, cattle, and goat homologs (76.21, 77.17, and 77.05%, respectively), and low identity with the mouse homolog (61.78%). Immunofluorescence analysis showed that the typical PML-NBs could be observed after overexpression of the five PML isoforms in PK15 cells. Quantitative reverse transcription PCR (RT-qPCR) analysis showed significant upregulation of PML isoforms and PML-NB-associated genes (Daxx and SP100) at 36 and 48 h post-infection (hpi). Western blotting analysis indicated that the PML isoforms were upregulated during the late stage of infection. Moreover, the number of PML-NBs was increased after JEV infection. These results suggest that porcine PML isoforms may play essential roles in JEV infection.

Japanese encephalitis virus perturbs PML-nuclear bodies by engaging in interactions with distinct porcine PML isoforms

“…In the infected group, viral antigen-negative cells exhibited a significantly higher quantity of PML-NBs compared to the cells in the uninfected group. This is consistent with our previous findings and the results observed in DENVinfected cells (Giovannoni et al, 2015;Zhu et al, 2021). A plausible explanation for this might be that the IFN secreted by JEV-infected cells triggers an increase in the number of PML-NBs in the adjacent cells (Giovannoni et al, 2015).…”

“…In our previous work, we identified seven alternative splicing variants of porcine PML. While all these variants share the same Nterminal sequence, they exhibit differences in their C-terminal sequences (Zhu et al, 2021). Each PML isoform has a unique Cterminal sequence, which results in distinct roles for each PML isoform within the cell (Nisole et al, 2013).…”

“…These amplified sequences were then ligated to the pmCherry-N1 vector (Clontech, Mountain View, CA, USA) to generate the Cherry-C, prM, E, NS1, NS2A, NS2B, NS3, NS4A, NS4B, NS5 expression vectors. Additionally, EGFP-PML1, PML2, PML3, PML4, and PML5 vectors were provided by our laboratory (Zhu et al, 2021). The pcDNA3.1 vector (Invitrogen, Carlsbad, CA, USA) expression plasmids for the PML isoforms were constructed using recombinant pEGFP-C1 plasmids.…”

“…We previously identified five alternative splice forms of the porcine PML gene, which we named as PML1, PML2, PML3, PML4, and PML5 (Zhu et al, 2021). To identify which of the five porcine PML isoforms could potentially recruit JEV NS2B, NS3, NS4A, NS4B, and NS5 proteins into PML-NBs.…”

“…We previously identified seven alternative splicing variants of porcine PML that encode five proteins and examined the expression of these five PML isoforms during JEV infection (Zhu et al, 2021). In this study, we investigated how JEV counteracts the structure of PML-NBs via its expressed proteins.…”