Seven Homologous Regions from a Novel Bombyx mori Nucleopolyhedrovirus (YT Strain) Isolate Enhance Early and Late Viral Promoters In Vitro and In Vivo.

Homologous regions (hrs) are repetitive DNA sequences widely distributed in baculovirus genomes, and are known to function as transcriptional enhancers. In this study, we systematically characterized the seven hrs (hr1, hr2L, hr2R, hr3, hr4L, hr4R, and hr5) from a novel isolate of Bombyx mori nucleopolyhedrovirus (strain BmNPV-YT). The seven hrs range from 390 to 880 bp in length and consist of 1-7 repeat units of approximately 72 bp, each containing a 30 bp palindrome with an EcoRI site. Structural diversity among the hrs includes variable numbers of stem-loop motifs, major late transcription factor (MLTF) sites, cAMP response elements (CRE-like), and 12-O-tetradecanoylphorbol 13-acetate (TPA)-response elements (TRE-like). Functional assays using luciferase reporter plasmids revealed that all seven hrs function as potent transcriptional enhancers for both homologous and heterologous promoters in vitro and in vivo. In BmN cells and fifth-instar silkworm larvae, the hrs enhanced the homologous ie1 promoter 11-1258-fold and 43-1545-fold, respectively, with hr2L showing the strongest activity (counts per minute: 4.82 × 108 in cells; 7.08 × 107 in larvae) and hr4R the weakest. Similarly, when driving the heterologous AcMNPV ie1 promoter in Sf21 cells and larvae, enhancement ranged from 19- to 1023-fold and 13- to 449-fold, respectively, again with hr2L and hr3 showing maximal effects and hr4R minimal enhancement. Notably, all the hrs, except hr4R, also stimulated the homologous late p10 promoter, with activation levels of up to 37.7-fold in vitro and 79.2-fold in vivo, significantly above baseline. However, hr4R showed negligible or no enhancement (0.84-1.05-fold). Among all the active hrs, hr5 emerged as the most potent enhancer of the p10 promoter. Spearman's correlation analyses demonstrated that enhancer efficiency for all three promoters (homologous ie1, heterologous ie1, and homologous p10) correlated significantly with the number of palindromic sequences, the combined count of CRE and TRE motifs, and the total number of characteristic sequences (r = 0.873-0.982, p < 0.05 or p < 0.01). Collectively, our findings clarify the structural features of hrs from the newly isolated strain BmNPV-YT, systematically identify their promoter preferences and host adaptability as transcriptional enhancers, and establish their structure-function relationships. These findings provide novel insights into the transcriptional regulation of baculoviruses and offer valuable tools for optimizing baculovirus expression vector systems.