Abstract

The engineering of viral genomes facilitates both fundamental and applied research on viruses. However, the multiloci manipulation of DNAs of viruses with large DNA genomes, such as baculoviruses, herpesviruses, and poxviruses, is technically challenging, particularly for highly homologous or repetitive sequences. Homologous regions (hrs) have multiple copies in many large DNA viruses and play pivotal roles in the viral life cycle. Here, we used synthetic biology to investigate the fundamental function of baculoviral hrs by conducting multiloci manipulation of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) DNA that contains eight hrs scattered in the genome. Using transformation-associated recombination in yeast, we generated recombinant AcMNPV genomes in which we deleted all hrs or retained a single hr (hr1, hr2, or hr3). Infectious viruses were rescued after transfecting the synthetic viral genomes into host cells, and their replication features were characterized. The results demonstrated that deletion of all hrs severely compromised viral DNA replication and progeny production, whereas retaining only a single hr was essential for efficient viral DNA replication and progeny production. The synthetic virus with hr2 or hr3 showed a growth curve similar to that of the parental virus. Transcriptomic analysis revealed that hr1, hr2, and hr3 could enhance gene transcription within a surrounding region of 14.6 kb, 13.8 kb, and 29.8 kb, respectively. Overall, this study revealed the advantages of synthetic biology in multiloci engineering and functional studies of large DNA viruses. In addition, our findings on hrs will be helpful for the design and improvement of baculovirus-based expression vectors.

Full Text
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