Guanine-rich sequences in the genomes of herpesviruses can fold into G-quadruplexes. Compared with the widely-studied G₃-quadruplexes, the dynamic G₂-quadruplexes are more sensitive to the cell microenvironment, but they attract less attention. Pseudorabies virus (PRV) is the model species for the study of the latency and reactivation of herpesvirus in the nervous system. A total of 1722 G₂-PQSs and 205 G₃-PQSs without overlap were identified in the PRV genome. Twelve G₂-PQSs from the CDS region exhibited high conservation in the genomes of the Varicellovirus genus. Eleven G₂-PQSs were 100% conserved in the repeated region of the annotated PRV genomes. There were 212 non-redundant G₂-PQSs in the 3' UTR and 19 non-redundant G₂-PQSs in the 5' UTR, which would mediate gene expression in the post-transcription and translation processes. The majority of examined G₂-PQSs formed parallel structures and exhibited different sensitivities to cations and small molecules in vitro. Two G₂-PQSs, respectively, from 3' UTR of UL5 (encoding helicase motif) and UL9 (encoding sequence-specific ori-binding protein) exhibited diverse regulatory activities with/without specific ligands in vivo. The G-quadruplex ligand, NMM, exhibited a potential for reducing the virulence of the PRV Ea strain. The systematic analysis of the distribution of G₂-PQSs in the PRV genomes could guide further studies of the G-quadruplexes' functions in the life cycle of herpesviruses.
Keywords: G-quadruplex; G-quadruplex ligand; alphaherpesviruses; genome; nucleic acids conformation; pseudorabies virus; regulatory element.