| Summary: | Guanine-rich sequences in the genomes of herpesviruses can fold into G-quadruplexes. Compared with the widely-studied G<sub>3</sub>-quadruplexes, the dynamic G<sub>2</sub>-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 G<sub>2</sub>-PQSs and 205 G<sub>3</sub>-PQSs without overlap were identified in the PRV genome. Twelve G<sub>2</sub>-PQSs from the CDS region exhibited high conservation in the genomes of the <i>Varicellovirus</i> genus. Eleven G<sub>2</sub>-PQSs were 100% conserved in the repeated region of the annotated PRV genomes. There were 212 non-redundant G<sub>2</sub>-PQSs in the 3′ UTR and 19 non-redundant G<sub>2</sub>-PQSs in the 5′ UTR, which would mediate gene expression in the post-transcription and translation processes. The majority of examined G<sub>2</sub>-PQSs formed parallel structures and exhibited different sensitivities to cations and small molecules in vitro. Two G<sub>2</sub>-PQSs, respectively, from 3′ UTR of <i>UL5</i> (encoding helicase motif) and <i>UL9</i> (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 G<sub>2</sub>-PQSs in the PRV genomes could guide further studies of the G-quadruplexes’ functions in the life cycle of herpesviruses.
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