[PubMed] [CrossRef] [Google Scholar] 46

[PubMed] [CrossRef] [Google Scholar] 46. E2-K111R mutant recruited E1 to the viral replication origin, surprisingly, unwinding of the duplex DNA did not occur. In contrast, the K111 glutamine (K111Q) mutant increased origin melting and stimulated replication compared to wild-type E2. These experiments reveal a novel activity of E2 necessary for denaturing ROCK inhibitor-2 the viral origin that likely depends on acetylation of highly conserved lysine 111. IMPORTANCE HPV is one of the most common sexually transmitted infections in the United States. Over 200 HPVs have been described, and they manifest in a variety of ways; they can be asymptomatic or can result in benign lesions (papillomas) or progress to IHG2 malignancy. Although 90% of infections are asymptomatic and resolve easily, HPV16 and -18 alone are responsible for 70% of all cervical cancers, which are almost entirely caused by HPV infection. Interestingly, 60 to 90% of other cancers have been linked to HPV. The goal of this research is to further elucidate the mechanisms that regulate and mediate viral replication. and regulate its transcriptional activation capability (31). These lysines at positions 111 and 112 (K111 and K112) are conserved in more than 90% of papillomaviruses, including BPV1 and cancer-associated high-risk HPV16, -18, and ROCK inhibitor-2 -31. Here we examined the role of these HPV31 E2 lysines in the initiation of DNA replication. We discovered that viral replication did not tolerate the conservative mutation of K111 to arginine (K111R). ROCK inhibitor-2 Unexpectedly, our data reveal that DNA unwinding by the E1 helicase depends on a ROCK inhibitor-2 PTM at lysine 111 in E2. RESULTS Mutation of K111 affects transient viral replication. Two lysine residues, K111 and K112, are very highly conserved among human and animal papillomaviruses (PVs) (Fig. 1A). We previously demonstrated the requirement of lysine 111 for BPV1 E2 transcriptional activation (31). While E2 is known to bind and localize the E1 helicase to high-affinity E2 binding sites at the viral origin, additional activities for E2 in DNA replication have not been elucidated. We began testing both BPV and HPV31 E2 lysine 111 and 112 mutants (E2-K111 and E2-K112) for stimulation of E1-dependent viral replication by cotransfection with the corresponding E1 gene and the PV origin-containing luciferase reporter construct. This di-lysine motif has been previously studied in BPV1, and mutations of each of these lysines resulted in decreased viral replication, with K111R causing a more severe reduction (32, 33). Compared to wild-type BPV E2, mutations of these lysines had deleterious effects on replication; K112R resulted in a 3-fold reduction, and K111R completely abrogated transient DNA replication, similarly to the replication-dead W145R mutant (Fig. 1B). The conservative arginine and alanine mutations were then tested in the context of HPV31 E2. The results indicate that mutations that prevent acetylation at K111 or mimic a deacetylated lysinearginine and alanine, respectivelyfailed to induce E1-dependent replication (Fig. 1C). However, the mutation of HPV31 K111 to glutamine (K111Q), which resembles an acetylated lysine, stimulated replication to levels that surpassed wild-type E2. We ensured that the abrogation of replication from the K111R mutant HPV31 E2 protein was not due to reduced cell viability (Fig. 1C). These experiments also demonstrated that while replication is affected by K112 in the context of BPV1 (31), mutations at this position in HPV31 E2 did not exhibit a similar dependence (Fig. 1C). Open in a separate window FIG 1 K111 is necessary for transient viral replication. Mutations of E2 lysine 111 (K111) alter transient replication. (A) Sequence alignment shows the highly conserved K111..