Human Papillomavirus Replication Regulation by Acetylation of a Conserved Lysine in the E2 Protein

The papillomavirus (PV) E2 protein is a sequence-specific DNA binding protein that recruits cellular factors to its genome in infected epithelial cells. E2 also binds to and loads the viral E1 DNA helicase at the origin of replication. Posttranslational modifications (PTMs) of PV E2 have been identified as potential regulators of E2 functions. We recently reported lysine 111 (K111) as a target of p300 acetylation in bovine PV (BPV). The di-lysines at 111 and 112 are conserved in almost all papillomaviruses. We pursued a mutational approach to query the functional significance of lysine in human PV (HPV) E2. Amino acid substitutions that prevent acetylation, including arginine, were unable to stimulate transcription and E1-mediated DNA replication. The arginine K111 mutant retained E2 transcriptional repression, nuclear localization, DNA and chromatin binding, and association with E2 binding partners involved in PV transcription and replication. While the replication-defective 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 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 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

Autophagy dysregulation in cell culture and animals models of Spinal Muscular Atrophy

Abnormal autophagy has become a central thread linking neurodegenerative diseases, particularly of the motor neuron. One such disease is spinal muscular atrophy (SMA), a genetic neuromuscular disorder caused by mutations in the SMN1 gene resulting in low levels of Survival Motor Neuron (SMN) protein. Despite knowing the causal protein, the exact intracellular processes that are involved in the selective loss of motor neurons remains unclear. Autophagy induction can be helpful or harmful depending on the situation, and we sought to understand the state of the autophagic response in SMA. We show that cell culture and animal models demonstrate induction of autophagy accompanied by attenuated autophagic flux, resulting in the accumulation of autophagosomes and their associated cargo. Expression of the SMN-binding protein a-COP, a known modulator of autophagic flux, can ameliorate this autophagic traffic jam

Small Molecules in Development for the Treatment of Spinal Muscular Atrophy

Spinal muscular atrophy (SMA) is an autosomal recessive neurodegenerative disease resulting from pathologically low levels of survival motor neuron (SMN) protein. The majority of mRNA from the SMN2 allele undergoes alternative splicing and excludes critical codons, causing an SMN protein deficiency. While there is currently no FDA-approved treatment for SMA, early therapeutic efforts have focused on testing repurposed drugs such as phenylbutyrate (2), valproic acid (3), riluzole (6), hydroxyurea (7), and albuterol (9), none of which has demonstrated clinical effectiveness. More recently, clinical trials have focused on novel small-molecule compounds identified from high-throughput screening and medicinal chemistry optimization such as olesoxime (11), CK-2127107, RG7800, LMI070, and RG3039 (17). In this paper, we review both repurposed drugs and small-molecule compounds discovered following medicinal chemistry optimization for the potential treatment of SMA

Differential regulation of the SMN2 gene by individual HDAC proteins

Spinal muscular atrophy (SMA) is an autosomal recessive neurodegenerative disorder that is the leading genetic cause of infantile death. SMA is caused by homozygous deletion or mutation of the survival of motor neuron 1 gene (SMN1). The SMN2 gene is nearly identical to SMN1, however is alternatively spliced. The close relationship to SMN1 results in SMN2 being a very power genetic modifier of SMA disease severity and a target for therapies. We sought to identify the regulatory role individual HDAC proteins use to control expression of full length protein from the SMN2 genes. We used quantitative PCR to determine the effects shRNA silencing of individual HDACs on the steady state levels of a SMN2-luciferase reporter transcripts. We determined that reduction of individual HDAC proteins was sufficient to increase SMN protein levels in a transgenic reporter system. Knockdown of class I HDAC proteins preferentially activated the reporter by increased promoter transcription. Silencing of class II HDAC proteins maintained transcriptional activity; however silencing of HDAC 5 and 6 also appeared to enhance inclusion of an alternatively spliced exon. This work highlights HDAC proteins 2 and 6 as excellent investigative targets. These data are important to the basic understanding of SMN expression regulation and the refinements of current therapeutic compounds as well as the development of novel SMA therapeutics

Modulating role of RNA structure in alternative splicing of a critical exon in the spinal muscular atrophy genes

Humans have two nearly identical copies of the survival motor neuron (SMN ) gene, SMN1 and SMN2. Homozygous loss of SMN1 causes spinal muscular atrophy (SMA). SMN2 is unable to prevent the disease due to skipping of exon 7. Using a systematic approach of in vivo selection, we have previously demonstrated that a weak 5′ splice site (ss) serves as the major cause of skipping of SMN2 exon 7. Here we show the inhibitory impact of RNA structure on the weak 5′ ss of exon 7. We call this structure terminal stem–loop 2 (TSL2). Confirming the inhibitory nature of TSL2, point mutations that destabilize TSL2 promote exon 7 inclusion in SMN2, whereas strengthening of TSL2 promotes exon 7 skipping even in SMN1. We also demonstrate that TSL2 negatively affects the recruitment of U1snRNP at the 5′ ss of exon 7. Using enzymatic structure probing, we confirm that the sequence at the junction of exon 7/intron 7 folds into TSL2 and show that mutations in TSL2 cause predicted structural changes in this region. Our findings reveal for the first time the critical role of RNA structure in regulation of alternative splicing of human SMN

Tax1BP1 interacts with papillomavirus E2 and regulates E2-dependent transcription and stability

The papillomavirus E2 proteins regulate viral replication, gene transcription, and genome maintenance by interacting with other viral and host proteins. From a yeast two-hybrid screen, we identified the cellular protein Tax1BP1 as a novel binding partner of human papillomavirus type 18 (HPV18) E2. Tax1BP1 also interacts with the HPV16 and bovine papillomavirus type 1 (BPV1) E2 proteins, with the C-terminal region of Tax1BP1 interacting with the N-terminal transactivation domain of BPV1 E2. Tax1BP1 complexes with p300 and acts synergistically as a coactivator with p300 to enhance E2-dependent transcription. Using chromatin immunoprecipitation assays, we show that Tax1BP1 and E2 localize to the long control region on the BPV1 genome. Tax1BP1 was recently reported to bind ubiquitin and to function as an essential component of an A20 ubiquitin-editing complex. We demonstrate that Tax1BP1 plays a role in the regulation of the steady-state level of E2 by preventing its proteasomal degradation. These studies provide new insights into the regulation of E2 functions

The SMC5/6 Complex Represses the Replicative Program of High-Risk Human Papillomavirus Type 31

The multi-subunit structural maintenance of chromosomes (SMC) 5/6 complex includes SMC6 and non-SMC element (NSE)3. SMC5/6 is essential for homologous recombination DNA repair and functions as an antiviral factor during hepatitis B (HBV) and herpes simplex-1 (HSV-1) viral infections. Intriguingly, SMC5/6 has been found to associate with high-risk human papillomavirus (HPV) E2 regulatory proteins, but the functions of this interaction and its role during HPV infection remain unclear. Here, we further characterize SMC5/6 interactions with HPV-31 E2 and its role in the HPV life cycle. Co-immunoprecipitation (co-IP) revealed that SMC6 interactions with HPV-31 E2 require the E2 transactivation domain, implying that SMC5/6 interacts with full-length E2. Using chromatin immunoprecipitation, we found that SMC6 is present on HPV-31 episomes at E2 binding sites. The depletion of SMC6 and NSE3 increased viral replication and transcription in keratinocytes maintaining episomal HPV-31, indicating that SMC5/6 restricts the viral replicative program. SMC6 interactions with E2 were reduced in the presence of HPV-31 E1, suggesting that SMC6 and E1 compete for E2 binding. Our findings demonstrate SMC5/6 functions as a repressor of the viral replicative program and this may involve inhibiting the initiation of viral replication

Levels of the E2 interacting protein TopBP1 modulate papillomavirus maintenance stage replication

The evolutionarily conserved DNA topoisomerase II beta-binding protein 1 (TopBP1) functions in DNA replication, DNA damage response, and cell survival. We analyzed the role of TopBP1 in human and bovine papillomavirus genome replication. Consistent with prior reports, TopBP1 co-localized in discrete nuclear foci and was in complex with papillomavirus E2 protein. Similar to E2, TopBP1 is recruited to the region of the viral origin of replication during G1/S and early S phase. TopBP1 knockdown increased, while over-expression decreased transient virus replication, without affecting cell cycle. Similarly, using cell lines harboring HPV-16 or HPV-31 genome, TopBP1 knockdown increased while over-expression reduced viral copy number relative to genomic DNA. We propose a model in which TopBP1 serves dual roles in viral replication: it is essential for initiation of replication yet it restricts viral copy number

Papillomavirus E2 protein is regulated by specific fibroblast growth factor receptors

The papillomavirus (PV) E2 protein activates transcription and replication by recruiting cellular proteins and the E1 DNA helicase to their binding sites in the viral genome. We recently demonstrated that phosphorylation of tyrosine 102 in the bovine papillomavirus (BPV-1) E2 protein restricts these activities and that fibroblast growth factor receptor-3 (FGFR3) tyrosine kinase binds PV E2. Expression of FGFR3 decreased viral replication with both wild-type and the phenylalanine substitution at position 102, inferring that another kinase targets Y102. Here we tested FGFR- 1, −2 and −4 for association with PV E2 proteins. FGFR2 but not FGFR1 or FGFR4 co-immunoprecipitated with BPV-1 E2. We found that FGFR2 suppressed replication but did not depend on phosphorylation of BPV-1 Y102. HPV-16 and −31 E2 interacted with FGFR1, −2, and −4. These results imply that the expression and activity of FGF receptors in epithelial cells can regulate the function of E2 in viral replication