Human polyomavirus JCV late leader peptide region contains important regulatory elements

Abstract

Transcription is a complex process that relies on the cooperative interaction between sequence-specific factors and the basal transcription machinery. The strength of a promoter depends on upstream or downstream cis-acting DNA elements, which bind transcription factors. In this study, we investigated whether DNA elements located downstream of the JCV late promoter, encompassing the late leader peptide region, which encodes agnoprotein, play regulatory roles in the JCV lytic cycle. For this purpose, the entire coding region of the leader peptide was deleted and the functional consequences of this deletion were analyzed. We found that viral gene expression and replication were drastically reduced. Gene expression also decreased from a leader peptide point mutant but to a lesser extent. This suggested that the leader peptide region of JCV might contain critical cis-acting DNA elements to which transcription factors bind and regulate viral gene expression and replication. We analyzed the entire coding region of the late leader peptide by a footprinting assay and identified three major regions (region I, II and III) that were protected by nuclear proteins. Further investigation of the first two protected regions by band shift assays revealed a new band that appeared in new infection cycles, suggesting that viral infection induces new factors that interact with the late leader peptide region of JCV. Analysis of the effect of the leader peptide region on the promoter activity of JCV by transfection assays demonstrated that this region has a positive and negative effect on the large T antigen (LT-Ag)-mediated activation of the viral early and late promoters, respectively. Furthermore, a partial deletion analysis of the leader peptide region encompassing the protected regions I and II demonstrated a significant down-regulation of viral gene expression and replication. More importantly, these results were similar to that obtained from a complete deletion of the late leader peptide region, indicating the critical importance of these two protected regions in JCV regulation. Altogether, these findings suggest that the late leader peptide region contains important regulatory elements to which transcription factors bind and contribute to the JCV gene regulation and replication.

Nuclear Magnetic Resonance Structure Revealed that the Human Polyomavirus JC Virus Agnoprotein Contains an α-Helix Encompassing the Leu/Ile/Phe-Rich Domain

“…We have also previously reported that alterations in the protein kinase C (PKC) phosphorylation sites of JCV agnoprotein (conversion of Ser7, Ser11, and Thr21 to Ala) resulted in phenotypes that are unable to sustain the viral replication cycle (19). Deletion of either the intrinsically disordered Cterminal region (the sequence from amino acids 51 to 71) (2,79) or internal sequences containing the ␣-helix region (amino acids 17 to 42) (7) resulted in a replication-incompetent virus. Recent fine mapping studies of the multimerization (␣-helix) domain of agnoprotein also revealed the involvement of this domain in determining the stability of agnoprotein as well as in regulation of the splicing of the viral late transcripts (1).…”

JC virus nucleotides 376-396 are critical for VP1 capsid protein expression

“…Figure 1 shows the locations of the additional mutants that were generated, as well as the locations of the predicted host cell protein binding sites (Akan et al, 2006). Mad-1 PtΔ300-349 encompasses the region from the beginning of the JCV CPN deletion to the start of binding site III.…”

“…Finally, Mad-1 PtΔ411-442 is the region between binding sites III and II. As controls, we used the previously characterized viruses Mad-1 Pt, a start codon deletion mutant, Mad-1 Del, a full agnogene deletion mutant (Akan et al, 2006), and Mad-1 C-Agno, JCV Mad-1 with the JCV CPN agnogene, which has a deletion of nucleotides 300-442 (Ellis et al, 2013). …”

“…Studies have shown that deleting the agnogene DNA is more detrimental to JCV replication than loss of agnoprotein expression without deletion of the DNA sequence, indicating that the agnogene may contain a cis-acting regulatory element (Akan et al, 2006; Ellis et al, 2013). Additionally, 3 potential host-factor binding sites have been identified in the agnogene (Akan et al, 2006).…”

“…Additionally, 3 potential host-factor binding sites have been identified in the agnogene (Akan et al, 2006). …”