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SummaryBK polyomavirus is the causative agent of several diseases in transplant patients and the immunosuppressed. In order to better understand the structure and life cycle of BK, we produced infectious virions and VP1-only virus-like particles in cell culture, and determined their three-dimensional structures using cryo-electron microscopy (EM) and single-particle image processing. The resulting 7.6-Å resolution structure of BK and 9.1-Å resolution of the virus-like particles are the highest-resolution cryo-EM structures of any polyomavirus. These structures confirm that the architecture of the major structural protein components of these human polyomaviruses are similar to previous structures from other hosts, but give new insight into the location and role of the enigmatic minor structural proteins, VP2 and VP3. We also observe two shells of electron density, which we attribute to a structurally ordered part of the viral genome, and discrete contacts between this density and both VP1 and the minor capsid proteins.

The serine-arginine-specific protein kinase SRPK1 is a common binding partner of the E1^E4 protein of diverse human papillomavirus types. We show here for the first time that the interaction between HPV1 E1^E4 and SRPK1 leads to potent inhibition of SRPK1 phosphorylation of host serine-arginine (SR) proteins that have critical roles in mRNA metabolism, including pre-mRNA processing, mRNA export, and translation. Furthermore, we show that SRPK1 phosphorylates serine residues of SR/RS dipeptides in the hinge region of the HPV1 E2 protein in in vitro kinase assays and that HPV1 E1^E4 inhibits this phosphorylation. After mutation of the putative phosphoacceptor serine residues, the localization of the E2 protein was altered in primary human keratinocytes; with a significant increase in the cell population showing intense E2 staining of the nucleolus. A similar effect was observed following coexpression of E2 and E1^E4 that is competent for inhibition of SRPK1 activity, suggesting that the nuclear localization of E2 is sensitive to E1^E4-mediated SRPK1 inhibition. Collectively, these data suggest that E1^E4-mediated inhibition of SRPK1 could affect the functions of host SR proteins and those of the virus transcription/replication regulator E2. We speculate that the novel E4 function identified here is involved in the regulation of E2 and SR protein function in posttranscriptional processing of viral transcripts. IMPORTANCEThe HPV life cycle is tightly linked to the epithelial terminal differentiation program, with the virion-producing phase restricted to differentiating cells. While the most abundant HPV protein expressed in this phase is the E4 protein, we do not fully understand the role of this protein. Few E4 interaction partners have been identified, but we had previously shown that E4 proteins from diverse papillomaviruses interact with the serine-arginine-specific protein kinase SRPK1, a kinase important in the replication cycles of a diverse range of DNA and RNA viruses. We show that HPV1 E4 is a potent inhibitor of this host cell kinase. We show that E4 inhibits SRPK1 phosphorylation, not only of cellular SR proteins involved in regulating alternative splicing of RNA but also the viral transcription/replication regulator E2. Our findings reveal a potential E4 function in regulation of viral late gene expression through the inhibition of a host cell kinase. H uman papillomaviruses (HPVs) cause hyperproliferative warts and papillomas of the squamous epithelium at different body sites. Infection of the anogenital tract and oropharynx can lead to benign and malignant disease. There are 13 HPV types defined as causative agents of cancers at these sites, the most common being HPV 16 (HPV16) and HPV18. Of the viruses that infect cutaneous surfaces, some, such as HPV1, cause only benign warts, while infection with others, such as HPV5 and HPV8, can, in immunocompromised individuals, cause the formation of lesions that are at risk of malignant conversion.Despite the heterogeneity in pathogenesis, HPVs show a ...

Mucosal human papillomaviruses express a small, hydrophobic, protein called E5, which plays an important role in the HPV life cycle by delaying normal epithelial cell differentiation while maintaining cell cycle progression. In addition, E5 exhibits transforming abilities in a number of cell culture systems and transgenic mouse models. Lacking any described enzymatic activity, E5 is thought to function by binding to host proteins and modulating their activities. In particular, members of the growth factor receptor family are known targets for subversion. This review article summarizes our latest understanding of this enigmatic oncoprotein, including its role in the HPV life cycle, interactions with host proteins and contribution toward tumorigenesis.

BK polyomavirus (BKPyV; hereafter referred to as BK) causes a lifelong chronic infection and is associated with debilitating disease in kidney transplant recipients. Despite its importance, aspects of the virus life cycle remain poorly understood. In addition to the structural proteins, the late region of the BK genome encodes for an auxiliary protein called agnoprotein. Studies on other polyomavirus agnoproteins have suggested that the protein may contribute to virion infectivity. Here, we demonstrate an essential role for agnoprotein in BK virus release. Viruses lacking agnoprotein fail to release from host cells and do not propagate to wild-type levels. Despite this, agnoprotein is not essential for virion infectivity or morphogenesis. Instead, agnoprotein expression correlates with nuclear egress of BK virions. We demonstrate that the agnoprotein binding partner α-soluble N-ethylmaleimide sensitive fusion (NSF) attachment protein (α-SNAP) is necessary for BK virion release, and siRNA knockdown of α-SNAP prevents nuclear release of wild-type BK virions. These data highlight a novel role for agnoprotein and begin to reveal the mechanism by which polyomaviruses leave an infected cell.

BK polyomavirus (BKPyV) causes a lifelong chronic infection and is associated with debilitating disease in kidney transplant recipients. Despite its importance, aspects of the virus life cycle remain poorly understood. In addition to the structural proteins, the late region of the BK genome encodes for an auxiliary protein called agnoprotein. Studies on other polyomavirus agnoproteins have suggested that the protein may contribute to virion infectivity. Here, we demonstrate an essential role for agnoprotein in BK virus release. Viruses lacking agnoprotein do not propagate to wild-type levels and fail to release from host cells. Despite this, loss of agnoprotein does not impair virion infectivity or morphogenesis. Instead, agnoprotein expression correlates with nuclear egress of BK virions. We demonstrate that the agnoprotein binding partner α-SNAP is necessary for BK virion release, and siRNA knockdown of α-SNAP prevents nuclear release of wild-type BK virions. These data highlight a novel role for agnoprotein and begin to reveal the mechanism by which polyomaviruses leave an infected cell.

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BK polyomavirus (BKPyV) is a common opportunistic pathogen and the causative agent of several diseases in transplant patients and the immunosuppressed. Despite its importance, aspects of the virus lifecycle such as how the virus exits an infected cells, remain poorly understood. The late region of the BKPyV genome encodes an auxillery protein called agnoprotein. We and others have shown that agnoprotein is an essential factor in virus release, and the loss of agnoprotein results in an accumulation of virus particles within the nucleus of an infected cell. The functions of agnoprotein necessary for this egress phenotype are not known. Here we demonstrate that agnoprotein shows properties associated with viroporins, a group of virus-encoded membrane spanning proteins that play key roles in virus infection and release. We demonstrate that agnoprotein oligomerises and perturbs membranes in cells. The development of a novel recombinant agnoprotein expression system permitted the identification of the first small molecules targeting agnoprotein. These compounds abrogated agnoprotein viroporin activity in vitro and reduced virus release, indicating that viroporin activity contributes to the phenotype observed in agnoprotein knockout viruses. The identification of channel activity should enhance the future understanding of the physiological function of agnoprotein and could represent an important target for antiviral intervention.