Abstract
The mechanism used by mouse polyomavirus (MPyV) to overcome the crowded cytosol to reach the nucleus has not been fully elucidated. Here, we investigated the involvement of importin α/β1 mediated transport in the delivery of MPyV genomes into the nucleus. Interactions of the virus with importin β1 were studied by co-immunoprecipitation and proximity ligation assay. For infectivity and nucleus delivery assays, the virus and its capsid proteins mutated in the nuclear localization signals (NLSs) were prepared and produced. We found that at early times post infection, virions bound importin β1 in a time dependent manner with a peak of interactions at 6 h post infection. Mutation analysis revealed that only when the NLSs of both VP1 and VP2/3 were disrupted, virus did not bind efficiently to importin β1 and its infectivity remarkably decreased (by 80%). Nuclear targeting of capsid proteins was improved when VP1 and VP2 were co-expressed. VP1 and VP2 were effectively delivered into the nucleus, even when one of the NLS, either VP1 or VP2, was disrupted. Altogether, our results showed that MPyV virions can use VP1 and/or VP2/VP3 NLSs in concert or individually to bind importins to deliver their genomes into the cell nucleus.
Highlights
Mouse polyomavirus belongs to the Polyomaviridae family, a group of tumorigenic non-enveloped double stranded DNA viruses
The pictures were taken 20× magnification; (C) The graph represents the mean values of two independent proximity ligation assay (PLA) experiments. These findings suggest that (i) mouse polyomavirus (MPyV) DNA delivery from the cytoplasm into the nucleus can be performed by the importins utilizing nuclear localization signals (NLSs) of both VP1 and VP2/VP3 capsid proteins; and (ii) capsid proteins accompany the viral DNA to the nucleus during productive infection
Years of study have revealed that the virus, being internalized into smooth vesicles, travels via early and late endocytic compartments into the endoplasmic reticulum (ER) from where it should be delivered into the cell nucleus [9,10]
Summary
Mouse polyomavirus belongs to the Polyomaviridae family, a group of tumorigenic non-enveloped double stranded DNA viruses. The number of newly discovered mammalian PyVs has increased dramatically in recent years. Simian virus 40 (SV40) and MPyV have served as model viruses for many years and are by far the best studied. The capsid of MPyV is composed of 360 molecules of the VP1 protein organized into 72 capsomeres, VP1 pentamers, forming a T7 icosahedral surface lattice. Each capsomere contains one molecule of the minor capsid protein, either VP2 or its shorter variant, VP3 [2,3]. The plasmid pVP1 encoding for MPyV VP1 from LID strain [37] and ph2p∆G encoding for MPyV VP2 from LID strain [38] were used to introduce mutations in the NLS coding sequences of the capsid proteins VP1 and VP2. The following protein variants were produced: VP1/K6Q, VP1/K6Q-S7R-G8R, VP2/K315A, VP2/K314A-K315A, and VP2/K314A-K315A-R317A. Mary’s, London, UK), mouse monoclonal anti-importin β1 antibody (Fisher Scientific), rabbit anti-importin β1 antibody (Bioss Antibodies, Woburn, MA, USA), normal mouse IgG (Upstate Biotechnology, Lake Placid, NY, USA), normal rabbit IgG
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