Interaction of the Vp3 Nuclear Localization Signal with the Importin α2/β Heterodimer Directs Nuclear Entry of Infecting Simian Virus 40

Abstract

For nuclear entry of large nucleoprotein complexes, it is thought that one key nuclear localization signal (NLS) of a protein component becomes exposed to mediate importin recognition. We show that the nuclear entry of simian virus 40 involves a dynamic interplay between two distinct interiorly situated capsid NLSs, the Vp1 NLS and the Vp3 NLS, and the selective exposure and importin recognition of the Vp3 NLS. The Vp3 NLS-null mutants assembled normally into virion-like particles (VLP) in mutant DNA-transfected cells. When used to infect a new host, the null VLP entered the cell normally but was impaired in viral DNA nuclear entry due to a lack of recognition by the importin ␣2/␤ heterodimer, leading to reduced viability. Both Vp3 and Vp1 NLSs directed importin interaction in vitro, but the Vp1 NLS, which overlaps the Vp1 DNA binding domain, did not bind importins in the presence of DNA. The results suggest that certain canonical NLSs within a nucleoprotein complex, such as the Vp1 NLS, can be masked from functioning by binding to the nucleic acid component and that the availability of an NLS that is not masked and can become exposed for importin binding, such as the Vp3 NLS, is a general feature of the nuclear entry of the nucleoprotein complexes, including those of other animal viruses.

Nuclear Entry Mechanism of the Human Polyomavirus JC Virus-like Particle

“…The transport of wtVLPs to the nucleus of digitonin-permeabilized HeLa or SVG cells in the presence of an ATP-regenerating system was restored not only by a cytosolic fraction but also by recombinant importins ␣ and ␤. We also demonstrated the ability of wtVLPs to bind to both importin ␣ and ␤ with an overlay assay, suggesting that both importins are required for the nuclear import of wtVLPs, as has previously been shown to be the case for SV40 VP3 (30).…”

Entry, infection, replication, and egress of human polyomaviruses: an update

“…In the SV40 LTAg, protein kinase CK2 phosphorylation increases the affinity of recognition of the NLS by IMP␣1 or IMP␤1, thereby accelerating the nuclear import rate, which can be further enhanced by phosphorylation by double-stranded DNA-dependent protein kinase (dsDNA-PK) at specific sites (Fulcher and Jans 2011). The SV40 virion may enter the nucleus through the NPC through the interactions between NLSs at the common C-terminal basic amino acids of VP2 and VP3 (VP1 and VP2 NLSs) and the canonical nuclear import machinery (Nakanishi et al 2002(Nakanishi et al , 2007. Anti-IMP␣ and anti-IMP␤ antibody-based co-immunoprecipitation assays with SV40 DNA postinfection in permissive cells indicated that minor capsid proteins must associate with virion DNA for nuclear entry (Nakanishi et al 2002(Nakanishi et al , 2007.…”

“…Anti-IMP␣ and anti-IMP␤ antibody-based co-immunoprecipitation assays with SV40 DNA postinfection in permissive cells indicated that minor capsid proteins must associate with virion DNA for nuclear entry (Nakanishi et al 2002(Nakanishi et al , 2007. Only a small fraction of virions that expose an NLS deliver their genomes into the nucleus; however, a complete dissociation of the virion within the cell cytoplasm prevented the nuclear entry of the viral DNA (Nakanishi et al 2002(Nakanishi et al , 2007. Kuksin and Norkin (2012) reported that VP2 and VP3 NLSs may function only in targeting SV40 disassembly intermediates to the nucleus, but SV40 genomes dissociate from VP2 and VP3 before or at the point of nuclear entry and thus enter the nucleus devoid of these viral proteins.…”

“…The SV40 virion may enter the nucleus through the NPC through the interactions between NLSs at the common C-terminal basic amino acids of VP2 and VP3 (VP1 and VP2 NLSs) and the canonical nuclear import machinery (Nakanishi et al 2002(Nakanishi et al , 2007. Anti-IMP␣ and anti-IMP␤ antibody-based co-immunoprecipitation assays with SV40 DNA postinfection in permissive cells indicated that minor capsid proteins must associate with virion DNA for nuclear entry (Nakanishi et al 2002(Nakanishi et al , 2007. Only a small fraction of virions that expose an NLS deliver their genomes into the nucleus; however, a complete dissociation of the virion within the cell cytoplasm prevented the nuclear entry of the viral DNA (Nakanishi et al 2002(Nakanishi et al , 2007.…”

Virus entry at a glance

“…Many associate with microtubule-based motors, such as dynein and dynactin, and move along microtubules towards the nucleus (Bremner et al, 2009;Dodding and Way, 2011;Döhner et al, 2002;Engelke et al, 2011;Leopold et al, 2000;Radtke et al, 2010;Sodeik et al, 1997;Suomalainen et al, 1999;Yamauchi et al, 2008). For transport into the nucleus, they bind nuclear import receptors, such as importins, by using them to target their capsids or genome to nuclear pore complexes (NPCs) (Darshan et al, 2004;Klucevsek et al, 2006;Nakanishi et al, 2002;Schmitz et al, 2010;Wodrich et al, 2006) (reviewed by Puntener and Greber, 2009).…”