Classic Nuclear Localization Signals and a Novel Nuclear Localization Motif Are Required for Nuclear Transport of Porcine Parvovirus Capsid Proteins

Abstract

Nuclear targeting of capsid proteins (VPs) is important for genome delivery and precedes assembly in the replication cycle of porcine parvovirus (PPV). Clusters of basic amino acids, corresponding to potential nuclear localization signals (NLS), were found only in the unique region of VP1 (VP1up, for VP1 unique part). Of the five identified basic regions (BR), three were important for nuclear localization of VP1up: BR1 was a classic Pat7 NLS, and the combination of BR4 and BR5 was a classic bipartite NLS. These NLS were essential for viral replication. VP2, the major capsid protein, lacked these NLS and contained no region with more than two basic amino acids in proximity. However, three regions of basic clusters were identified in the folded protein, assembled into a trimeric structure. Mutagenesis experiments showed that only one of these three regions was involved in VP2 transport to the nucleus. This structural NLS, termed the nuclear localization motif (NLM), is located inside the assembled capsid and thus can be used to transport trimers to the nucleus in late steps of infection but not for virions in initial infection steps. The two NLS of VP1up are located in the N-terminal part of the protein, externalized from the capsid during endosomal transit, exposing them for nuclear targeting during early steps of infection. Globally, the determinants of nuclear transport of structural proteins of PPV were different from those of closely related parvoviruses. IMPORTANCEMost DNA viruses use the nucleus for their replication cycle. Thus, structural proteins need to be targeted to this cellular compartment at two distinct steps of the infection: in early steps to deliver viral genomes to the nucleus and in late steps to assemble new viruses. Nuclear targeting of proteins depends on the recognition of a stretch of basic amino acids by cellular transport proteins. This study reports the identification of two classic nuclear localization signals in the minor capsid protein (VP1) of porcine parvovirus. The major protein (VP2) nuclear localization was shown to depend on a complex structural motif. This motif can be used as a strategy by the virus to avoid transport of incorrectly folded proteins and to selectively import assembled trimers into the nucleus. Structural nuclear localization motifs can also be important for nuclear proteins without a classic basic amino acid stretch, including multimeric cellular proteins.

Identification of three PPV1 VP2 protein-specific B cell linear epitopes using monoclonal antibodies against baculovirus-expressed recombinant VP2 protein

“…Of the three structural proteins, VP2 is the major capsid protein. It automatically assembles into virus-like particles (VLPs) and contains a novel nuclear localization motif, which is critical for the assembly of newly virions during viral proliferation (Boisvert et al 2014;Maranga et al 2003). Most importantly, the PPV1 VP2 protein plays a dominant role in eliciting neutralizing antibodies against viral infection (Antonis et al 2006;Rueda et al 2000;Xu and Li 2007).…”

Role of capsid proteins in parvoviruses infection

“…The sequences of the BCs are highly conserved among parvoviruses; thus, we inferred that the BC elements, which contain the NLS, most likely participate in the nuclear transport in other representative parvoviruses, as well. A recent study showed that the PPV was distinctly different from the closely related parvoviruses because the PPV had more than one activated NLS, including a novel nuclear localization motif (NLM) [ 42 ]. One of the NLSs is typically located on the VP1 N-terminal and plays a role in the early steps of infection, whereas the other NLS is a novel NLM that targets the VP2 trimers to the nucleus late in the infection [ 42 ].…”

Heterogeneous nuclear ribonucleoprotein K is overexpressed and associated with poor prognosis in gastric cancer

“…hnRNP K contains three repeats of a motif termed the KH domain (for K homology) (19), which could recognize single-stranded DNA or RNA. In addition, hnRNP K has a nuclear-localization signal (NLS) and a nuclear shuttling domain (KNS) which allow transport from the cytoplasm to the nucleus (20). Due to these different structures, hnRNP K is involved in translational modifications, including methylation, sumoylation and phosphorylation.…”