Characterization of Hantavirus N Protein Intracellular Dynamics and Localization

Abstract

Hantaviruses are enveloped viruses that possess a tri-segmented, negative-sense RNA genome. The viral S-segment encodes the multifunctional nucleocapsid protein (N), which is involved in genome packaging, intracellular protein transport, immunoregulation, and several other crucial processes during hantavirus infection. In this study, we generated fluorescently tagged N protein constructs derived from Puumalavirus (PUUV), the dominant hantavirus species in Central, Northern, and Eastern Europe. We comprehensively characterized this protein in the rodent cell line CHO-K1, monitoring the dynamics of N protein complex formation and investigating co-localization with host proteins as well as the viral glycoproteins Gc and Gn. We observed formation of large, fibrillar PUUV N protein aggregates, rapidly coalescing from early punctate and spike-like assemblies. Moreover, we found significant spatial correlation of N with vimentin, actin, and P-bodies but not with microtubules. N constructs also co-localized with Gn and Gc albeit not as strongly as the glycoproteins associated with each other. Finally, we assessed oligomerization of N constructs, observing efficient and concentration-dependent multimerization, with complexes comprising more than 10 individual proteins.

Diverse susceptibilities and responses of human and rodent cells to orthohantavirus infection reveal different levels of cellular restriction

“…Our findings and confirmation by immunofluorescence co-staining of interaction of N protein with tubulin subunits, support the importance of microtubule components for cytoskeleton integrity and the production of infectious orthohantavirus particles [ 22 ]. Also, the interaction of N protein with DDX6 a marker of P-bodies is comforted by results from the literature showing that SNV N protein co-localize with P-bodies suspected to be important for cap snatching [ 23 ] and has been recently shown to occur in cells transfected with PUUV-N encoding plasmid [ 77 ]. P-bodies are cytoplasmic structures, which are implicated in cellular mRNA turnover.…”

P-Body and Cytoskeleton Remodeling by Orthohantaviruses

“…Previous studies have extensively investigated the role of cytoskeletal factors in orthohantavirus infection (5,7,11,(16)(17)(18). At this point, it is well established that virus proteins colocalize with microtubules, actin and the intermediate filament protein vimentin (5,11,16,18).…”

“…The actin remodelling we have described here and previously (7) could likely be involved in this manipulation of cellular functions. Interestingly, N protein has been reported to strongly co-localize with actin and P-bodies, even in absence of other viral components (18), thus one could speculate whether direct interactions between N and these cellular factors is contributing to their redistribution. It remains elusive however, how exactly cellular protein and mRNA homeostasis are affected by such virus-induced host cell rearrangements.…”

Relating the Biogenesis and Function of P Bodies in Drosophila to Human Disease

“…Due to the role of P bodies in RNA metabolism, they appear to be a target for a variety of viruses, which collectively have a devastating impact on human health worldwide [ 171 , 172 , 173 , 174 , 175 , 176 , 177 , 178 , 179 , 180 , 181 , 182 ]. Typical P body functions are often perturbed as a consequence of viral activity in human cell lines [ 171 ], and the modulation of P bodies by viruses is conserved to Drosophila [ 172 , 173 , 174 , 175 ].…”

“…Typical P body functions are often perturbed as a consequence of viral activity in human cell lines [ 171 ], and the modulation of P bodies by viruses is conserved to Drosophila [ 172 , 173 , 174 , 175 ]. Viral proteins and RNAs can localise to and interact with P bodies, resulting in their disassembly or aggregation [ 173 , 174 , 175 , 176 , 177 , 178 , 179 , 180 , 181 ]. In addition viruses can disrupt P bodies by hijacking components for their own replication [ 182 , 183 , 184 ] or transcription [ 172 ].…”