The RNA Replication Site of Tula Orthohantavirus Resides within a Remodelled Golgi Network

Abstract

<jats:p>The family Hantaviridae within the Bunyavirales order comprises tri-segmented negative sense RNA viruses, many of which are rodent-borne emerging pathogens associated with fatal human disease. In contrast, hantavirus infection of corresponding rodent hosts results in inapparent or latent infections, which can be recapitulated in cultured cells that become persistently infected. In this study, we used Tula virus (TULV) to investigate the location of hantavirus replication during early, peak and persistent phases of infection, over a 30-day time course. Using immunofluorescent (IF) microscopy, we showed that the TULV nucleocapsid protein (NP) is distributed within both punctate and filamentous structures, with the latter increasing in size as the infection progresses. Transmission electron microscopy of TULV-infected cell sections revealed these filamentous structures comprised aligned clusters of filament bundles. The filamentous NP-associated structures increasingly co-localized with the Golgi and with the stress granule marker TIA-1 over the infection time course, suggesting a redistribution of these cellular organelles. The analysis of the intracellular distribution of TULV RNAs using fluorescent in-situ hybridization revealed that both genomic and mRNAs co-localized with Golgi-associated filamentous compartments that were positive for TIA. These results show that TULV induces a dramatic reorganization of the intracellular environment, including the establishment of TULV RNA synthesis factories in re-modelled Golgi compartments.</jats:p>

Hantavirus Replication Cycle—An Updated Structural Virology Perspective

“…Additional open questions relate to the exact cellular localization of viral genome replication and transcription as well as their regulation. Several studies present contradictory results regarding the replication and transcription site with the involvement of stress granules, P bodies, and different subcellular locations [ 86 , 89 , 100 ]. Importantly, most of the current studies used the overexpression of the N protein [ 71 , 86 , 100 ], which likely results in artificial distributions of the protein and the induction of cellular stress.…”

“…Notably, RVFV N protein localization partially overlaps with P body-resident proteins [ 88 ]. In contrast, a recent study found no significant colocalization of TULV N protein and P body markers but reported significant colocalization of TULV RNA and N protein with stress granule-resident T-cell restricted intracellular antigen 1 (TIA-1) as well as an increase in the number of stress granules in TULV-infected cells [ 89 ]. Stress granules, similar to P bodies, are non-membranous compartments but rather serve as storage sites for translation-stalled mRNAs under cellular stress more than as sites of RNA decay.…”

“…These structures were of tubular nature and stained positive for Golgi markers and vRNA as well as for cRNA. Furthermore, they observed the recruitment of stress granules to these structures, suggesting the formation of viral factories within a structurally remodeled Golgi associated with stress granules ( Figure 3 c) [ 89 ].…”

“…Via interaction with the cytoplasmic tail of Gn, it confers recruitment of the genomic RNA segments into virions, but the exact sequence of these assembly steps is not yet clear. Some evidence suggest that the vRNA, the N protein, and the glycoproteins accumulate at the Golgi [ 89 , 116 ] and that microtubules are necessary for the correct movement of the viral components to the assembly site [ 100 ]. After budding into the Golgi apparatus, the viral particle is transported to the plasma membrane where it is then released via exocytosis.…”

“…Of note, although the N protein can be detected in infected cells as early as 4 h post infection [ 100 ], putative viral factories were not perceptible at 36 h post infection, at which point the N protein was mostly localized in the perinuclear puncta. Larger, tubular structures of N protein assemblies became apparent at 7 days post infection, although no further time points between 36 h and 7 days post infection have been tested [ 89 ]. This is in accordance with reports noting the slow growth of hantaviruses [ 14 ], and hence, the assembly of N protein and other components into viral factories and recruitment of cellular structures as described in Section 2.2 may be a rate-determining step in the hantavirus replication cycle.…”