Subcellular Localization and Functional Characterization of GII.4 Norovirus-Encoded NTPase

Abstract

The genotype II.4 (GII.4) variants of human noroviruses (HuNVs) are recognized as the major agent of global gastroenteritis outbreaks. Due to the lack of an efficient cell culture system for HuNV propagation, the exact roles of HuNV-encoded nonstructural proteins (including Nterm, NTPase, P22, VPg, Pro, and RdRp) in viral replication or pathogenesis have not yet been fully understood. Here, we report the molecular characterization of the GII.4 HuNV-encoded NTPase (designated GII-NTPase). Results from our studies showed that GII-NTPase forms vesicular or nonvesicular textures in the cell cytoplasm, and the nonvesicular fraction of GII-NTPase significantly localizes to the endoplasmic reticulum (ER) or mitochondria. Deletion analysis revealed that the N-terminal 179-amino-acid (aa) region of GII-NTPase is required for vesicle formation and for ER colocalization, whereas the C-terminal region is involved in mitochondrial colocalization. In particular, two mitochondrion-targeting domains were identified in the C-terminal region of GII-NTPase which perfectly colocalized with mitochondria when the N-terminal region of GII-NTPase was deleted. However, the corresponding C-terminal portions of NTPase derived from the GI HuNV did not show mitochondrial colocalization. We also found that GII-NTPase physically interacts with itself as well as with Nterm and P22, but not VPg, Pro, and RdRp, in cells. The Nterm- and P22-interacting region was mapped to the N-terminal 179-aa region of GII-NTPase, whereas the self-assembly of GII-NTPase could be achieved via a head-to-head, tail-to-tail, or head-to-tail configuration. More importantly, we demonstrate that GII-NTPase possesses a proapoptotic activity, which can be further enhanced by coexpression with Nterm or P22.IMPORTANCE Despite the importance of human norovirus GII.4 variants in global gastroenteritis outbreaks, the basic biological functions of the viral nonstructural proteins in cells remain rarely investigated. In this report, we focus our studies on characteristics of the GII.4 norovirus-encoded NTPase (GII-NTPase). We unexpectedly find that GII-NTPase can perfectly colocalize with mitochondria after its N-terminal region is deleted. However, such a phenomenon is not observed for NTPase encoded by a GI strain. We further reveal that the N-terminal 179-aa region of GII-NTPase is sufficient to mediate (i) vesicle formation, (ii) ER colocalization, (iii) the interaction with two other nonstructural proteins, including Nterm and P22, (iv) the formation of homodimers or homo-oligomers, and (v) the induction of cell apoptosis. Taken together, our findings emphasize that the virus-encoded NTPase must have multiple activities during viral replication or pathogenesis; however, these activities may vary somewhat among different genogroups.

Nucleotide triphosphatase and RNA chaperone activities of murine norovirus NS3

“…All were reported to form homooligomeric ring-like structures in vitro, combined with high activity. More recently, a cellular localization study for GII.4 norovirus NTPase demonstrated that this protein can form homodimers and homooligomers through a head-to-head, head-to-tail, or tail-to-tail configuration [41]. These reports support our finding that MNV NS3 forms homooligomers.…”

Mouse Norovirus Infection Reduces the Surface Expression of Major Histocompatibility Complex Class I Proteins and Inhibits CD8 + T Cell Recognition and Activation

“…4). NS3 has been characterized as a nucleoside triphosphatase (NTPase), and we and others have recently shown that it is associated with the viral replication complex, the ER, mitochondria, lipids, and microtubules (36)(37)(38). NTPases have diverse activities and functions, including an association with microtubule dynamics, intracellular transport, and protein degradation (39)(40)(41)(42)(43).…”

Human Norovirus Proteins: Implications in the Replicative Cycle, Pathogenesis, and the Host Immune Response

“…Furthermore, NV NTPase possesses fundamental enzymatic activities that play a significant role in viral replication, such as (a) NTP-dependent helicase activity for unrolling of RNA helices, (b) NTP-independent chaperone activity for remodeling of the RNA structure and facilitating the annealing of the RNA chains, and (c) collaboration in RNA synthesis carried out by NS7 (62). Significantly, the subcellular localization of the GII.4 NTPase protein was determined through a confocal immunofluorescence analysis in human melanoma A7 cells, determining that this protein can form vesicular and nonvesicular structures in the cell cytoplasm (63). Besides that, the non-vesicular form of NTPase can be located in the ER or the membrane of the cellular mitochondria.…”

“…Besides that, the non-vesicular form of NTPase can be located in the ER or the membrane of the cellular mitochondria. The amino acid 179 of the N-terminal region is necessary for the formation and the localization of vesicles in the ER (63). On the other hand, for mitochondrial localization, the NTPase C-terminal region of GII plays a significant role, even when the N-terminal NTPase region is completely removed (63).…”

“…The amino acid 179 of the N-terminal region is necessary for the formation and the localization of vesicles in the ER (63). On the other hand, for mitochondrial localization, the NTPase C-terminal region of GII plays a significant role, even when the N-terminal NTPase region is completely removed (63). However, this activity is not observed in the GI viruses (NV); thus, the NTPase mitochondrial location differs between HuNoV genogroups (63).…”