Respiratory Syncytial Virus Matrix (M) Protein Interacts with Actin In Vitro and in Cell Culture

Shahriari, Shadi; Wei, Kejun; Ghildyal, Reena

doi:10.3390/v10100535

Cited by 37 publications

(26 citation statements)

References 36 publications

(67 reference statements)

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“…Moreover, Lamp1, a marker of late endosomes and lysosomes, was very little detected in fractions corresponding to HRSV inclusion bodies, suggesting that these organelles are not components of HRSV inclusion bodies. It is noteworthy that actin was concentrated in cell fractions corresponding to HRSV inclusion bodies, in agreement with previous findings by Shahriari et al (40).…”

Section: Discussionsupporting

confidence: 92%

Host Retromer Protein Sorting Nexin 2 Interacts with Human Respiratory Syncytial Virus Structural Proteins and is Required for Efficient Viral Production

Cardoso

Tavares

Jesus

et al. 2020

mBio

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Human respiratory syncytial virus (HRSV) envelope glycoproteins traffic to assembly sites through the secretory pathway, while nonglycosylated proteins M and N are present in HRSV inclusion bodies but must reach the plasma membrane, where HRSV assembly happens. Little is known about how nonglycosylated HRSV proteins reach assembly sites. Here, we show that HRSV M and N proteins partially colocalize with the Golgi marker giantin, and the glycosylated F and nonglycosylated N proteins are closely located in the trans-Golgi, suggesting their interaction in that compartment. Brefeldin A compromised the trafficking of HRSV F and N proteins and inclusion body sizes, indicating that the Golgi is important for both glycosylated and nonglycosylated HRSV protein traffic. HRSV N and M proteins colocalized and interacted with sorting nexin 2 (SNX2), a retromer component that shapes endosomes in tubular structures. Glycosylated F and nonglycosylated N HRSV proteins are detected in SNX2-laden aggregates with intracellular filaments projecting from their outer surfaces, and VPS26, another retromer component, was also found in inclusion bodies and filament-shaped structures. Similar to SNX2, TGN46 also colocalized with HRSV M and N proteins in filamentous structures at the plasma membrane. Cell fractionation showed enrichment of SNX2 in fractions containing HRSV M and N proteins. Silencing of SNX1 and 2 was associated with reduction in viral proteins, HRSV inclusion body size, syncytium formation, and progeny production. The results indicate that HRSV structural proteins M and N are in the secretory pathway, and SNX2 plays an important role in the traffic of HRSV structural proteins toward assembly sites. IMPORTANCE The present study contributes new knowledge to understand HRSV assembly by providing evidence that nonglycosylated structural proteins M and N interact with elements of the secretory pathway, shedding light on their intracellular traffic. To the best of our knowledge, the present contribution is important given the scarcity of studies about the traffic of HRSV nonglycosylated proteins, especially by pointing to the involvement of SNX2, a retromer component, in the HRSV assembly process.

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Section: Discussionsupporting

confidence: 92%

Host Retromer Protein Sorting Nexin 2 Interacts with Human Respiratory Syncytial Virus Structural Proteins and is Required for Efficient Viral Production

Cardoso

Tavares

Jesus

et al. 2020

mBio

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“…The sub-IB localisation of bRSV N and P was similar to that previously described for hRSV, with N and P being found on the periphery of the organelle (26). The significant intra-IB localisation of the M protein at 24 h p.i., as well as its partial nuclear localisation, is consistent with previously reported IF in RSV-infected cells (37,38). However, the role of M in RNA virus IBs reflects an interesting point of divergence; with some viral IBs being M positive (e.g.…”

Section: The Nf-κb Subunit P65 Co-localises With Viral Inclusion Bodisupporting

confidence: 90%

Respiratory Syncytial Virus Sequesters NF-κB Subunit p65 to Cytoplasmic Inclusion Bodies To Inhibit Innate Immune Signaling

Jobe

Simpson

Hawes

et al. 2020

J Virol

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Viruses routinely employ strategies to prevent the activation of innate immune signalling in infected cells. RSV is no exception, encoding two accessory proteins (NS1 and NS2) which are well established to block Interferon signalling. However, RSV-encoded mechanisms for inhibiting NF-κB signalling are less well characterised. In this study we identified RSV-mediated antagonism of this pathway, independent of the NS1 and NS2 proteins, and indeed distinct from other known viral mechanisms of NF-κB inhibition. In both human and bovine RSV infected cells we demonstrated that the P65 subunit of NF-κB is rerouted to perinuclear puncta in the cytoplasm, puncta which are synonymous with viral inclusion bodies (IBs), the site for viral RNA replication. Captured P65 was unable to translocate to the nucleus or transactivate a NF-κB reporter following TNF-α stimulation, confirming the immune-antagonistic nature of this sequestration. Subsequently, we used correlative light electron microscopy (CLEM) to colocalise RSV N protein and P65 within bRSV IBs; granular, membraneless regions of cytoplasm with liquid organelle-like properties. Additional characterisation of bRSV IBs indicated that although they are likely formed by liquid-liquid phase separation (LLPS), they have a differential sensitivity to hypotonic shock proportional to their size. Together, these data identify a novel mechanism for viral antagonism of innate immune signalling which relies on sequestration of the NF-κB subunit p65 to a biomolecular condensate – a mechanism conserved across the Orthopneumovirus genus and not host-cell specific. More generally they provide additional evidence that RNA virus IBs are important immunomodulatory complexes within infected cells. Importance Many viruses replicate almost entirely in the cytoplasm of infected cells; however, how these pathogens are able to compartmentalise their life cycle to provide favourable conditions for replication and to avoid the litany of antiviral detection mechanisms in the cytoplasm remains relatively uncharacterised. In this paper we show that bovine RSV (bRSV), which infects cattle, does this by generating inclusion bodies in the cytoplasm of infected cells. We confirm that both bRSV and human RSV viral RNA replication takes place in these inclusion bodies, likely meaning these organelles are a functionally conserved feature of this group of viruses (the orthopneumoviruses). Importantly, we also showed that these organelles are able to capture important innate immune transcription factors (in this case NF-KB), blocking the normal signalling processes that tell the nucleus the cell is infected, which may help us to understand how these viruses cause disease.

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“…The M protein in particular is a bridge between the RNPs and the lipid bilayer envelope. It also serves as an inhibitor of virus transcription in the late stages of infection and facilitates virion assembly and budding by coating the RNPs [ 48 ] and modifying the actin cytoskeleton [ 49 ].…”

Section: General Features Of These Two Respiratory Virusesmentioning

confidence: 99%

Innate Immune Components That Regulate the Pathogenesis and Resolution of hRSV and hMPV Infections

Andrade

Pacheco

Gálvez

et al. 2020

Viruses

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The human respiratory syncytial virus (hRSV) and human Metapneumovirus (hMPV) are two of the leading etiological agents of acute lower respiratory tract infections, which constitute the main cause of mortality in infants. However, there are currently approved vaccines for neither hRSV nor hMPV. Moreover, despite the similarity between the pathology caused by both viruses, the immune response elicited by the host is different in each case. In this review, we discuss how dendritic cells, alveolar macrophages, neutrophils, eosinophils, natural killer cells, innate lymphoid cells, and the complement system regulate both pathogenesis and the resolution of hRSV and hMPV infections. The roles that these cells play during infections by either of these viruses will help us to better understand the illnesses they cause. We also discuss several controversial findings, relative to some of these innate immune components. To better understand the inflammation in the lungs, the role of the respiratory epithelium in the recruitment of innate immune cells is briefly discussed. Finally, we review the main prophylactic strategies and current vaccine candidates against both hRSV and hMPV.

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Respiratory Syncytial Virus Matrix (M) Protein Interacts with Actin In Vitro and in Cell Culture

Cited by 37 publications

References 36 publications

Host Retromer Protein Sorting Nexin 2 Interacts with Human Respiratory Syncytial Virus Structural Proteins and is Required for Efficient Viral Production

Host Retromer Protein Sorting Nexin 2 Interacts with Human Respiratory Syncytial Virus Structural Proteins and is Required for Efficient Viral Production

Respiratory Syncytial Virus Sequesters NF-κB Subunit p65 to Cytoplasmic Inclusion Bodies To Inhibit Innate Immune Signaling

Innate Immune Components That Regulate the Pathogenesis and Resolution of hRSV and hMPV Infections

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