Quantitative live cell imaging reveals influenza virus manipulation of Rab11A transport through reduced dynein association

Bhagwat, Amar R.; Sage, Valerie Le; Nturibi, Eric; Kulej, Katarzyna; Jones, Jennifer E.; Guo, Min; Kim, Eui Tae; García, Benjamin A.; Weitzman, Matthew D.; Shroff, Hari; Lakdawala, Seema S.

doi:10.1038/s41467-019-13838-3

Cited by 38 publications

(61 citation statements)

References 67 publications

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“…Additionally, C proteins have been found to be tightly associated with nucleocapsid structures both inside the cell as well as when isolated from SeV virions, with approximately 40 C proteins per vRNP (52). The observed role of polymerase components in assembly are consistent with what was observed in infections with the orthomyxovirus influenza, where influenza virus RNPs interact with Rab11a via one of its polymerase components PB2 (53, 54). As has been suggested for influenza, having the SeV polymerase complex initiate packaging via interactions with Rab11a could be a mechanism to ensure that only vRNPs that contain a RdRP, and are thus infectious, can be packaged into virions.…”

Section: Discussionsupporting

confidence: 85%

“…A549 (human type II pneumocytes, ATCC CCL-185), HEK 293T/17 (ATCC ® CRL-11268) and LLCMK-2 (monkey kidney epithelial cells, ATTC CCL-7) were maintained in Tissue Culture Medium (DMEM (Invitrogen) supplemented with 10% Fetal Bovine Serum (Sigma), Gentamicin (ThermoFisher), Sodium Pyruvate (Invitrogen), L-glutamine (Invitrogen)) at 7% CO2, 37°C. A549 Rab11a-GFP and A549 Rab11a-mCherry cells were generated as described in (54). Cells were tested monthly for mycoplasma MycoAlert Mycoplasma detection kit (Lonza) and treated with Mycoplasma Removal Agent (MP Biomedical) upon thawing.…”

Section: Methodsmentioning

confidence: 99%

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The Viral Polymerase Complex Mediates the Interaction of vRNPs with Recycling Endosomes During SeV Assembly

Genoyer

Kulej

Hung

et al. 2020

Preprint

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31Paramyxoviruses are negative sense single-stranded RNA viruses that comprise many important 32 human and animal pathogens, including human parainfluenza viruses. These viruses bud from 33 the plasma membrane of infected cells after the viral ribonucleoprotein complex (vRNP) is 34 transported from the cytoplasm to the cell membrane via The viral proteins that are critical for mediating this important initial step in viral assembly are 36 unknown. Here we use the model paramyxovirus, murine parainfluenza virus 1, or Sendai virus 37 (SeV), to investigate the roles of viral proteins in Rab11a-driven virion assembly. We previously 38 reported that infection with SeV containing high levels of copy-back defective viral genomes 39 (DVGs) generates heterogenous populations of cells. Cells enriched in full-length virus produce 40 viral particles containing standard or defective viral genomes, while cells enriched in DVGs do 41 not, despite high levels of defective viral genome replication. Here we take advantage of this 42 heterogenous cell phenotype to identify proteins that mediate interaction of vRNPs with Rab11a. 43We examine the role of matrix protein and nucleoprotein and determine that they are not 44 sufficient to drive interaction of vRNPs with recycling endosomes. Using a combination of mass 45 spectrometry and comparative protein abundance and localization in DVG-and FL-high cells, 46we identify viral polymerase complex components L and, specifically, its cofactor C proteins as 47Paramyxoviruses are single-stranded negative sense RNA viruses that include viruses of 65 clinical and global health significance, such as human parainfluenza viruses (HPIV). HPIV1 and 66 2 are the leading cause of croup in young children and HPIV3 is associated with bronchiolitis, 67 bronchitis, and pneumonia (1). These infections have also been associated with the development 68 or exacerbation of asthma and chronic airway disorders (2). Additionally, HPIVs are the 69 etiological agent of 7% of hospitalized pneumonia cases in children in the U.S. (3), as well as in 70Africa and Asia (4). Although HPIVs pose a significant health burden, there are no direct acting 71 antivirals or preventative vaccines. In order to identify targets for antiviral development, there is 72 a need to better understand fundamental aspects of paramyxovirus biology, including 73 mechanisms of virion assembly and particle production. 74Sendai virus (SeV), or murine parainfluenza virus 1, is closely related to human 75 parainfluenza viruses 1 and 3 with 69% homology at the RNA level between SeV and HPIV1. 76SeV has long served as a well-established model for understanding paramyxovirus replication. 77SeV genome replication occurs in the cytoplasm of infected cells (5) and virions bud from the 78 plasma membrane. The viral genomes are tightly coated in the viral nucleocapsid protein (NP) 79 with one NP molecule for every six nucleotides of genome (6). This viral RNA-protein complex 80 is referred to as the viral ribonucleoprotein (vRNP) (7, 8). Viral genomes a...

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Section: Discussionsupporting

confidence: 85%

Section: Methodsmentioning

confidence: 99%

The Viral Polymerase Complex Mediates the Interaction of vRNPs with Recycling Endosomes During SeV Assembly

Genoyer

Kulej

Hung

et al. 2020

Preprint

Self Cite

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“…Three-dimensional movement of fluorescently tagged Rab11A-RE and IAV vRNPs was tracked in infected A549 cells using dual-view inverted selective-plane illumination microscopy (diSPIM). Although Rab11A motion was dependent on microtubules in A549 cells, depletion of microtubule filaments by nocodazole treatment had little impact on vRNP movement (Bhagwat et al 2020). In addition, a large reduction in the amount of dynein, the minus-enddirected microtubule motor, associated with Rab11A was observed in IAV-infected cells.…”

Section: Progeny Vrnp Transport To the Plasma Membranementioning

confidence: 88%

Host Cell Factors That Interact with Influenza Virus Ribonucleoproteins

Staller

Barclay

2020

Cold Spring Harb Perspect Med

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Influenza viruses hijack host cell factors at each stage of the viral life cycle. After host cell entry and endosomal escape, the influenza viral ribonucleoproteins (vRNPs) are released into the cytoplasm where the classical cellular nuclear import pathway is usurped for nuclear translocation of the vRNPs. Transcription takes place inside the nucleus at active host transcription sites, and cellular mRNA export pathways are subverted for export of viral mRNAs. Newly synthesized RNP components cycle back into the nucleus using various cellular nuclear import pathways and host-encoded chaperones. Replication of the negative-sense viral RNA (vRNA) into complementary RNA (cRNA) and back into vRNA requires complex interplay between viral and host factors. Progeny vRNPs assemble at the host chromatin and subsequently exit from the nucleus-processes orchestrated by sets of host and viral proteins. Finally, several host pathways appear to play a role in vRNP trafficking from the nuclear envelope to the plasma membrane for egress.

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“…From previous studies on influenza A it is known that cellular Rab11a-containing endosomes colocalize with vRNPs. 31 Using our methodology, we can now characterise these vesicles in size and cellular distribution. This is important because these vesicles are essential for the transport of vRNPs to the plasma membrane where the mature virions form and are thought to mediate vRNA-vRNA interactions which according to recent studies are crucial for influenza virus assembly.…”

Section: Discussionmentioning

confidence: 99%

“…This is interesting and in line with observations for influenza A virus which suggests that the mode of transport for vRNP complexes can be microtubule-independent. 19 Moreover, there was no identifiable DNA compaction inside the nucleus, which is typical of other viruses such as herpes. 20 In the future, we aim to use this technique for studying the interplay between the viral proteins and the cell compartments in order to dissect the whole replication cycle of LAIV.…”

Section: Expansion Microscopy Highlights the Vesicular Structure Of Nmentioning

confidence: 99%

Combining sample expansion and light sheet microscopy for the volumetric imaging of virus-infected cells with optical super-resolution

Mascheroni

Scherer

Ward

et al. 2020

Preprint

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Expansion microscopy is a sample preparation technique that enables the optical imaging of biological specimens at super-resolution owing to their physical magnification, which is achieved through water-absorbing polymers. The technique uses readily available chemicals and does not require sophisticated equipment, thus offering super-resolution to laboratories that are not microscopy-specialised. Here we present a protocol combining sample expansion with light sheet microscopy to generate high-contrast, high-resolution 3D reconstructions of whole virus-infected cells. The results are superior to those achievable with comparable imaging modalities and reveal details of the infection cycle that are not discernible before expansion. An image resolution of approximately 95 nm could be achieved in samples labelled in 3 colours. We clearly resolve the concentration of viral nucleoprotein on the surface of vesicular structures within the cell and their positioning relative to cellular organelles. We provide detailed guidance and a video protocol for the optimal application of the method and demonstrate its potential to study virus-host cell interactions.

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Quantitative live cell imaging reveals influenza virus manipulation of Rab11A transport through reduced dynein association

Cited by 38 publications

References 67 publications

The Viral Polymerase Complex Mediates the Interaction of vRNPs with Recycling Endosomes During SeV Assembly

The Viral Polymerase Complex Mediates the Interaction of vRNPs with Recycling Endosomes During SeV Assembly

Host Cell Factors That Interact with Influenza Virus Ribonucleoproteins

Combining sample expansion and light sheet microscopy for the volumetric imaging of virus-infected cells with optical super-resolution

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