Respiratory Syncytial Virus Assembles into Structured Filamentous Virion Particles Independently of Host Cytoskeleton and Related Proteins

Shaikh, Fyza Y.; Utley, Thomas J.; Craven, Ryan E.; Rogers, Meredith C.; Lapierre, Lynne A.; Goldenring, James R.; Crowe, James E.

doi:10.1371/journal.pone.0040826

Cited by 34 publications

(37 citation statements)

References 41 publications

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“…A role of actin and myosin in the transport of the viral ribonucleoprotein (RNP) complex has also been described previously, and the role of the ARP2/3 complex in the terminal stage of virus budding and in the process of pinching off from the cell surface has been proposed [6]. Conversely, a previous study showed that F protein trafficking to the cell surface and assembly of RSV filaments appeared to take place even when cytoskeleton rearrangement is blocked by actin inhibitors [45]. Our results suggest that efficient RSV budding indeed depends on functional ARP2/3 nucleation and actin rearrangement.…”

Section: Discussionmentioning

confidence: 96%

Actin-Related Protein 2 (ARP2) and Virus-Induced Filopodia Facilitate Human Respiratory Syncytial Virus Spread

et al. 2016

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Human respiratory syncytial virus (RSV) is an enveloped RNA virus that is the most important viral cause of acute pediatric lower respiratory tract illness worldwide, and lacks a vaccine or effective antiviral drug. The involvement of host factors in the RSV replicative cycle remains poorly characterized. A genome-wide siRNA screen in human lung epithelial A549 cells identified actin-related protein 2 (ARP2) as a host factor involved in RSV infection. ARP2 knockdown did not reduce RSV entry, and did not markedly reduce gene expression during the first 24 hr of infection, but decreased viral gene expression thereafter, an effect that appeared to be due to inhibition of viral spread to neighboring cells. Consistent with reduced spread, there was a 10-fold reduction in the release of infectious progeny virions in ARP2-depleted cells at 72 hr post-infection. In addition, we found that RSV infection induced filopodia formation and increased cell motility in A549 cells and that this phenotype was ARP2 dependent. Filopodia appeared to shuttle RSV to nearby uninfected cells, facilitating virus spread. Expression of the RSV F protein alone from a plasmid or heterologous viral vector in A549 cells induced filopodia, indicating a new role for the RSV F protein, driving filopodia induction and virus spread. Thus, this study identified roles for ARP2 and filopodia in RSV-induced cell motility, RSV production, and RSV cell-to-cell spread.

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Section: Discussionmentioning

confidence: 96%

Actin-Related Protein 2 (ARP2) and Virus-Induced Filopodia Facilitate Human Respiratory Syncytial Virus Spread

et al. 2016

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“…Structural information of the CT and the targets with which it may interact will be required to understand this differential impact. A candidate for interaction is filamin-A, which was recently identified in a yeast two-hybrid screen of a lung cDNA library using CT peptide as bait (17). The second most influential region was CT-R1, with a maximum decrease in infectivity of 85%.…”

Section: Discussionmentioning

confidence: 99%

“…F is critically involved in entry steps, such as attachment and membrane fusion (14)(15)(16), and also plays a role in viral assembly (3,4,17). Using a virus lacking the F protein CT, we previously demonstrated that the CT plays a role in F protein distribution and membrane fusion function and is critical for the production of infectious progeny (3).…”

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confidence: 99%

“…Whereas F572 thus appears to play a major role in viral filament formation, the impact of this and other CT residues on infectivity has not been determined. The mechanisms by which the CT imparts its function(s) are not known but may involve filamin-A, which was recently identified as a putative interacting partner (17), or lipid rafts (18)(19)(20). The F protein was shown to independently associate with lipid rafts (19).…”

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confidence: 99%

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The Respiratory Syncytial Virus Fusion Protein Targets to the Perimeter of Inclusion Bodies and Facilitates Filament Formation by a Cytoplasmic Tail-Dependent Mechanism

Baviskar

Hotard

Moore

et al. 2013

J Virol

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The human respiratory syncytial virus (HRSV) fusion (F) protein cytoplasmic tail (CT) and matrix (M) protein are key mediators of viral assembly, but the underlying mechanisms are poorly understood. A complementation assay was developed to systematically examine the role of the F protein CT in infectious virus production. The ability of F mutants with alanine substitutions in the CT to complement an F-null virus in generating infectious progeny was quantitated by flow cytometry. Two CT regions with impact on infectious progeny production were identified: residues 557 to 566 (CT-R1) and 569 to 572 (CT-R2). Substitutions in CT-R1 decreased infectivity by 40 to 85% and increased the level of F-induced cell-cell fusion but had little impact on assembly of viral surface filaments, which are believed to be virions. Substitutions in CT-R2, as well as deletion of the entire CT, abrogated infectious progeny production and impaired viral filament formation. However, CT-R2 mutations did not block but rather delayed the formation of viral filaments, which continued to form at a low rate and contained the viral M protein and nucleoprotein (N). Microscopy analysis revealed that substitutions in CT-R2 but not CT-R1 led to accumulation of M and F proteins within and at the perimeter of viral inclusion bodies (IBs), respectively. The accumulation of M and F at IBs and coincident strong decrease in filament formation and infectivity upon CT-R2 mutations suggest that F interaction with IBs is an important step in the virion assembly process and that CT residues 569 to 572 act to facilitate release of M-ribonucleoprotein complexes from IBs.

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“…We know from previous work that the M protein and the F protein cytoplasmic tail (CT) are important in the organization of virion assembly (5,24,(33)(34)(35). M is a nonglycosylated phosphorylated protein of 256 amino acids (36)(37)(38).…”

mentioning

confidence: 99%

The Respiratory Syncytial Virus Phosphoprotein, Matrix Protein, and Fusion Protein Carboxy-Terminal Domain Drive Efficient Filamentous Virus-Like Particle Formation

Meshram

Baviskar

Ognibene

et al. 2016

J Virol

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Virus-like particles (VLPs) are attractive as a vaccine concept. For human respiratory syncytial virus (hRSV), VLP assembly is poorly understood and appears inefficient. Hence, hRSV antigens are often incorporated into foreign VLP systems to generate anti-RSV vaccine candidates. To better understand the assembly, and ultimately to enable efficient production, of authentic hRSV VLPs, we examined the associated requirements and mechanisms. In a previous analysis in HEp-2 cells, the nucleoprotein (N), phosphoprotein (P), matrix protein (M), and fusion protein (F) were required for formation of filamentous VLPs, which, similar to those of wild-type virus, were associated with the cell surface. Using fluorescence and electron microscopy combined with immunogold labeling, we examined the surfaces of transfected HEp-2 cells and further dissected the process of filamentous VLP formation. Our results show that N is not required. Coexpression of P plus M plus F, but not P plus M, M plus F, or P plus F, induced both viral protein coalescence and formation of filamentous VLPs that resembled wild-type virions. Despite suboptimal coalescence in the absence of P, the M and F proteins, when coexpressed, formed cell surface-associated filaments with abnormal morphology, appearing longer and thinner than wild-type virions. For F, only the carboxy terminus (Fstem) was required, and addition of foreign protein sequences to Fstem allowed incorporation into VLPs. Together, the data show that P, M, and the F carboxy terminus are sufficient for robust viral protein coalescence and filamentous VLP formation and suggest that M-F interaction drives viral filament formation, with P acting as a type of cofactor facilitating the process and exerting control over particle morphology. IMPORTANCEhRSV is responsible for >100,000 deaths in children worldwide, and a vaccine is not available. Among the potential anti-hRSV approaches are virus-like particle (VLP) vaccines, which, based on resemblance to virus or viral components, can induce protective immunity. For hRSV, few reports are available concerning authentic VLP production or testing, in large part because VLP production is inefficient and the mechanisms underlying particle assembly are poorly understood. Here, we took advantage of the cell-associated nature of RSV particles and used high-resolution microscopy analyses to examine the viral proteins required for formation of wild-type-virus-resembling VLPs, the contributions of these proteins to morphology, and the domains involved in incorporation of the antigenically important viral F protein. The results provide new insights that will facilitate future production of hRSV VLPs with defined shapes and compositions and may translate into improved manufacture of live-attenuated hRSV vaccines. H uman respiratory syncytial virus (hRSV) is an important viral agent of respiratory tract disease in infants, children, immunosuppressed individuals, and the elderly (1-3). In pursuit of a vaccine to prevent hRSV disease, a virus-like particle (...

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Respiratory Syncytial Virus Assembles into Structured Filamentous Virion Particles Independently of Host Cytoskeleton and Related Proteins

Cited by 34 publications

References 41 publications

Actin-Related Protein 2 (ARP2) and Virus-Induced Filopodia Facilitate Human Respiratory Syncytial Virus Spread

Actin-Related Protein 2 (ARP2) and Virus-Induced Filopodia Facilitate Human Respiratory Syncytial Virus Spread

The Respiratory Syncytial Virus Fusion Protein Targets to the Perimeter of Inclusion Bodies and Facilitates Filament Formation by a Cytoplasmic Tail-Dependent Mechanism

The Respiratory Syncytial Virus Phosphoprotein, Matrix Protein, and Fusion Protein Carboxy-Terminal Domain Drive Efficient Filamentous Virus-Like Particle Formation

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