Matrix Proteins of Nipah and Hendra Viruses Interact with Beta Subunits of AP-3 Complexes

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Hendra virus (HeV) is a zoonotic paramyxovirus that causes deadly illness in horses and humans. An intriguing feature of HeV is the utilization of endosomal protease for activation of the viral fusion protein (F). Here we investigated how endosomal F trafficking affects HeV assembly. We found that the HeV matrix (M) and F proteins each induced particle release when they were expressed alone but that their coexpression led to coordinated assembly of virus-like particles (VLPs) that were morphologically and physically distinct from M-only or F-only VLPs. Mutations to the F protein transmembrane domain or cytoplasmic tail that disrupted endocytic trafficking led to failure of F to function with M for VLP assembly. Wild-type F functioned normally for VLP assembly even when its cleavage was prevented with a cathepsin inhibitor, indicating that it is endocytic F trafficking that is important for VLP assembly, not proteolytic F cleavage. Under specific conditions of reduced M expression, we found that M could no longer induce significant VLP release but retained the ability to be incorporated as a passenger into F-driven VLPs, provided that the F protein was competent for endocytic trafficking. The F and M proteins were both found to traffic through Rab11-positive recycling endosomes (REs), suggesting a model in which F and M trafficking pathways converge at REs, enabling these proteins to preassemble before arriving at plasma membrane budding sites.IMPORTANCE Hendra virus and Nipah virus are zoonotic paramyxoviruses that cause lethal infections in humans. Unlike that for most paramyxoviruses, activation of the henipavirus fusion protein occurs in recycling endosomal compartments. In this study, we demonstrate that the unique endocytic trafficking pathway of Hendra virus F protein is required for proper viral assembly and particle release. These results advance our basic understanding of the henipavirus assembly process and provide a novel model for the interplay between glycoprotein trafficking and paramyxovirus assembly.

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

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Mutations in the Transmembrane Domain and Cytoplasmic Tail of Hendra Virus Fusion Protein Disrupt Virus-Like-Particle Assembly

Cifuentes-Muñoz

Sun

Ray

et al. 2017

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“…Supporting the idea of a critical role in virus particle formation and budding, NiV M protein forms virus-like particles (VLPs) when expressed on its own (12,13), and it drives apical assembly and budding of NiV virions in polarized epithelial cells (14). Trafficking of NiV M is a complex process involving transit through the nucleus (15)(16)(17)(18), despite replication occurring exclusively in the cytoplasm. When NiV M protein nuclear localization or export signals are interrupted, or if the endosomal sorting complexes required for transport (ESCRT) pathway-interacting late domains are disrupted, NiV M proteins lose their ability to accumulate at the plasma membrane and no longer generate virus-like particles (12,17,19).…”

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confidence: 99%

Nipah Virus Matrix Protein Influences Fusogenicity and Is Essential for Particle Infectivity and Stability

Dietzel

Kolesnikova

Sawatsky

et al. 2016

Nipah virus (NiV) causes fatal encephalitic infections in humans. To characterize the role of the matrix (M) protein in the viral life cycle, we generated a reverse genetics system based on NiV strain Malaysia. Using an enhanced green fluorescent protein (eGFP)-expressing M protein-deleted NiV, we observed a slightly increased cell-cell fusion, slow replication kinetics, and significantly reduced peak titers compared to the parental virus. While increased amounts of viral proteins were found in the supernatant of cells infected with M-deleted NiV, the infectivity-to-particle ratio was more than 100-fold reduced, and the particles were less thermostable and of more irregular morphology. Taken together, our data demonstrate that the M protein is not absolutely required for the production of cell-free NiV but is necessary for proper assembly and release of stable infectious NiV particles. IMPORTANCEHenipaviruses cause a severe disease with high mortality in human patients. Therefore, these viruses can be studied only in biosafety level 4 (BSL-4) laboratories, making it more challenging to characterize their life cycle. Here we investigated the role of the Nipah virus matrix protein in virus-mediated cell-cell fusion and in the formation and release of newly produced particles. We found that even though low levels of infectious viruses are produced in the absence of the matrix protein, it is required for the release of highly infectious and stable particles. Fusogenicity of matrixless viruses was slightly enhanced, further demonstrating the critical role of this protein in different steps of Nipah virus spread.

“…M proteins of Sendai virus (6,7), measles virus (8,9), Nipah virus (10,11), Hendra virus (12), Newcastle disease virus (13), and human parainfluenza virus 1 (HPIV1) (14) are all capable of directing VLP production and release from transfected cells when expressed alone. In these cases, additional viral components, including the viral glycoproteins and the nucleocapsid-like structures that form upon expression of paramyxovirus N/NP proteins, can be efficiently packaged into the VLPs if they are coexpressed along with the M proteins (4).…”

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confidence: 99%

C-Terminal DxD-Containing Sequences within Paramyxovirus Nucleocapsid Proteins Determine Matrix Protein Compatibility and Can Direct Foreign Proteins into Budding Particles

Ray

Schmitt

2016

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T he paramyxoviruses comprise a group of enveloped viruses that harbor nonsegmented, negative-sense RNA genomes (1). Included among the paramyxoviruses are a number of human and animal pathogens, including measles virus, mumps virus, Nipah virus, respiratory syncytial virus (RSV), and Newcastle disease virus (NDV). Paramyxovirus infections are spread via particles which bud from plasma membranes of infected cells. Formation of these particles is driven by the viral matrix (M) proteins which can self-assemble to form ordered yet flexible arrays (2, 3) that likely play key roles in generating the membrane curvature required for budding. M proteins also organize the particle assembly process by interacting with the viral glycoproteins via their cytoplasmic tails and also with the viral ribonucleoprotein (vRNP) complexes via the nucleocapsid (N or NP) proteins (reviewed in references 4 and 5). These interactions bring together and concentrate all of the viral structural components onto specific sites underlying infected cell plasma membranes, enabling infectious virions to subsequently bud from these locations.For many paramyxoviruses, expression of M protein in the absence of any other viral components is sufficient to induce the assembly and release of virus-like particles (VLPs) from transfected cells. M proteins of Sendai virus (6, 7), measles virus (8, 9), Nipah virus (10, 11), Hendra virus (12), Newcastle disease virus (13), and human parainfluenza virus 1 (HPIV1) (14) are all capable of directing VLP production and release from transfected cells when expressed alone. In these cases, additional viral components, including the viral glycoproteins and the nucleocapsid-like structures that form upon expression of paramyxovirus N/NP proteins, can be efficiently packaged into the VLPs if they are coexpressed along with the M proteins (4). For other paramyxoviruses, including mumps virus (15) and parainfluenza virus 5 (PIV5) (16), the