Quantitative analysis of Nipah virus proteins released as virus-like particles reveals central role for the matrix protein

Department of Microbiology and Immunology, Uniformed Services University, Bethesda, Maryland 20814, USA.
Virology Journal (Impact Factor: 2.18). 02/2007; 4:1. DOI: 10.1186/1743-422X-4-1
Source: PubMed


Nipah virus (NiV) is an emerging paramyxovirus distinguished by its ability to cause fatal disease in both animal and human hosts. Together with Hendra virus (HeV), they comprise the genus Henipavirus in the Paramyxoviridae family. NiV and HeV are also restricted to Biosafety Level-4 containment and this has hampered progress towards examining details of their replication and morphogenesis. Here, we have established recombinant expression systems to study NiV particle assembly and budding through the formation of virus-like particles (VLPs).
When expressed by recombinant Modified Vaccinia virus Ankara (rMVA) or plasmid transfection, individual NiV matrix (M), fusion (F) and attachment (G) proteins were all released into culture supernatants in a membrane-associated state as determined by sucrose density gradient flotation and immunoprecipitation. However, co-expression of F and G along with M revealed a shift in their distribution across the gradient, indicating association with M in VLPs. Protein release was also altered depending on the context of viral proteins being expressed, with F, G and nucleocapsid (N) protein reducing M release, and N release dependent on the co-expression of M. Immunoelectron microscopy and density analysis revealed VLPs that were similar to authentic virus. Differences in the budding dynamics of NiV proteins were also noted between rMVA and plasmid based strategies, suggesting that over-expression by poxvirus may not be appropriate for studying the details of recombinant virus particle assembly and release.
Taken together, the results indicate that NiV M, F, and G each possess some ability to bud from expressing cells, and that co-expression of these viral proteins results in a more organized budding process with M playing a central role. These findings will aid our understanding of paramyxovirus particle assembly in general and could help facilitate the development of a novel vaccine approach for henipaviruses.

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Available from: Lin-Fa Wang, Oct 05, 2015
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    • "Another virus protein that could be involved in molecular virus - host interactions is the structural matrix protein M. According to the well-known function of M proteins in paramyxovirus assembly, henipavirus M proteins are essentially involved in membrane envelopment and budding [8]. In NiV M, a late domain like YMYL and a YPLGVG motif contribute to budding activity. "
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    ABSTRACT: Membrane envelopment and budding of negative strand RNA viruses (NSVs) is mainly driven by viral matrix proteins (M). In addition, several M proteins are also known to be involved in host cell manipulation. Knowledge about the cellular targets and detailed molecular mechanisms, however, is poor for many M proteins. For instance, Nipah Virus (NiV) M protein trafficking through the nucleus is essential for virus release, but nuclear targets of NiV M remain unknown. To identify cellular interactors of henipavirus M proteins, tagged Hendra Virus (HeV) M proteins were expressed and M-containing protein complexes were isolated and analysed. Presence of acidic leucine-rich nuclear phosphoprotein 32 family member B (ANP32B) in the complex suggested that this protein represents a direct or indirect interactor of the viral matrix protein. Over-expression of ANP32B led to specific nuclear accumulation of HeV M, providing a functional link between ANP32B and M protein. ANP32B-dependent nuclear accumulation was observed after plasmid-driven expression of HeV and NiV matrix proteins and also in NiV infected cells. The latter indicated that an interaction of henipavirus M protein with ANP32B also occurs in the context of virus replication. From these data we conclude that ANP32B is a nuclear target of henipavirus M that may contribute to virus replication. Potential effects of ANP32B on HeV nuclear shuttling and host cell manipulation by HeV M affecting ANP32B functions in host cell survival and gene expression regulation are discussed.
    PLoS ONE 05/2014; 9(5):e97233. DOI:10.1371/journal.pone.0097233 · 3.23 Impact Factor
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    • "The F, G and M genes were amplified from the HMPV A2 positive Nasopharyngeal washings (SIN06-NTU271) using the primers F271pCAGGf F271pCAGGmycr, G271pCAGGf G271pCAGGflagr, M84pCAGGf and M84pCAGGr (Additional file 2: Table S1). The PCR products were then inserted into the vector pCAGGS [17]. "
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    ABSTRACT: Human metapneumovirus (HMPV) is now a major cause of lower respiratory infection in children. Although primary isolation of HMPV has been achieved in several different cell lines, the low level of virus replication and the subsequent recovery of low levels of infectious HMPV have hampered biochemical studies on the virus. These experimental methodologies usually require higher levels of biological material that can be achieved following HMPV infection. In this study we demonstrate that expression of the HMPV F, G and M proteins in mammalian cells leads to HMPV virus-like particles (VLP) formation. This experimental strategy will serve as a model system to allow the process of HMPV virus assembly to be examined. The HMPV F, G and M proteins were expressed in mammalian cell lines. Protein cross-linking studies, sucrose gradient centrifugation and in situ imaging was used to examine interactions between the virus proteins. VLP formation was examined using sucrose density gradient centrifugation and electron microscopy analysis. Analysis of cells co-expressing of the F, G and M proteins demonstrated that these proteins interacted. Furthermore, in cells co-expression the three HMPV proteins the formation VLPs was observed. Image analysis revealed the VLPs had a similar morphology to the filamentous virus morphology that we observed on HMPV-infected cells. The capacity of each protein to initiate VLP formation was examined using a VLP formation assay. Individual expression of each virus protein showed that the G protein was able to form VLPs in the absence of the other virus proteins. Furthermore, co-expression of the G protein with either the M or F proteins facilitated their incorporation into the VLP fraction. Co-expression of the F, G and M proteins lead to the formation of VLPs, and that incorporation of the F and M proteins into VLPs is facilitated by their interaction with the G protein. Our data suggests that the G protein plays a central role in VLP formation, and further suggests that the G protein may also play a role in the recruitment of the F and M proteins to sites of virus particle formation during HMPV infection.
    Virology Journal 09/2013; 10(1):294. DOI:10.1186/1743-422X-10-294 · 2.18 Impact Factor
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    • "Furthermore , there are precedents for the use of VLP-based vaccines in humans: Cervarix (GlaxoSmithKline, Rixensart , Belgium; Merck, Whitehouse station, New Jersey, USA.) and Gardasil (Merck) vaccines for human papilloma virus and Recombivax HB (Merck) for hepatitis B are Food and Drug Administration approved VLP-based vaccines (Ellis, 1993; Monie et al., 2008; Harper, 2009; Lu et al., 2011). In addition, promising VLP-based vaccine candidates for emerging viral pathogens such as Chikungunya virus (Akahata et al., 2010), Ebola and Marburg viruses (Warfield et al., 2005, 2007), H1N1 and H5N1 influenza viruses (Kang et al., 2009; Landry et al., 2010; Pushko et al., 2010; Ding et al., 2011a,b; Giles and Ross, 2011), Hantavirus (Li et al., 2010), arenaviruses (Casabona et al., 2009; Branco et al., 2010) and Nipah virus (Patch et al., 2007; Walpita et al., 2011) are in clinical trials or various stages of development (Roldao et al., 2010). "
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    ABSTRACT: Rift Valley fever virus (RVFV) is an arbovirus that causes significant morbidity and mortality in both humans and livestock. With increased world travel and the threat of bioterrorism, there is a real risk of RVFV spreading to naïve geographical areas (Trans. R. Soc. Trop. Med. Hyg., 73, 1979, 618; MMWR Morb. Mortal. Wkly Rep., 49, 2000, 905). The introduction of RVFV would cause critical public health, agricultural and economic damage. Despite the clear need for an efficacious vaccine, there are no United States (US) Food and Drug Administration or US Department of Agriculture approved vaccines against RVFV. To address this need, a virus-like particle (VLP)-based vaccine candidate was developed. First, a non-replicating chimeric RVF VLP vaccine candidate was generated that protected mice and rats against a lethal RVFV challenge. This was followed by the development and optimization of conditions for production of RVF VLPs in insect and mammalian cells. Immunological studies demonstrated that VLP-based vaccine candidates elicit both humoral and cellular immune responses. Subsequent challenge studies using a lethal wild-type RVFV strain under high-containment conditions showed that RVF VLP vaccine candidates can completely protect mice and rats.
    Zoonoses and Public Health 09/2012; 59((Suppl 1)):1-9. DOI:10.1111/j.1863-2378.2012.01478.x · 2.37 Impact Factor
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