Nipah virus (NiV) is a newly recognized, emerging paramyxovirus capable of causing lethal infections in a number of mammalian species including humans [1]. Together, with Hendra virus (HeV), they are members of the recently created Henipavirus genus within the Paramyxoviridae family [2]. The type species HeV [3] appeared first in eastern Australia in 1994 and was transmitted to humans from infected horses (reviewed [4]). NiV was identified later during an outbreak of severe encephalitis in Malaysia and Singapore that began in 1998 and continued into 1999 and was primarily transmitted to humans from infected pigs, although several additional animal species were also noted to be infected (reviewed [5]). Subsequent outbreaks of NiV in Bangladesh [6-10] and India [11] have been smaller in scope but associated with higher mortality and some human-to-human transmission [8]. Their broad species tropism coupled with their highly pathogenic characteristics has distinguished the henipaviruses from all other paramyxoviruses (reviewed in [12]).
The natural hosts of HeV and NiV appear to be several species of flying foxes, bats in the genus Pteropus [13]. Evidence of henipavirus infection of bats has been obtained in Australia, Malaysia, Cambodia, and Thailand [14-17] and virus has been isolated from bat urine and partially eaten fruit [16,18]. Because of their availability from natural sources and relative ease of propagation and dissemination, NiV and HeV have been classified as priority pathogens by the Centers for Disease Control and Prevention (CDC) and the National Institute of Allergy and Infectious Diseases (NIAID). There are currently no approved vaccines or effective therapeutics for the prevention or treatment of NiV or HeV infection.
Like other paramyxoviruses, NiV and HeV are enveloped with single-stranded negative-sense RNA genomes that replicate in the cytoplasm [1,19]. Members of this family include several well-known viruses such as measles virus (MeV), Sendai virus (SeV), human parainfluenza viruses (hPIV) types 1–4, simian virus 5 (SV5), Newcastle disease virus (NDV), mumps virus, and respiratory syncytial virus [19]. The genome encodes six principal viral proteins: nucleocapsid (N) protein, phosphoprotein (P), matrix (M), the fusion (F) and attachment (H, HN, G) envelope glycoproteins, along with the viral RNA-dependent RNA polymerase (L). Additional viral proteins include V, C, and others that vary according to species [19]. Among the paramyxoviruses, comparatively little is known about the cell biology of the henipaviruses, but there have been several significant advances made in recent years through the analysis of the structure and function of several henipavirus proteins expressed from cloned genes, particularly the polycistronic P gene which encodes four proteins: P, V, C and W that have been shown to modulate virulence by abrogatingthe cellular interferon response [20-23]. Other studies on the F and G envelope glycoproteins, which together determine host range and cellular tropism [24,25], have identified EphrinB2 as a key cellular receptor for both NiV and HeV [26,27], and have also revealed a unique F precursor cleavage and maturation process [28-30]. However, an examination of henipavirus particle assembly and roles the various viral proteins may play in that biological process has not been described.
The assembly and morphogenesis of progeny virions requires that viral proteins, including the envelope glycoproteins and ribonucleoprotein (RNP) complex, associate at the plasma membrane for inclusion into budding virions. This association is thought to be mediated by the M protein; however the details of this process are poorly understood and seem to vary among viral species [31]. Recombinant MeV [32], SeV [33] and rabies virus [34] that lack M are impaired in budding ability but remain infectious as demonstrated by increased cell-cell fusion. Recombinant expression of the M protein of SeV [35,36], hPIV-1 [37], or NDV [38], in the absence of other viral proteins, leads to budding of virus-like particles (VLPs). Similar results have been observed for vesicular stomatitis virus (VSV) [39-41] and Ebola virus (EBOV) [42-46]. In addition, certain envelope glycoproteins also appear to have intrinsic budding activity, as has been shown for SeV F [35,36], the G protein of rabies virus (RV) and VSV [47,48], and the envelope glycoprotein (Gp) of Ebola virus [44,45,49]. In contrast, SV5 requires expression of M along with N and at least one of its envelope glycoproteins in order for efficient budding to occur [50].
NiV culture is restricted to BSL-4 containment and this imparts significant limitations on experimentation aimed at exploring the cell biology of the virus. To circumvent this, we used recombinant gene expression systems, both plasmid transfection-based and recombinant Modified Vaccinia virus Anakara (MVA), to safely study the viral proteins individually and together through the generation of VLPs in cell culture. Both vaccinia virus and MVA have been used in reverse genetics systems to generate negative-sense RNA viruses, including paramyxoviruses, from cDNA [51]. Vaccinia virus has also been employed in budding assays for both rhabdoviruses and filoviruses [39,41,42,49], however certain features of MVA suggested it may be a better platform for such assays. MVA is an attenuated deletion mutant of Vaccinia virus that cannot replicate in most mammalian cells [52]. The block in replication occurs during viral assembly, which allows for a high level of gene expression without progeny virus production and with less cytopathic effect [53]. Here we describe the generation and characterization of NiV VLPs. Our results demonstrate that NiV M possesses intrinsic budding activity and can facilitate the inclusion of other viral proteins into VLPs. Both the F and G envelope glycoproteins could also be independently released from expressing cells in association with membrane, however co-expression of these viral proteins resulted in a reduction of the level of M budding perhaps by virtue of a more organized assembly process with M playing a central role. Sucrose density gradient analysis and immunoelectron microscopy revealed particles consistent in density and size with authentic NiV. These findings will aid our understanding of paramyxovirus particle assembly in general and could help facilitate the development of novel vaccine approaches for henipaviruses.
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