A luciferase-based budding assay for Ebola virus

The VP40 matrix protein of Ebola virus (EBOV) is capable of budding from mammalian cells as a virus-like particle (VLP) and is the major protein involved in virus egress. A functional budding assay has been developed based upon this characteristic of VP40 to assess the contributions of VP40 sequences as well as host proteins to the budding process. This well-defined assay has been modified for potential use in a high-throughput format in which the detection and quantification of firefly luciferase protein in VLPs represents a direct measure of VP40 budding efficiency. Luciferase was found to be incorporated into budding VP40 VLPs. Furthermore, co-expression of EBOV glycoprotein (GP) enhances release of VLPs containing VP40 and luciferase. In contrast, when luciferase is co-expressed with a budding deficient mutant of VP40, luciferase levels in the VLP fraction decrease significantly. This assay represents a promising high-throughput approach to identify inhibitors of EBOV budding.     

Filovirus-like particles as vaccines and discovery tools

Ebola and Marburg viruses are members of the family Filoviridae, which cause severe hemorrhagic fevers in humans. Filovirus outbreaks have been sporadic, with mortality rates currently ranging from 30 to 90%. Unfortunately, there is no efficacious human therapy or vaccine available to treat disease caused by either Ebola or Marburg virus infection. Expression of the filovirus matrix protein, VP40, is sufficient to drive spontaneous production and release of virus-like particles (VLPs) that resemble the distinctively filamentous infectious virions. The addition of other filovirus proteins, including virion proteins (VP)24, 30 and 35 and glycoprotein, increases the efficiency of VLP production and results in particles containing multiple filovirus antigens. Vaccination with Ebola or Marburg VLPs containing glycoprotein and VP40 completely protects rodents from lethal challenge with the homologous virus. These candidate vaccines are currently being tested for immunogenicity and efficacy in nonhuman primates. Furthermore, the Ebola and Marburg VLPs are being used as a surrogate model to further understand the filovirus life cycle, with the goal of developing rationally designed vaccines and therapeutics. Thus, in addition to their use as a vaccine, VLPs are currently being used as tools to learn lessons about filovirus pathogenesis, immunology, replication and assembly requirements.     

Mechanisms for enveloped virus budding: can some viruses do without an ESCRT?

Many enveloped viruses complete their replication cycle by forming vesicles that bud from the plasma membrane. Some viruses encode "late" (L) domain motifs that are able to hijack host proteins involved in the vacuolar protein sorting (VPS) pathway, a cellular budding process that gives rise to multivesicular bodies and that is topologically equivalent to virus budding. Although many enveloped viruses share this mechanism, examples of viruses that require additional viral factors and viruses that appear to be independent of the VPS pathway have been identified. Alternative mechanisms for virus budding could involve other topologically similar process such as cell abscission, which occurs following cytokinesis, or virus budding could proceed spontaneously as a result of lipid microdomain accumulation of viral proteins. Further examination of novel virus-host protein interactions and characterization of other enveloped viruses for which budding requirements are currently unknown will lead to a better understanding of the cellular processes involved in virus assembly and budding.     

The creation of stable cell lines expressing Ebola virus glycoproteins and the matrix protein VP40 and generating Ebola virus-like particles utilizing an ecdysone inducible mammalian expression system

Ebola virus is a filovirus that causes hemorrhagic fever in humans and is associated with case fatality rates of up to 90%. The lack of therapeutic interventions in combination with the threat of weaponizing this organism has enhanced research investigations. The expression of key viral proteins and the production of virus-like particles in mammalian systems are often pursued for characterization and functional studies. Common practice is to express these proteins through transient transfection of mammalian cells. Unfortunately the transfection reagents are expensive and the process is time consuming and labour intensive. This work describes utilizing an ecdysone inducible mammalian expression system to create stable cell lines that express the Ebola virus transmembrane glycoprotein (GP), the soluble glycoprotein (sGP) and the matrix protein (VP40) individually as well as GP and VP40 simultaneously (for the production of virus like particles). These products were the same as those expressed by the transient system, by Western blot analysis and electron microscopy. The inducible system proved to be an improvement of the current technology by enhancing the cost effectiveness and simplifying the process.     

Filovirus assembly and budding

Filoviruses belong to the order of negative-stranded non-segmented RNA viruses and are classified into two genera, Ebola and Marburg viruses. They have a characteristic filamentous shape, which is largely determined by the matrix protein VP40. Although VP40 is the main driving force for assembly and budding from the host cell, the production of infectious virus involves an intricate interplay between all viral structural proteins in addition to cellular factors, e.g., those that normally function in multi-vesicular body biogenesis. As a consequence, assembly and budding steps are defined to specific cellular compartments, and the recent progress in understanding how the different components are assembled into stable enveloped virus particles is reviewed.     

Oligomerization and polymerization of the filovirus matrix protein VP40

The matrix protein VP40 from Ebola virus plays an important role in the assembly process of virus particles by interacting with cellular factors, cellular membranes, and the ribonuclearprotein particle complex. Here we show that the N-terminal domain of VP40 folds into a mixture of two different oligomeric states in vitro, namely hexameric and octameric ringlike structures, as detected by gel filtration chromatography, chemical cross-linking, and electron microscopy. Octamer formation depends largely on the interaction with nucleic acids, which in turn confers in vitro SDS resistance. Refolding experiments with a nucleic acid free N-terminal domain preparation reveal a mostly dimeric form of VP40, which is transformed into an SDS resistant octamer upon incubation with E. coli nucleic acids. In addition, we demonstrate that the N-terminal domain of Marburg virus VP40 also folds into ringlike structures, similar to Ebola virus VP40. Interestingly, Marburg virus VP40 rings reveal a high tendency to polymerize into rods composed of stacked rings. These results may suggest distinct roles for different oligomeric forms of VP40 in the filovirus life cycle.     

Role of multivesicular bodies and their components in the egress of enveloped RNA viruses

As an enveloped virus buds, the nascent viral capsid becomes wrapped in a plasma membrane-derived lipid envelope, and a membrane fission event is thus necessary to separate the virion from the host cell. This membrane fission event is well characterised in the case of enveloped RNA viruses, where it is promoted by late assembly domains (L-domains) present at the level of specific viral structural proteins. Research conducted over the past 10 years has demonstrated that L-domains represent docking sites for cellular proteins essential for the biogenesis of a cellular organelle, the multivesicular body (MVB). In this way, enveloped RNA viruses hijack the MVB components to the cellular site where the budding is executed. This review will focus on the cellular machinery exploited by enveloped RNA viruses in order to be released from infected cells. The role of ubiquitin and lipids in viral budding will also be discussed.     

Construction of green fluorescent protein-tagged recombinant iridovirus to assess viral replication

Green fluorescent protein-tagged recombinant virus has been successfully applied to observing the infective dynamics and evaluating viral replication. Here, we identified soft-shelled turtle iridovirus (STIV) ORF55 as an envelope protein (VP55), and developed a recombinant STIV expressing an enhanced green fluorescent protein (EGFP) fused to VP55 (EGFP-STIV). Recombinant EGFP-STIV shared similar single-step growth curves and ultrastructural morphology with wild type STIV (wt-STIV). The green fluorescence distribution during EGFP-STIV infection was consistent with the intracellular distribution of VP55 which was mostly co-localized with virus assembly sites. Furthermore, EGFP-STIV could be used to evaluate viral replication conveniently under drug treatment, and the result showed that STIV replication was significantly inhibited after the addition of antioxidant pyrrolidine dithiocarbamate (PDTC). Thus, the EGFP-tagged recombinant iridovirus will not only be useful for further investigations on the viral replicative dynamics, but also provide an alternative simple strategy to screen for antiviral substances.     

Tetramerization of white spot syndrome virus envelope protein VP33 and its interaction with VP24

VP33, also termed VP281, VP37 or VP36B, is a minor envelope protein of white spot syndrome virus (WSSV). Because of its low abundance and lack of a transmembrane domain, we hypothesized that VP33 is likely to be attached to the viral envelope by interaction with other envelope proteins. In this study, we employed far-western blotting and pull-down assays to demonstrate that VP33 interacts with itself, as well as with VP24, which is one of the four major viral envelope proteins. Moreover, a gel-filtration analysis was performed to show that this self-interaction led to the formation of stable VP33 tetramers. These results implied that VP33 tetramers were anchored to the viral envelope through interaction with VP24, suggesting that VP33 may participate in the formation of the WSSV envelope.     

Purification and characterization of vaccinia virus structural protein VP8

A major vaccinia virus core protein, designated VP8, has been purified from virions to homogeneity through DEAE-cellulose, CM-cellulose, and hydroxyapatite chromatography. VP8 migrates as a 25-kDa band in SDS-polyacrylamide gel electrophoresis and sediments as a monomeric species in neutral sucrose gradient centrifugation. This protein is a significant constitutent of the virion, comprising about 6.5% of the total viral polypeptides by mass. Analysis by filter binding and by sucrose gradient centrifugation shows that VP8 binds to double-stranded as well as to single-stranded DNA at low salt concentrations (25 mM NaCl). At higher salt concentrations (100 mM NaCl), the protein binds with a relatively greater affinity to single-stranded DNA. The results from sucrose gradient centrifugation indicate that VP8 probably binds noncooperatively to all structural forms of DNA. The protein is likely to be a component of the viral nucleoprotein complex.     

Immunochemical identification of viral and nonviral proteins of the respiratory syncytial virus virion.

We identified by immunochemical methods 13 polypeptides associated with the infectious respiratory syncytial virus virion. Eight of these polypeptides (VP200, VP84, VP66, VP43, VP40, VP37, VP28, and VP19) were identified as virus specific. Two other polypeptides, (VP) 22 and (VP) 12, are provisionally considered to be of viral origin. Three nonviral proteins are also intimately associated with the infectious virion. These nonviral proteins were identified as cellular actin and two proteins with bovine serum albumin immunospecificity. VP40 was identified as the major ribonucleoprotein. Based on biochemical and biophysical similarities with paramyxovirus proteins, other respiratory syncytial virus proteins are believed to have these specific viral functions: VP84, "hemagglutinin"; VP66, undissociated fusion protein, F1,2; VP43, F1; and VP19, F2, VP66 contains a major determinant involved in viral infectivity since all neutralizing antibodies tested, including a monoclone, precipitated this protein.     

Detection of all known filovirus species by reverse transcription-polymerase chain reaction using a primer set specific for the viral nucleoprotein gene

The filoviruses, Marburg virus (MARV) and Ebola virus (EBOV), are causative agents of severe hemorrhagic fever with high mortality rates in humans and non-human primates. Sporadic outbreaks of filovirus infection have occurred in Central Africa and parts of Asia. Identification of the natural reservoir animals that are unknown yet and epidemiological investigations are current challenges to forestall outbreaks of filovirus diseases. The filovirus species identified currently include one in the MARV group and five in the EBOV group, with large genetic variations found among the species. Therefore, it has been difficult to develop a single sensitive assay to detect all filovirus species, which would advance laboratory diagnosis greatly in endemic areas. In this study, a highly sensitive universal RT-PCR assay targeting the nucleoprotein (NP) gene of filoviruses was developed. The genomic RNAs of all known MARV and EBOV species were detected by using an NP-specific primer set. In addition, this RT-PCR procedure was verified further for its application to detect viral RNAs in tissue samples of animals infected experimentally and blood specimens of infected patients. This assay will be a useful method for diagnostics and epidemiological studies of filovirus infections.     

Influence of calcium/calmodulin on budding of Ebola VLPs: implications for the involvement of the Ras/Raf/MEK/ERK pathway

The VP40 matrix protein of Ebola virus is able to bud from mammalian cells as a virus-like particle (VLP). Interactions between L-domain motifs of VP40 and host proteins such as Tsg101 and Nedd4 serve to facilitate budding of VP40 VLPs. Since intracellular levels of calcium are known to influence localization and function of host proteins involved in virus budding, we sought to determine, whether alterations of calcium or calmodulin levels in cells would affect budding of VP40 VLPs. VP40 VLP release was assessed in cells treated with BAPTA/AM, a calcium ion chelator, or with ionomycin, a calcium ionophore. In addition, VLP budding was assessed in cells treated with W7, W13, or TFP; all calmodulin antagonists. Results from these experiments indicated that: (i) budding of VP40 VLPs was reduced in a dose-dependent manner in the presence of BAPTA/AM, and slightly enhanced in the presence of ionomycin, (ii) VP40 VLP budding was reduced in a dose-dependent manner in the presence of W7, whereas VP40 VLP budding was unaffected in the presence of cyclosporine-A, (iii) budding of VSV-WT and a VSV recombinant (M40 virus) possessing the L-domains of Ebola VP40 was inhibited in the presence of W7, W13, or TFP, (iv) inhibition of virus budding by W7, W13, and TFP appears to be L-domain independent, and (v) the mechanism of calcium/calmodulin-mediated inhibition of Ebola VLP budding may involve the Ras/Raf/MEK/ERK signaling pathway.     

Identification of two major virion protein genes of white spot syndrome virus of shrimp

White Spot Syndrome Virus (WSSV) is an invertebrate virus, causing considerable mortality in shrimp. Two structural proteins of WSSV were identified. WSSV virions are enveloped nucleocapsids with a bacilliform morphology with an approximate size of 275 x 120 nm, and a tail-like extension at one end. The double-stranded viral DNA has an approximate size 290 kb. WSSV virions, isolated from infected shrimps, contained four major proteins: 28 kDa (VP28), 26 kDa (VP26), 24 kDa (VP24), and 19 kDa (VP19) in size, respectively. VP26 and VP24 were found associated with nucleocapsids; the others were associated with the envelope. N-terminal amino acid sequences of nucleocapsid protein VP26 and the envelope protein VP28 were obtained by protein sequencing and used to identify the respective genes (vp26 and vp28) in the WSSV genome. To confirm that the open reading frames of WSSV vp26 (612) and vp28 (612) are coding for the putative major virion proteins, they were expressed in insect cells using baculovirus vectors and analyzed by Western analysis. A polyclonal antiserum against total WSSV virions confirmed the virion origin of VP26 and VP28. Both proteins contained a putative transmembrane domain at their N terminus and many putative N- and O-glycosylation sites. These major viral proteins showed no homology to baculovirus structural proteins, suggesting, together with the lack of DNA sequence homology to other viruses, that WSSV may be a representative of a new virus family, Whispoviridae.     

Mutations in the FPIV motif of Newcastle disease virus matrix protein attenuate virus replication and reduce virus budding

The FPIV-like late domains identified in the matrix (M) proteins of parainfluenza virus 5 and mumps virus have been demonstrated to be critical for virus budding. In this study, we found that the same FPIV sequence motif is present in the N-terminus of the Newcastle disease virus (NDV) M protein. Mutagenesis experiments demonstrated that mutation of either phenylalanine (F) or proline (P) to alanine led to a more obvious decrease in viral virulence and replication and resulted in poor budding of the mutant viruses. Additionally, evidence for the involvement of cellular multivesicular body (MVB) proteins was obtained, since NDV production was inhibited upon expression of dominant-negative versions of the VPS4A-E228Q protein. Together, these results demonstrate that the FPIV motif, especially the residues F and P, within the NDV M protein, plays a critical role in NDV replication and budding, and this budding process likely involves the cellular MVB pathway.     

Influenza virus morphogenesis and budding

Influenza viruses are enveloped, negative stranded, segmented RNA viruses belonging to Orthomyxoviridae family. Each virion consists of three major sub-viral components, namely (i) a viral envelope decorated with three transmembrane proteins hemagglutinin (HA), neuraminidase (NA) and M2, (ii) an intermediate layer of matrix protein (M1), and (iii) an innermost helical viral ribonucleocapsid [vRNP] core formed by nucleoprotein (NP) and negative strand viral RNA (vRNA). Since complete virus particles are not found inside the cell, the processes of assembly, morphogenesis, budding and release of progeny virus particles at the plasma membrane of the infected cells are critically important for the production of infectious virions and pathogenesis of influenza viruses as well. Morphogenesis and budding require that all virus components must be brought to the budding site which is the apical plasma membrane in polarized epithelial cells whether in vitro cultured cells or in vivo infected animals. HA and NA forming the outer spikes on the viral envelope possess apical sorting signals and use exocytic pathways and lipid rafts for cell surface transport and apical sorting. NP also has apical determinant(s) and is probably transported to the apical budding site similarly via lipid rafts and/or through cortical actin microfilaments. M1 binds the NP and the exposed RNAs of vRNPs, as well as to the cytoplasmic tails (CT) and transmembrane (TM) domains of HA, NA and M2, and is likely brought to the budding site on the piggy-back of vRNP and transmembrane proteins.Budding processes involve bud initiation, bud growth and bud release. The presence of lipid rafts and assembly of viral components at the budding site can cause asymmetry of lipid bilayers and outward membrane bending leading to bud initiation and bud growth. Bud release requires fusion of the apposing viral and cellular membranes and scission of the virus buds from the infected cellular membrane. The processes involved in bud initiation, bud growth and bud scission/release require involvement both viral and host components and can affect bud closing and virus release in both positive and negative ways. Among the viral components, M1, M2 and NA play important roles in bud release and M1, M2 and NA mutations all affect the morphology of buds and released viruses. Disassembly of host cortical actin microfilaments at the pinching-off site appears to facilitate bud fission and release. Bud scission is energy dependent and only a small fraction of virus buds present on the cell surface is released. Discontinuity of M1 layer underneath the lipid bilayer, absence of outer membrane spikes, absence of lipid rafts in the lipid bilayer, as well as possible presence of M2 and disassembly of cortical actin microfilaments at the pinching-off site appear to facilitate bud fission and bud release. We provide our current understanding of these important processes leading to the production of infectious influenza virus particles.     

HIV-1 matrix protein: a mysterious regulator of the viral life cycle

Significant progress has been achieved in the last few years concerning the human immunodeficiency virus (HIV-1) life cycle, mostly in the fields of cellular receptors for the virus, virus assembly and budding of virus particles from the cell surface. Meanwhile, some aspects, such as postentry events, virus maturation and the regulatory role of individual viral proteins remain poorly defined. This review summarizes some recent findings concerning the role of Gag Pr55 and its proteolytic processing in the HIV-1 life cycle with particular emphasis on the functions of matrix protein p17 (MA), the protein which plays a key role in regulation of the early and late steps of viral morphogenesis. Based on our recent observations, the possibility is discussed that two subsets of MA exist, one cleaved from the Gag precursor in the host cell (cMA), and the other cleaved in the virions (vMA). It is suggested that two MA fractions possess diverse functions and are involved in different stages of virus morphogenesis as key regulators of the viral life cycle.     

Assembly of five bluetongue virus proteins expressed by recombinant baculoviruses: Inclusion of the largest protein VP1 in the core and virus-like particles

Bluetongue virus (BTV) VP1 protein, a component of the viral RNA-directed RNA polymerase, but not the VP4 or VP6 proteins, was specifically incorporated into baculovirus expressed BTV core-like particles (composed of VP3 and VP7) and BTV virus-like particles (composed of VP2, VP3, VP5, and VP7). The VP1 protein has been shown to be associated with subcore particles composed of VP3. The data suggest that the VP1 protein of BTV has both enzymatic and structural roles in the virus life cycle.