The nucleocapsid protein (NP) of Newcastle disease virus expressed in E. coli assembled as ring- and herringbone-like particles. In order to identify the contiguous NP sequence essential for assembly of these particles, 11 N- or C-terminally deleted NP mutants were constructed and their ability to self-assemble was tested. The results indicate that a large part of the NP N-terminal end, encompassing amino acids 1 to 375, is required for proper folding to form a herringbone-like structure. In contrast, the C-terminal end covering amino acids 376 to 489 was dispensable for the formation of herringbone-like particles. A region located between amino acids 375 to 439 may play a role in regulating the length of the herringbone-like particles. Mutants with amino acid deletions further from the C-terminal end (84, 98, 109 and 114 amino acids) tended to form longer particles compared to mutants with shorter deletions (25 and 49 amino acids).
Encapsidation by nucleocapsid (N) protein is crucial for viral RNA to serve as a functional template for virus replication. However, the potential region that is vital for RNA encapsidation of Nipah virus (NiV) is still unknown. Thus, this study was aimed to identify these regions using a NiV minireplicon system. A series of broad range internal deletion mutations was generated in the 5' non-translated region (NTR) of the N gene mRNA region of NiV leader promoter via site-directed overlapping PCR-mediated mutagenesis. The mutation effects on synthesis and encapsidation of antigenome RNA, transcription, and RNA binding affinity of N protein were evaluated. The deletions of nucleotides 73-108, 79-108, and 85-108 from NiV leader promoter inhibited the encapsidation of antigenome RNA, while the deletion of nucleotides 103-108 suppressed the synthesis and encapsidation of antigenome RNA, implying that these regions are required for genome replication. Surprisingly, none of the mutations had detrimental effect on viral transcription. Using isothermal titration calorimetry, the binding of NiV N protein to genome or antigenome RNA transcript lacking of nucleotides 73-108 was found to be suppressed. Additionally, in silico analysis on secondary structure of genome RNA further supported the plausible cause of inefficient encapsidation of antigenome RNA by the loss of encapsidation signal in genome template. In conclusion, this study suggests that the nucleotides 73-90 within 5' NTR of the N gene mRNA region in NiV leader promoter contain cis-acting RNA element that is important for efficient encapsidation of antigenome RNA.
The long surface antigen (L-HBsAg) of hepatitis B virus (HBV) plays a central role in the production of infectious virions. During HBV morphogenesis, both the PreS and S domains of L-HBsAg form docking sites for the viral nucleocapsids. Thus, a compound that disrupts the interaction between the L-HBsAg and nucleocapsids could serve as a therapeutic agent against the virus based upon inhibition of morphogenesis. Synthetic peptides correspond to the binding sites in L-HBsAg inhibited the association of L-HBsAg with core antigen (HBcAg). A synthetic peptide carrying the epitope for a monoclonal antibody to the PreS1 domain competed weakly with L-HBsAg for HBcAg, but peptides corresponding to a linear sequence at the tip of the nucleocapsid spike did not, showing that the competing peptide does not resemble the tip of the spike.
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.
White tail disease (WTD) kills prawn larvae and causes drastic losses to the freshwater prawn (Macrobrachium rosenbergii) industry. The main causative agent of WTD is Macrobrachium rosenbergii nodavirus (MrNV). The N-terminal end of the MrNV capsid protein is very rich in positively charged amino acids and is postulated to interact with RNA molecules. N-terminal and internal deletion mutagenesis revealed that the RNA-binding region is located at positions 20-29, where 80 % of amino acids are positively charged. Substitution of all these positively charged residues with alanine abolished the RNA binding. Mutants without the RNA-binding region still assembled into virus-like particles, suggesting that this region is not a part of the capsid assembly domain. This paper is, to the best of our knowledge, the first to report the specific RNA-binding region of MrNV capsid protein.
Nipah virus (NiV) causes fatal respiratory illness and encephalitis in humans and animals. The matrix (M) protein of NiV plays an important role in the viral assembly and budding process. Thus, an access to the NiV M protein is vital to the design of viral antigens as diagnostic reagents. In this study, recombinant DNA technology was successfully adopted in the cloning and expression of NiV M protein. A recombinant expression cassette (baculovirus expression vector) was used to encode an N-terminally His-tagged NiV M protein in insect cells. A time-course study demonstrated that the highest yield of recombinant M protein (400-500 μg) was expressed from 107 infected cells 3 days after infection. A single-step purification method based on metal ion affinity chromatography was established to purify the NiV M protein, which successfully yielded a purity level of 95.67% and a purification factor of 3.39. The Western blotting and enzyme-linked immunosorbent assay (ELISA) showed that the purified recombinant M protein (48 kDa) was antigenic and reacted strongly with the serum of a NiV infected pig.
Annexin A2 is a membrane scaffolding and binding protein, which mediated various cellular events. Its functions are generally affected by cellular localization. In the cytoplasm, they interacted with different phospholipid membranes in Ca2+ -dependent manner and play vital roles including actin binding, remodeling and dynamics, cytoskeletal rearrangement, and lipid-raft microdomain formation. However, upon cell exposure to certain stimuli, annexin A2 translocates to the external leaflets of the plasma membrane where annexin A2 was recently reported to serve as a virus receptor, play an important role in the formation of virus replication complex, or implicated in virus assembly and budding. Here, we review some of annexin A2 roles in virus infections and the potentiality of targeting annexin A2 in the design of novel and promising antivirus agent that may have a broader consequence in virus therapy.
Certain proteins have demonstrated proficient human immunodeficiency virus (HIV-1) life cycle disturbance. Recently, the ankyrin repeat protein targeting the HIV-1 capsid, AnkGAG1D4, showed a negative effect on the viral assembly of the HIV-1NL4-3 laboratory strain. To extend its potential for future clinical application, the activity of AnkGAG1D4 in the inhibition of other HIV-1 circulating strains was evaluated. Chimeric NL4-3 viruses carrying patient-derived Gag/PR-coding regions were generated from 131 antiretroviral drug-naïve HIV-1 infected individuals in northern Thailand during 2001⁻2012. SupT1, a stable T-cell line expressing AnkGAG1D4 and ankyrin non-binding control (AnkA32D3), were challenged with these chimeric viruses. The p24CA sequences were analysed and classified using the K-means clustering method. Among all the classes of virus classified using the p24CA sequences, SupT1/AnkGAG1D4 demonstrated significantly lower levels of p24CA than SupT1/AnkA32D3, which was found to correlate with the syncytia formation. This result suggests that AnkGAG1D4 can significantly interfere with the chimeric viruses derived from patients with different sequences of the p24CA domain. It supports the possibility of ankyrin-based therapy as a broad alternative therapeutic molecule for HIV-1 gene therapy in the future.
Tioman virus (TioPV) and Menangle virus (MenPV) are two antigenically and genetically related paramyxoviruses (genus: Rubulavirus, family: Paramyxoviridae) isolated from Peninsular Malaysia (2001) and Australia (1997), respectively. Both viruses are potential zoonotic agents. In the present study, the infectivity, growth kinetics, morphology and morphogenesis of these two paramyxoviruses in a human neuronal cell (SK-N-SH) line were investigated. Sub-confluent SK-N-SH cells were infected with TioPV and MenPV at similar multiplicity of infection. These cells were examined by conventional and immunoelectron microscopy, and virus titres in the supernatants were assayed. Syncytia were observed for both infections in SK-N-SH cells and were more pronounced during the early stages of TioPV infection. The TioPV titre increased consistently (10(1)) every 12 h after infection. In MenPV-infected cells, cellular material was frequently observed within budding virions, and microfilaments and microtubules were abundant. Viral budding was common, and extracellular MenPVs tended to be more pleomorphic compared to TioPVs, which appeared to be more spherical in appearance. The MenPV cytoplasmic viral inclusion appeared to be comparatively smaller, loose and interspersed with randomly scattered circle-like particles, whereas huge tubule-like cytoplasmic inclusions were observed in TioPV-infected cells. Both viruses also displayed different cellular pathology in the SK-N-SH cells. The intracellular ultrastructural characteristics of these two viruses in infected neuronal cells may allow them to be differentiated by electron microscopy.
Nipah virus, which was first recognized during an outbreak of encephalitis with high mortality in Peninsular Malaysia during 1998-1999, is most closely related to Hendra virus, another emergent paramyxovirus first recognized in Australia in 1994. We have studied the morphologic features of Nipah virus in infected Vero E6 cells and human brain by using standard and immunogold electron microscopy and ultrastructural in situ hybridization. Nipah virions are enveloped particles composed of a tangle of filamentous nucleocapsids and measured as large as 1900 nm in diameter. The nucleocapsids measured up to 1.67 microm in length and had the herringbone structure characteristic for paramyxoviruses. Cellular infection was associated with multinucleation, intracytoplasmic nucleocapsid inclusions (NCIs), and long cytoplasmic tubules. Previously undescribed for other members of the family Paramyxoviridae, infected cells also contained an inclusion formed of reticular structures. Ultrastructural ISH studies suggest these inclusions play an important role in the transcription process.
Macrobrachium rosenbergii nodavirus (MrNV) is a pathogen of freshwater prawns that poses a threat to food security and causes significant economic losses in the aquaculture industries of many developing nations. A detailed understanding of the MrNV virion structure will inform the development of strategies to control outbreaks. The MrNV capsid has also been engineered to display heterologous antigens, and thus knowledge of its atomic resolution structure will benefit efforts to develop tools based on this platform. Here, we present an atomic-resolution model of the MrNV capsid protein (CP), calculated by cryogenic electron microscopy (cryoEM) of MrNV virus-like particles (VLPs) produced in insect cells, and three-dimensional (3D) image reconstruction at 3.3 Å resolution. CryoEM of MrNV virions purified from infected freshwater prawn post-larvae yielded a 6.6 Å resolution structure, confirming the biological relevance of the VLP structure. Our data revealed that unlike other known nodavirus structures, which have been shown to assemble capsids having trimeric spikes, MrNV assembles a T = 3 capsid with dimeric spikes. We also found a number of surprising similarities between the MrNV capsid structure and that of the Tombusviridae: 1) an extensive network of N-terminal arms (NTAs) lines the capsid interior, forming long-range interactions to lace together asymmetric units; 2) the capsid shell is stabilised by 3 pairs of Ca2+ ions in each asymmetric unit; 3) the protruding spike domain exhibits a very similar fold to that seen in the spikes of the tombusviruses. These structural similarities raise questions concerning the taxonomic classification of MrNV.
The nucleocapsid (NP) protein of Newcastle disease virus (NDV) self-assembled in Escherichia coli as ring-like and herringbone-like particles. Several chimeric NP proteins were constructed in which the antigenic regions of the hemagglutinin-neuraminidase (HN) and fusion (F) proteins of NDV, myc epitope, and six histidines (a hexa-His tag) were linked to the C-terminus of the NP monomer. These chimeric proteins were expressed efficiently in soluble form in E. coli as detected by Western blot analysis. Electron microscopy of the purified products revealed that they self-assembled into ring-like particles. These chimeric particles exhibited antigenicity of the myc epitope, suggesting that the foreign sequences were exposed on the surface of the particles. Chickens inoculated with the chimeric particles mounted an immune response against NDV, suggesting the possibility of use of the ring-like particle as a carrier of immunogens in subunit vaccines and immunological reagents.