A small fragmented P protein of respiratory syncytial virus inhibits virus infection by targeting P protein
Koyu HaraKenichiro YaitaPattara KhamrinKattareeya KumthipTakahito KashiwagiJean‐François EléouëtMarie‐Anne Rameix‐WeltiHiroshi Watanabe
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Abstract:
Peptide-based inhibitors hold promising potential in the development of antiviral therapy. Here, we investigated the antiviral potential of fragmented viral proteins derived from ribonucleoprotein (RNP) components of the human respiratory syncytial virus (HRSV). Based on a mimicking approach that targets the functional domains of viral proteins, we designed various fragments of nucleoprotein (N), matrix protein M2-1 and phosphoprotein (P) and tested the antiviral activity in an RSV mini-genome system. We found that the fragment comprising residues 130-180 and 212-241 in the C-terminal region of P (81 amino acid length), denoted as P Fr, significantly inhibited the polymerase activity through competitive binding to the full-length P. Further deletion analysis of P Fr suggested that three functional domains in P Fr (oligomerization, L-binding and nucleocapsid binding) are required for maximum inhibitory activity. More importantly, a purified recombinant P Fr displayed significant antiviral activity at low nanomolar range in RSV-infected HEp-2 cells. These results highlight P as an important target for the development of antiviral compounds against RSV and other paramyxoviruses.Keywords:
Nucleoprotein
Phosphoprotein
Mononegavirales
The matrix protein (M) of respiratory syncytial virus (RSV), the prototype viral member of the Pneumovirinae (family Paramyxoviridae, order Mononegavirales), has been crystallized and the structure determined to a resolution of 1.6 A. The structure comprises 2 compact beta-rich domains connected by a relatively unstructured linker region. Due to the high degree of side-chain order in the structure, an extensive contiguous area of positive surface charge covering approximately 600 A(2) can be resolved. This unusually large patch of positive surface potential spans both domains and the linker, and provides a mechanism for driving the interaction of the protein with a negatively-charged membrane surface or other virion components such as the nucleocapsid. This patch is complemented by regions of high hydrophobicity and a striking planar arrangement of tyrosine residues encircling the C-terminal domain. Comparison of the RSV M sequence with other members of the Pneumovirinae shows that regions of divergence correspond to surface exposed loops in the M structure, with the majority of viral species-specific differences occurring in the N-terminal domain.
Mononegavirales
Pneumovirinae
Linker
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Mononegavirales
VP40
Pneumovirus
Pneumovirinae
RNA virus
Protein primary structure
Matrix (chemical analysis)
Conserved sequence
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ABSTRACT The first nested reverse transcription (RT)-PCR based on the nucleoprotein gene (n RT-PCR-N) of the bovine respiratory syncytial virus (BRSV) has been developed and optimized for the detection of BRSV in bronchoalveolar lavage fluid cells of calves. This test is characterized by a low threshold of detection (0.17 PFU/ml), which is 506 times lower than that obtained by an enzyme immunosorbent assay (EIA) test (RSV TESTPACK ABBOTT). During an experimental infection of 17 immunocompetent calves less than 3 months old, BRSV RNA could be detected up to 13 days after the onset of symptoms whereas isolation in cell culture was possible only up to 5 days. Compiling results obtained by conventional techniques (serology, antigen detection, and culture isolation) for 132 field samples collected from calves with acute respiratory signs revealed that n RT-PCR-N showed the highest diagnostic sensitivity and very good specificity. This n RT-PCR-N with its long period of detection during BRSV infection thus provides a valuable tool for diagnostic and epidemiological purposes.
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Mononegavirales
Pneumovirinae
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A recombinant baculovirus expressing the nucleocapsid gene (NP) of Newcastle disease virus (NDV), a member of the genus Rubulavirus, has been generated and shown to express the native protein to high levels in insect cells. In contrast to the NP protein of the rubulavirus human parainfluenza virus 2, the NDV protein has been demonstrated by electron microscopy and caesium chloride gradient analysis to be capable of self-assembly in vivo to form nucleocapsid-like structures in the absence of other NDV proteins. These structures, which contained RNA that was resistant to micrococcal nuclease digestion, were also observed when the protein was expressed in E. coli, a phenomenon which was not inhibited by the presence of a 40 amino acid fusion region at the amino terminus of the protein. Further, the formation of these structures was inhibited by the co-expression of the phosphoprotein (P). Therefore, we conclude that the P protein acts as a chaperone, preventing uncontrolled encapsidation of non-viral RNA by NP protein.
Phosphoprotein
Newcastle Disease
Mononegavirales
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Micrococcal nuclease
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Morbillivirus infections have been known for a long time to be associated with an acute immunosuppression in their natural hosts. Here, we show that recombinant Morbillivirus nucleoproteins from canine distemper virus, peste-des-petits-ruminants virus, and Rinderpest virus bind B-lymphocytes from dogs, goats, and cattle, respectively, similarly to measles virus nucleoprotein in humans. The use of surface plasmon resonance imaging allowed the real time detection of differential interactions between Morbillivirus nucleoproteins and FcγRIIb (CD32). Moreover, those nucleoproteins which bind murine Fcγ receptor inhibited the inflammatory immune responses in mice in a Fc receptor– dependent manner. In contrast, nucleoprotein from closely related Henipavirus genus, belonging to the Paramyxoviridae family as Morbillivirus, was devoid of capacity either to bind FcγRIIb or to inhibit inflammatory response. Altogether, these results suggest that nucleoprotein-FcR interaction is a common mechanism used by different Morbilliviruses to modulate the immune response.
Morbillivirus
Nucleoprotein
Rinderpest virus
Mononegavirales
Canine distemper
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All paramyxoviruses, which include the mumps virus, measles virus, Nipah virus, Newcastle disease virus, and Sendai virus, have non-segmented single-stranded negative-sense RNA genomes. These RNA genomes are enwrapped throughout the viral life cycle by nucleoproteins, forming helical nucleocapsids. In addition to these helical structures, recombinant paramyxovirus nucleocapsids may occur in other assembly forms such as rings, clam-shaped structures, and double-headed nucleocapsids; the latter two are composed of two single-stranded helices packed in a back-to-back pattern. In all of these assemblies, the neighboring nucleoprotein protomers adopt the same domain-swapping mode via the N-terminal arm, C-terminal arm, and recently disclosed N-hole. An intrinsically disordered region in the C-terminal domain of the nucleoproteins, called the N-tail, plays an unexpected role in regulating the transition among the different assembly forms that occurs with other viral proteins, especially phosphoprotein. These structures, together with the helical nucleocapsids, significantly enrich the structural diversity of the paramyxovirus nucleocapsids and help explain the functions of these diverse assemblies, including RNA genome protection, transcription, and replication, as well as encapsulation.
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Sendai virus
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Serological relationships among measles virus (MV), canine distemper virus (CDV), and rinderpest virus (RV), which constitute morbillivirus subgroup of paramyxoviridae, were investigated by immunoprecipitation and SDSpolyacrylamlde gel electrophoresis for their major structural proteins, i.e., hemagglutinin (H), nucleocapsid (NC), fusion (F), and matrix (M) proteins. The molecular weights of the four structural proteins of MV and CDV were confirmed to correspond to those previously reported by several investigators. Structural proteins of RV were analyzed for the first time in the present study and found to have molecular weights of 74, 000, 62, 000, 44, 000, and 40, 000 for H, NC, F, and M proteins, respectively. By labeling with glucosamine, the presence of carbohydrate moiety was found in H protein for all the three viruses and in F protein of CDV. The serums from the convalescent animals infected with respective virus disclosed one-way cross pattern depending on the combinations of virus and antiserums, but failed to show the reciprocal cross reactivity. On the other hand, hyperimmune serums to respective virus showed the reciprocal cross-reactivity with the four structural proteins indicating that each of the major structural proteins possesses the antigen common to all three morbilliviruses.
Rinderpest virus
Canine distemper
Morbillivirus
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Cross-reactivity
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Paramyxoviruses are a family of negative sense RNA viruses whose members cause serious diseases in humans, such as measles virus, mumps virus and respiratory syncytial virus; and in animals, such as Newcastle disease virus and rinderpest virus. Paramyxovirus particles form by assembly of the viral matrix protein, the ribonucleoprotein complex and the surface glycoproteins at the plasma membrane of infected cells and subsequent viral budding. Two major glycoproteins expressed on the viral envelope, the attachment protein and the fusion protein, promote attachment of the virus to host cells and subsequent virus-cell membrane fusion. Incorporation of the surface glycoproteins into infectious progeny particles requires coordinated interplay between the three viral structural components, driven primarily by the matrix protein. In this review, we discuss recent progress in understanding the contributions of the matrix protein and glycoproteins in driving paramyxovirus assembly and budding while focusing on the viral protein interactions underlying this process and the intracellular trafficking pathways for targeting viral components to assembly sites. Differences in the mechanisms of particle production among the different family members will be highlighted throughout.
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Mononegavirales
Budding
Mumps virus
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Newcastle disease virus (NDV) is an enveloped paramyxovirus. The matrix protein of the virus (M-NDV) has an innate propensity to produce virus-like particles budding from the plasma membrane of the expressing cell without recruiting other viral proteins. The virus predominantly infects the host cell via fusion with the host plasma membrane or, alternatively, can use receptor-mediated endocytic pathways. The question arises as to what are the mechanisms supporting such diversity, especially concerning the assembling and membrane binding properties of the virus protein scaffold under both neutral and acidic pH conditions. Here, we suggest a novel method of M-NDV isolation in physiological ionic strength and employ a combination of small-angle X-ray scattering, atomic force microscopy with complementary structural techniques, and membrane interaction measurements to characterize the solution behavior/structure of the protein as well as its binding to lipid membranes at pH 4.0 and pH 7.0. We demonstrate that the minimal structural unit of the protein in solution is a dimer that spontaneously assembles in a neutral milieu into hollow helical oligomers by repeating the protein tetramers. Acidic pH conditions decrease the protein oligomerization state to the individual dimers, tetramers, and octamers without changing the density of the protein layer and lipid membrane affinity, thus indicating that the endocytic pathway is a possible facilitator of NDV entry into a host cell through enhanced scaffold disintegration.
VP40
Mononegavirales
Newcastle Disease
Pneumovirinae
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