Requirement of Cysteines and Length of the Human Respiratory Syncytial Virus M2-1 Protein for Protein Function and Virus Viability

Tang, Roderick S.; Nguyen, Nick; Cheng, Xing; Ji, Hong

doi:10.1128/jvi.75.23.11328-11335.2001

Cited by 38 publications

(56 citation statements)

References 30 publications

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“…M2-1 appears to be an essential protein, and deletion of the complete gene has not been possible. However, it was possible to recover virus in which up to 67 amino acids were deleted from the C-terminus of M2-1 (29). These viruses were attenuated in rodents.…”

Section: Identifying Mutations That Attenuate Rsvmentioning

confidence: 99%

New Generation Live Vaccines against Human Respiratory Syncytial Virus Designed by Reverse Genetics

Collins

Murphy²

2005

Proceedings of the American Thoracic Society

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Development of a live pediatric vaccine against human respiratory syncytial virus (RSV) is complicated by the need to immunize young infants and the difficulty in balancing attenuation and immunogenicity. The ability to introduce desired mutations into infectious virus by reverse genetics provides a method for identifying and designing highly defined attenuating mutations. These can be introduced in combinations as desired to achieve gradations of attenuation. Attenuation is based on several strategies: multiple independent temperature-sensitive point mutations in the polymerase, a temperature-sensitive point mutation in a transcription signal, a set of non-temperature-sensitive mutations involving several genes, deletion of a viral RNA synthesis regulatory protein, and deletion of viral IFN ␣/␤ antagonists. The genetic stability of the live vaccine can be increased by judicious choice of mutations. The virus also can be engineered to increase the level of expression of the protective antigens. Protective antigens from antigenically distinct RSV strains can be added or swapped to increase the breadth of coverage. Alternatively, the major RSV protective antigens can be expressed from transcription units added to an attenuated parainfluenza vaccine virus, making a bivalent vaccine. This would obviate the difficulties inherent in the fragility and inefficient in vitro growth of RSV, simplifying vaccine design and use.

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Section: Identifying Mutations That Attenuate Rsvmentioning

confidence: 99%

New Generation Live Vaccines against Human Respiratory Syncytial Virus Designed by Reverse Genetics

Collins

Murphy²

2005

Proceedings of the American Thoracic Society

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“…The HRSV M2-1 protein also has been shown to be an RNA-binding protein and binds to the N protein (9,15). Expression of the HRSV M2-1 protein, either from an added support plasmid or by promiscuous expression from the antigenome cDNA, appears to be essential for the recovery of recombinant HRSV from transfected cDNA, and it has not been possible to recover infectious HRSV in which the integrity of the M2-1 protein has been drastically disturbed by introduced mutations or by extensive deletion (10,12,28,34).…”

mentioning

confidence: 99%

Deletion of M2 Gene Open Reading Frames 1 and 2 of Human Metapneumovirus: Effects on RNA Synthesis, Attenuation, and Immunogenicity

Buchholz

Biacchesi

Pham

et al. 2005

J Virol

128

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The M2 gene of human metapneumovirus (HMPV) contains two overlapping open reading frames (ORFs), M2-1 and M2-2. The expression of separate M2-1 and M2-2 proteins from these ORFs was confirmed, and recombinant HMPVs were recovered in which expression of M2-1 and M2-2 was ablated individually or together [r⌬M2-1, r⌬M2-2, and r⌬M2(1؉2)]. Each M2 mutant virus directed efficient multicycle growth in Vero cells. The ability to recover HMPV lacking M2-1 contrasts with human respiratory syncytial virus, for which M2-1 is an essential transcription factor. Expression of the downstream HMPV M2-2 ORF was not reduced when translation of the upstream M2-1 ORF was silenced, indicating that it is initiated separately. The r⌬M2-2 mutants exhibited a two-to fivefold increase in the accumulation of mRNA, normalized to the genome template, suggesting that M2-2 has a role in regulating RNA synthesis. Replication and immunogenicity were tested in hamsters. Animals infected intranasally with r⌬M2-1 or r⌬M2(1؉2) did not have recoverable virus in the lungs or nasal turbinates on days 3 or 5 postinfection and did not develop HMPV-neutralizing serum antibodies or resistance to HMPV challenge. Thus, M2-1 appears to be essential for significant virus replication in vivo. In animals infected with r⌬M2-2, virus was recovered from only 1 of 12 animals and only in the nasal turbinates on a single day. However, all of the animals developed a high titer of HMPV-neutralizing serum antibodies and were highly protected against challenge with wild-type HMPV. The HMPV r⌬M2-2 virus is a promising and highly attenuated HMPV vaccine candidate.Human metapneumovirus (HMPV) was first identified in 2001 in The Netherlands from infants and children with acute respiratory tract disease (38) and is now recognized to be worldwide in prevalence (22,41). HMPV resembles human respiratory syncytial virus (HRSV) with regard to disease signs and the ability to infect and cause disease in infants as well as in individuals of all ages (7,18,20,29,32,39,41). The contribution of HMPV to respiratory tract disease remains to be fully defined but appears to be sufficient to warrant the development of a vaccine, especially for the pediatric population. Reverse genetic systems were recently developed for HMPV, allowing the generation of infectious virus from cDNA and providing an important tool for characterizing HMPV biology and for designing live-attenuated HMPV vaccines (5, 25).HMPV has a negative-strand RNA genome of approximately 13 kb (4, 37). It has been classified, together with avian metapneumovirus, in the Metapneumovirus genus, Pneumovirus subfamily, Paramyxovirus family, of the order Mononegavirales. The Pneumovirus subfamily also contains the genus Pneumovirus, represented by HRSV. The Metapneumovirus gene order is N-P-M-F-M2-SH-G-L. By analogy to HRSV, the predicted HMPV proteins are the following: the nucleocapsid protein N, which encapsidates the RNA genome and, together with the phosphoprotein P and the RNA polymerase protein L, forms the ribonucleoprotein co...

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“…By analogy, a homologue of VP30, the M2-1 protein of human respiratory syncytial virus (hRSV), has a Cys 3 -His motif that binds hRSV leader RNA with an apparent dissociation constant of 90 nM (8,14). Mutation of three cysteine residues of the Cys 3 -His motif led to a significant reduction of hRSV recovery (39).…”

mentioning

confidence: 99%

Ebola Virus VP30 Is an RNA Binding Protein

John

Wang

Steffen

et al. 2007

J Virol

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The Ebola virus (EBOV) genome encodes for several proteins that are necessary and sufficient for replication and transcription of the viral RNAs in vitro; NP, VP30, VP35, and L. VP30 acts in trans with an RNA secondary structure upstream of the first transcriptional start site to modulate transcription. Using a bioinformatics approach, we identified a region within the N terminus of VP30 with sequence features that typify intrinsically disordered regions and a putative RNA binding site. To experimentally assess the ability of VP30 to directly interact with the viral RNA, we purified recombinant EBOV VP30 to >90% homogeneity and assessed RNA binding by UV cross-linking and filter-binding assays. VP30 is a strongly acidophilic protein; RNA binding became stronger as pH was decreased. Zn Ebola viruses (EBOV) are nonsegmented, negative-stranded RNA viruses, which together with Marburg virus, constitute the family Filoviridae (18). Filoviruses cause severe and lethal hemorrhagic fevers in humans and nonhuman primates and, as such, are classified as biosafety level 4 (BSL-4) agents (11). Of the eight proteins encoded by the EBOV genome, the L protein, VP35, the N protein (NP), and VP30 are proposed to form a ribonucleocapsid complex, along with the genomic RNA (10, 28). The L protein bears the enzymatic activities required for transcription and replication, whereas VP30 and VP35 are implicated in regulation of transcription and replication (28,41). Although the NP is necessary for transcription, its role is still unknown (28). Phosphorylation of VP30 positively regulates its binding to NP and negatively regulates its transcriptional activity (26). In addition, enhancement of transcription by VP30 requires a putative RNA secondary structure located within nucleotides (nt) 54 to 80 of the leader region (44). Deletion of the predicted RNA secondary structure permits VP30-independent transcription of viral messengers (44). These reported activities of VP30 suggest the possibility of a direct interaction of VP30 with EBOV RNA in its role in transcription. In addition, the N terminus of VP30 contains a Zn 2ϩ binding Cys 3 -His motif (25) and is rich in basic amino acids (34), which can interact with nucleic acid, and provided impetus for our studies here. By analogy, a homologue of VP30, the M2-1 protein of human respiratory syncytial virus (hRSV), has a Cys 3 -His motif that binds hRSV leader RNA with an apparent dissociation constant of 90 nM (8,14). Mutation of three cysteine residues of the Cys 3 -His motif led to a significant reduction of hRSV recovery (39).Recent publications with the minigenome system for EBOV suggest at least two possible mechanisms that VP30 may use in its transcriptional regulatory role. One possible mechanism could be that VP30 interacts with one or more of the other nucleocapsid proteins, polymerase, NP or VP35, and promotes increased stability of the transcriptional complex. An increase in the number of active transcription initiation complexes or the increased stability of the transcriptional...

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Requirement of Cysteines and Length of the Human Respiratory Syncytial Virus M2-1 Protein for Protein Function and Virus Viability

Cited by 38 publications

References 30 publications

New Generation Live Vaccines against Human Respiratory Syncytial Virus Designed by Reverse Genetics

New Generation Live Vaccines against Human Respiratory Syncytial Virus Designed by Reverse Genetics

Deletion of M2 Gene Open Reading Frames 1 and 2 of Human Metapneumovirus: Effects on RNA Synthesis, Attenuation, and Immunogenicity

Ebola Virus VP30 Is an RNA Binding Protein

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