Zinc Binding Activity of Human Metapneumovirus M2-1 Protein Is Indispensable for Viral Replication and PathogenesisIn Vivo

ABSTRACTHuman metapneumovirus (hMPV) is a member of thePneumovirinaesubfamily in theParamyxoviridaefamily that causes respiratory tract infections in humans. Unlike members of theParamyxovirinaesubfamily, the polymerase complex of pneumoviruses requires an additional cofactor, the M2-1 protein, which functions as a transcriptional antitermination factor. The M2-1 protein was found to incorporate zinc ions, although the specific role(s) of the zinc binding activity in viral replication and pathogenesis remains unknown. In this study, we found that the third cysteine (C21) and the last histidine (H25) in the zinc binding motif (CCCH) of hMPV M2-1 were essential for zinc binding activity, whereas the first two cysteines (C7 and C15) play only minor or redundant roles in zinc binding. In addition, the zinc binding motif is essential for the oligomerization of M2-1. Subsequently, recombinant hMPVs (rhMPVs) carrying mutations in the zinc binding motif were recovered. Interestingly, rhMPV-C21S and -H25L mutants, which lacked zinc binding activity, had delayed replication in cell culture and were highly attenuated in cotton rats. In contrast, rhMPV-C7S and -C15S strains, which retained 60% of the zinc binding activity, replicated as efficiently as rhMPV in cotton rats. Importantly, rhMPVs that lacked zinc binding activity triggered high levels of neutralizing antibody and provided complete protection against challenge with rhMPV. Taken together, these results demonstrate that zinc binding activity is indispensable for viral replication and pathogenesisin vivo. These results also suggest that inhibition of zinc binding activity may serve as a novel approach to rationally attenuate hMPV and perhaps other pneumoviruses for vaccine purposes.IMPORTANCEThe pneumoviruses include many important human and animal pathogens, such as human respiratory syncytial virus (hRSV), hMPV, bovine RSV, and avian metapneumovirus (aMPV). Among these viruses, hRSV and hMPV are the leading causes of acute respiratory tract infection in infants and children. Despite major efforts, there is no antiviral or vaccine to combat these diseases. All known pneumoviruses encode a zinc binding protein, M2-1, which is a transcriptional antitermination factor. In this work, we found that the zinc binding activity of M2-1 is essential for virus replication and pathogenesisin vivo. Recombinant hMPVs that lacked zinc binding activity were not only defective in replication in the upper and lower respiratory tract but also triggered a strong protective immunity in cotton rats. Thus, inhibition of M2-1 zinc binding activity can lead to the development of novel, live attenuated vaccines, as well as antiviral drugs for pneumoviruses.

Download Full-text

Phosphorylation of Human Metapneumovirus M2-1 Protein Upregulates Viral Replication and Pathogenesis

Journal of Virology ◽

10.1128/jvi.00755-16 ◽

2016 ◽

Vol 90 (16) ◽

pp. 7323-7338 ◽

Cited By ~ 6

Author(s):

Hui Cai ◽

Yu Zhang ◽

Mijia Lu ◽

Xueya Liang ◽

Ryan Jennings ◽

...

Keyword(s):

Respiratory Tract ◽

Viral Replication ◽

Lower Respiratory Tract ◽

Rna Synthesis ◽

Binding Activity ◽

Human Metapneumovirus ◽

Zinc Binding ◽

Recombinant Viruses ◽

Cotton Rats

ABSTRACTHuman metapneumovirus (hMPV) is a major causative agent of upper- and lower-respiratory-tract infections in infants, the elderly, and immunocompromised individuals worldwide. Like all pneumoviruses, hMPV encodes the zinc binding protein M2-1, which plays important regulatory roles in RNA synthesis. The M2-1 protein is phosphorylated, but the specific role(s) of the phosphorylation in viral replication and pathogenesis remains unknown. In this study, we found that hMPV M2-1 is phosphorylated at amino acid residues S57 and S60. Subsequent mutagenesis found that phosphorylation is not essential for zinc binding activity and oligomerization, whereas inhibition of zinc binding activity abolished the phosphorylation and oligomerization of the M2-1 protein. Using a reverse genetics system, recombinant hMPVs (rhMPVs) lacking either one or both phosphorylation sites in the M2-1 protein were recovered. These recombinant viruses had a significant decrease in both genomic RNA replication and mRNA transcription. In addition, these recombinant viruses were highly attenuated in cell culture and cotton rats. Importantly, rhMPVs lacking phosphorylation in the M2-1 protein triggered high levels of neutralizing antibody and provided complete protection against challenge with wild-type hMPV. Collectively, these data demonstrated that phosphorylation of the M2-1 protein upregulates hMPV RNA synthesis, replication, and pathogenesisin vivo.IMPORTANCEThe pneumoviruses include many important human and animal pathogens, such as human respiratory syncytial virus (hRSV), hMPV, bovine RSV, and avian metapneumovirus (aMPV). Among these viruses, hRSV and hMPV are the leading causes of acute respiratory tract infection in infants and children. Currently, there is no antiviral or vaccine to combat these diseases. All known pneumoviruses encode a zinc binding protein, M2-1, which is a transcriptional antitermination factor. In this work, we found that phosphorylation of M2-1 is essential for virus replication and pathogenesisin vivo. Recombinant hMPVs lacking phosphorylation in M2-1 exhibited limited replication in the upper and lower respiratory tract and triggered strong protective immunity in cotton rats. This work highlights the important role of M2-1 phosphorylation in viral replication and that inhibition of M2-1 phosphorylation may serve as a novel approach to develop live attenuated vaccines as well as antiviral drugs for pneumoviruses.

Download Full-text

Synthetic enhancement of a TFIIB defect by a mutation in SSU72, an essential yeast gene encoding a novel protein that affects transcription start site selection in vivo.

Molecular and Cellular Biology ◽

10.1128/mcb.16.4.1557 ◽

1996 ◽

Vol 16 (4) ◽

pp. 1557-1566 ◽

Cited By ~ 58

Author(s):

Z W Sun ◽

M Hampsey

Keyword(s):

Site Selection ◽

Zinc Binding ◽

Binding Motif ◽

Start Site ◽

Transcription Start ◽

Gene Encoding ◽

N Terminus ◽

Zinc Binding Motif ◽

Novel Protein

An ssu72 mutant of Saccharomyces cerevisiae was identified as an enhancer of a TFIIB defect (sua7-1) that confers both a cold-sensitive growth defect and a downstream shift in transcription start site selection. The ssu72-1 allele did not affect cold sensitivity but, in combination with sua7-1, created a heat-sensitive phenotype. Moreover, start site selection at the ADH1 gene was dramatically shifted further downstream of the normal sites. Both of these effects could be rescued by either SUA7 or SSU72, thereby defining a functional relationship between the two genes. SSU72 is a single-copy, essential gene encoding a novel protein of 206 amino acids. The ssu72-1 allele is the result of a 30-bp duplication creating a sequence encoding a Cys-X2-Cys-X6-Cys-X2-Cys zinc binding motif near the N terminus of Ssu72p. Mutational analysis demonstrated that the N terminus of Ssu72p is essential for function and that cysteine residues in both the normal and mutant proteins are critical. We discuss the possibility that the potential zinc binding motif of Ssu72 facilitates assembly of the transcription preinitiation complex and that this effect is important for accurate start site selection in vivo.

Download Full-text

Zn2+Ions Selectively Induce Antimicrobial Salivary Peptide Histatin-5 To Fuse Negatively Charged Vesicles. Identification and Characterization of a Zinc-Binding Motif Present in the Functional Domain†

Biochemistry ◽

10.1021/bi990212c ◽

1999 ◽

Vol 38 (30) ◽

pp. 9626-9633 ◽

Cited By ~ 51

Author(s):

Sonia Melino ◽

Stefano Rufini ◽

Marco Sette ◽

Roberto Morero ◽

Alessandro Grottesi ◽

...

Keyword(s):

Zinc Binding ◽

Binding Motif ◽

Functional Domain ◽

Histatin 5 ◽

Salivary Peptide ◽

Identification And Characterization ◽

Zinc Binding Motif

Download Full-text

Effect of mutations in a zinc-binding domain of yeast RNA polymerase C (III) on enzyme function and subunit association

Molecular and Cellular Biology ◽

10.1128/mcb.12.3.1087-1095.1992 ◽

1992 ◽

Vol 12 (3) ◽

pp. 1087-1095

Author(s):

M Werner ◽

S Hermann-Le Denmat ◽

I Treich ◽

A Sentenac ◽

P Thuriaux

Keyword(s):

Rna Polymerase ◽

Zinc Binding ◽

Enzyme Function ◽

Directed Mutagenesis ◽

Binding Motif ◽

Zinc Ions ◽

Amino Terminal ◽

Zinc Binding Domain ◽

Zinc Binding Motif

The conserved amino-terminal region of the largest subunit of yeast RNA polymerase C is capable of binding zinc ions in vitro. By oligonucleotide-directed mutagenesis, we show that the putative zinc-binding motif CX2CX6-12CXGHXGX24-37CX2C, present in the largest subunit of all eukaryotic and archaebacterial RNA polymerases, is essential for the function of RNA polymerase C. All mutations in the invariant cysteine and histidine residues conferred a lethal phenotype. We also obtained two conditional thermosensitive mutants affecting this region. One of these produced a form of RNA polymerase C which was thermosensitive and unstable in vitro. This instability was correlated with the loss of three of the subunits which are specific to RNA polymerase C: C82, C34, and C31.

Download Full-text

Recombinant subtype A and B human respiratory syncytial virus clinical isolates co-infect the respiratory tract of cotton rats

Journal of General Virology ◽

10.1099/jgv.0.001471 ◽

2020 ◽

Vol 101 (10) ◽

pp. 1056-1068

Author(s):

Linda J. Rennick ◽

Sham Nambulli ◽

Ken Lemon ◽

Grace Y. Olinger ◽

Nicholas A. Crossland ◽

...

Keyword(s):

Respiratory Syncytial Virus ◽

Respiratory Tract ◽

Reverse Genetics ◽

Fluorescent Protein ◽

Human Respiratory Syncytial Virus ◽

Subtype B ◽

Cotton Rats ◽

Syncytial Virus ◽

Subtype A

Human respiratory syncytial virus (HRSV) is an important respiratory pathogen causing a spectrum of illness, from common cold-like symptoms, to bronchiolitis and pneumonia requiring hospitalization in infants, the immunocompromised and the elderly. HRSV exists as two antigenic subtypes, A and B, which typically cycle biannually in separate seasons. There are many unresolved questions in HRSV biology regarding the interactions and interplay of the two subtypes. Therefore, we generated a reverse genetics system for a subtype A HRSV from the 2011 season (A11) to complement our existing subtype B reverse genetics system. We obtained the sequence (HRSVA11) directly from an unpassaged clinical sample and generated the recombinant (r) HRSVA11. A version of the virus expressing enhanced green fluorescent protein (EGFP) from an additional transcription unit in the fifth (5) position of the genome, rHRSVA11EGFP(5), was also generated. rHRSVA11 and rHRSVA11EGFP(5) grew comparably in cell culture. To facilitate animal co-infection studies, we derivatized our subtype B clinical isolate using reverse genetics toexpress the red fluorescent protein (dTom)-expressing rHRSVB05dTom(5). These viruses were then used to study simultaneous in vivo co-infection of the respiratory tract. Following intranasal infection, both rHRSVA11EGFP(5) and rHRSVB05dTom(5) infected cotton rats targeting the same cell populations and demonstrating that co-infection occurs in vivo. The implications of this finding on viral evolution are important since it shows that inter-subtype cooperativity and/or competition is feasible in vivo during the natural course of the infection.

Download Full-text

Phosphonamidates are the first phosphorus-based zinc binding motif to show inhibition of β-class carbonic anhydrases from bacteria, fungi, and protozoa

Journal of Enzyme Inhibition and Medicinal Chemistry ◽

10.1080/14756366.2019.1681987 ◽

2019 ◽

Vol 35 (1) ◽

pp. 59-64 ◽

Cited By ~ 3

Author(s):

Siham A. Alissa ◽

Hanan A. Alghulikah ◽

Zeid A. Alothman ◽

Sameh M. Osman ◽

Sonia Del Prete ◽

...

Keyword(s):

Zinc Binding ◽

Binding Motif ◽

Carbonic Anhydrases ◽

Zinc Binding Motif

Download Full-text

Conformational dynamics and allosteric effect modulated by the unique zinc-binding motif in class IIa HDACs

Physical Chemistry Chemical Physics ◽

10.1039/c9cp02261a ◽

2019 ◽

Vol 21 (23) ◽

pp. 12173-12183 ◽

Cited By ~ 2

Author(s):

Huawei Liu ◽

Fan Zhang ◽

Kai Wang ◽

Xiaowen Tang ◽

Ruibo Wu

Keyword(s):

Histone Deacetylases ◽

Conformational Dynamics ◽

Zinc Binding ◽

Binding Motif ◽

Allosteric Effect ◽

Class Iia Hdacs ◽

Zinc Binding Motif

Class IIa histone deacetylases (HDACs) have been considered as potential targets for the treatment of several diseases.

Download Full-text

Effect of mutations in a zinc-binding domain of yeast RNA polymerase C (III) on enzyme function and subunit association.

Molecular and Cellular Biology ◽

10.1128/mcb.12.3.1087 ◽

1992 ◽

Vol 12 (3) ◽

pp. 1087-1095 ◽

Cited By ~ 40

Author(s):

M Werner ◽

S Hermann-Le Denmat ◽

I Treich ◽

A Sentenac ◽

P Thuriaux

Keyword(s):

Rna Polymerase ◽

Zinc Binding ◽

Enzyme Function ◽

Directed Mutagenesis ◽

Binding Motif ◽

Zinc Ions ◽

Amino Terminal ◽

Zinc Binding Domain ◽

Zinc Binding Motif

Download Full-text

Mutational analysis of the proteolytic domain of pregnancy-associated plasma protein-A (PAPP-A): classification as a metzincin

Biochemical Journal ◽

10.1042/bj3580359 ◽

2001 ◽

Vol 358 (2) ◽

pp. 359-367 ◽

Cited By ~ 70

Author(s):

Henning B. BOLDT ◽

Michael T. OVERGAARD ◽

Lisbeth S. LAURSEN ◽

Kathrin WEYER ◽

Lars SOTTRUP-JENSEN ◽

...

Keyword(s):

Plasma Protein ◽

Active Site ◽

Mammalian Cells ◽

Mutational Analysis ◽

Protein A ◽

Residual Activity ◽

Zinc Binding ◽

Binding Motif ◽

Dependent Manner ◽

Zinc Binding Motif

The bioavailability of insulin-like growth factor (IGF)-I and -II is controlled by six IGF-binding proteins (IGFBPs 1–6). Bound IGF is not active, but proteolytic cleavage of the binding protein causes release of IGF. Pregnancy-associated plasma protein-A (PAPP-A) has recently been found to cleave IGFBP-4 in an IGF-dependent manner. To experimentally support the hypothesis that PAPP-A belongs to the metzincin superfamily of metalloproteinases, all containing the elongated zinc-binding motif HEXXHXXGXXH (His-482–His-492 in PAPP-A), we expressed mutants of PAPP-A in mammalian cells. Substitution of Glu-483 with Ala causes a complete loss of activity, defining this motif as part of the active site of PAPP-A. Interestingly, a mutant with Glu-483 replaced by Gln shows residual activity. Known metzincin structures contain a so-called Met-turn, whose strictly conserved Met residue is thought to interact directly with residues of the active site. By further mutagenesis we provide experimental evidence that Met-556 of PAPP-A, 63 residues from the zinc-binding motif, is located in a Met-turn of PAPP-A. Our hypothesis is also supported by secondary-structure prediction, and the ability of a 55-residue deletion mutant (d[S498-Y552]) to express and retain antigenecity. However, because PAPP-A differs in the features defining the individual established metzincin families, we suggest that PAPP-A belongs to a separate family. We also found that PAPP-A can undergo autocleavage, and that autocleaved PAPP-A is inactive. A lack of unifying elements in the sequences around the found cleavage sites of PAPP-A and a variant suggests steric regulation of substrate specificity.

Download Full-text