scholarly journals Specific Phosphorylated Forms of Glyceraldehyde 3-Phosphate Dehydrogenase Associate with Human Parainfluenza Virus Type 3 and Inhibit Viral Transcription In Vitro

2000 ◽  
Vol 74 (8) ◽  
pp. 3634-3641 ◽  
Author(s):  
Suresh Choudhary ◽  
Bishnu P. De ◽  
Amiya K. Banerjee
Keyword(s):  
Molecular Species ◽  
Cell Extract ◽  
Parainfluenza Virus ◽  
Viral Transcription ◽  
Cytoplasmic Fraction ◽  
La Protein ◽  
Transcription In Vitro ◽  
Human Parainfluenza Virus ◽  
Type 3

ABSTRACT We previously reported specific interaction of cellular glyceraldehyde 3-phosphate dehydrogenase (GAPDH), the key glycolytic enzyme, and La protein, the RNA polymerase III transcription factor, with the cis-acting RNAs of human parainfluenza virus type 3 (HPIV3) and packaging of these proteins within purified virions (B. P. De, S. Gupta, H. Zhao, J. Z. Drazba, and A. K. Banerjee, J. Biol. Chem. 271:24728–24735, 1996). To gain further insight into these molecular interactions, we analyzed the virion-associated GAPDH and La protein using two-dimensional gel electrophoresis and immunoblotting. The GAPDH was resolved into two major and one minor molecular species migrating in the pI range of 7.6 to 8.3, while the La protein was resolved into five molecular species in the pI range of 6.8 to 7.5. The GAPDH isoforms present in the virions were also detected in the cytoplasmic fraction of CV-1 cell extract, albeit as minor species. On the other hand, the multiple molecular forms of La protein as seen within the virions were readily detected in the total CV-1 cell extract. Further analysis of virion-associated GAPDH by in vivo labeling with [32P]orthophosphate revealed the presence of multiple phosphorylated species. The phosphorylated species were able to bind specifically to the viral cis-acting 3′ genome sense RNA but failed to bind to the leader sense RNA, as determined by gel mobility shift assay. In contrast, the La protein isoforms present within the virions were not phosphorylated and bound to the viralcis-acting RNAs in a phosphorylation-independent manner. The GAPDH isoforms purified from the CV-1 cell cytoplasmic fraction inhibited viral transcription in vitro. Consistent with this, flag-tagged recombinant GAPDH synthesized by using the vaccinia virus expression system also inhibited viral transcription. Together, these data indicate that specific phosphorylated forms of GAPDH associate with HPIV3 and are involved in the regulation of virus gene expression.

scholarly journals Protective Antibodies Against Human Parainfluenza Virus Type 3 (HPIV3) Infection

2020 ◽  
Author(s):  
Jim Boonyaratanakornkit ◽  
Suruchi Singh ◽  
Connor Weidle ◽  
Justas Rodarte ◽  
Ramasamy Bakthavatsalam ◽  
...  
Keyword(s):  
Virus Type ◽  
Neutralizing Antibodies ◽  
Parainfluenza Virus ◽  
Respiratory Pathogen ◽  
Effective Vaccine ◽  
Healthy Children ◽  
Protective Antibodies ◽  
Human Parainfluenza Virus ◽  
Type 3

ABSTRACTHuman parainfluenza virus type III (HPIV3) is a common respiratory pathogen that afflicts children and can be fatal in vulnerable populations, including the immunocompromised. Unfortunately, an effective vaccine or therapeutic is not currently available, resulting in tens of thousands of hospitalizations per year. In an effort to discover a protective antibody against HPIV3, we screened the B cell repertoires from peripheral blood, tonsils, or spleen from healthy children and adults. These analyses yielded five monoclonal antibodies that potently neutralized HPIV3 in vitro. These HPIV3 neutralizing antibodies targeted two non-overlapping epitopes of the HPIV3 F protein, with most targeting the apex. Importantly, prophylactic administration of one of these antibodies, named PI3-E12, resulted in potent protection against HPIV3 infection in cotton rats. Additionally, PI3-E12 could also be used therapeutically to suppress HPIV3 in immunocompromised animals. These results demonstrate the potential clinical utility of PI3-E12 for the prevention or treatment of HPIV3 in both immunocompetent and immunocompromised individuals.

scholarly journals Inhibiting Human Parainfluenza Virus Infection by Preactivating the Cell Entry Mechanism

mBio ◽  
2019 ◽  
Vol 10 (1) ◽  
Author(s):  
S. F. Bottom-Tanzer ◽  
K. Rybkina ◽  
J. N. Bell ◽  
C. A. Alabi ◽  
C. Mathieu ◽  
...  
Keyword(s):  
Receptor Binding ◽  
Conformational Changes ◽  
Host Cells ◽  
Parainfluenza Virus ◽  
Viral Fusion ◽  
Specific Antibodies ◽  
F Protein ◽  
Human Parainfluenza Virus ◽  
Type 3

ABSTRACTParamyxoviruses, specifically, the childhood pathogen human parainfluenza virus type 3, are internalized into host cells following fusion between the viral and target cell membranes. The receptor binding protein, hemagglutinin (HA)-neuraminidase (HN), and the fusion protein (F) facilitate viral fusion and entry into the cell through a coordinated process involving HN activation by receptor binding, which triggers conformational changes in the F protein to activate it to reach its fusion-competent state. Interfering with this process through premature activation of the F protein has been shown to be an effective antiviral strategyin vitro.Conformational changes in the F protein leading to adoption of the postfusion form of the protein—prior to receptor engagement of HN at the host cell membrane—render the virus noninfectious. We previously identified a small compound (CSC11) that implements this antiviral strategy through an interaction with HN, causing HN to activate F in an untimely process. To assess the functionality of such compounds, it is necessary to verify that the postfusion state of F has been achieved. As demonstrated by Melero and colleagues, soluble forms of the recombinant postfusion pneumovirus F proteins and of their six helix bundle (6HB) motifs can be used to generate postfusion-specific antibodies. We produced novel anti-HPIV3 F conformation-specific antibodies that can be used to assess the functionality of compounds designed to induce F activation. In this study, using systematic chemical modifications of CSC11, we synthesized a more potent derivative of this compound, CM9. Much like CSC11, CM9 causes premature triggering of the F protein through an interaction with HN prior to receptor engagement, thereby preventing fusion and subsequent infection. In addition to validating the potency of CM9 using plaque reduction, fusion inhibition, and binding avidity assays, we confirmed the transition to a postfusion conformation of F in the presence of CM9 using our novel anti-HPIV3 conformation-specific antibodies. We present both CM9 and these newly characterized postfusion antibodies as novel tools to explore and develop antiviral approaches. In turn, these advances in both our molecular toolset and our understanding of HN-F interaction will support development of more-effective antivirals. Combining the findings described here with our recently described physiologically relevantex vivosystem, we have the potential to inform the development of therapeutics to block viral infection.IMPORTANCEParamyxoviruses, including human parainfluenza virus type 3, are internalized into host cells by fusion between viral and target cell membranes. The receptor binding protein, hemagglutinin-neuraminidase (HN), and the fusion protein (F) facilitate viral fusion and entry into cells through a process involving HN activation by receptor binding, which triggers conformational changes in F to activate it to reach its fusion-competent state. Interfering with this process through premature activation of the F protein may be an effective antiviral strategyin vitro. We identified and optimized small compounds that implement this antiviral strategy through an interaction with HN, causing HN to activate F in an untimely fashion. To address that mechanism, we produced novel anti-HPIV3 F conformation-specific antibodies that can be used to assess the functionality of compounds designed to induce F activation. Both the novel antiviral compounds that we present and these newly characterized postfusion antibodies are novel tools for the exploration and development of antiviral approaches.

The human parainfluenza virus type 3 (HPIV 3) C protein inhibits viral transcription

2004 ◽  
Vol 99 (2) ◽  
pp. 199-204 ◽  
Author(s):  
Achut G. Malur ◽  
Michael A. Hoffman ◽  
Amiya K. Banerjee
Keyword(s):  
Virus Type ◽  
Parainfluenza Virus ◽  
Viral Transcription ◽  
C Protein ◽  
Human Parainfluenza Virus ◽  
Type 3

scholarly journals Involvement of Actin Microfilaments in the Replication of Human Parainfluenza Virus Type 3

1998 ◽  
Vol 72 (4) ◽  
pp. 2655-2662 ◽  
Author(s):  
Sanhita Gupta ◽  
Bishnu P. De ◽  
Judith A. Drazba ◽  
Amiya K. Banerjee
Keyword(s):  
Virus Type ◽  
Parainfluenza Virus ◽  
Actin Microfilaments ◽  
Viral Mrnas ◽  
Polymeric Form ◽  
Genomic Rnas ◽  
Human Parainfluenza Virus ◽  
Type 3

ABSTRACT Several studies indicate that paramyxoviruses require a specific cellular factor(s) for transcription of their genomic RNAs. We previously reported that the cellular cytoskeletal protein actin, in its polymeric form, participates in the transcription of human parainfluenza virus type 3 (HPIV3) in vitro. In the present study, we investigated the role of the polymeric form of actin, i.e., the actin microfilaments of the cytoskeletal framework, in the reproduction of HPIV3 in vivo. Pulse-chase labeling analyses indicate that the viral nucleocapsid-associated proteins, NP and P, are present predominantly in the cytoskeletal framework during infection. By in situ hybridization, we found that viral mRNAs and genomic RNA were synthesized from the nucleocapsids that were bound to the cytoskeletal framework. Double immunofluorescent labeling and confocal microscopy of the cytoarchitecture revealed that the viral nucleocapsids are specifically localized on the actin microfilaments. Treatment of cells with the actin-depolymerizing agent, cytochalasin D, resulted in the inhibition of viral RNA synthesis and ribonucleoprotein accumulation. These results strongly suggest that actin microfilaments play an important role in the replication of HPIV3.

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