Structural Basis of Human Parainfluenza Virus 3 Unassembled Nucleoprotein in Complex with Its Viral Chaperone

2021 ◽  
Author(s):  
Xiaofei Dong ◽  
Xue Wang ◽  
Mengjia Xie ◽  
Wei Wu ◽  
Zhongzhou Chen
Keyword(s):  
Drug Development ◽  
Rna Polymerase ◽  
Respiratory Diseases ◽  
Complex Structure ◽  
Viral Proteins ◽  
Parainfluenza Virus ◽  
Structural Basis ◽  
Free Form ◽  
Nucleoprotein N ◽  
Human Parainfluenza Virus

Human parainfluenza virus 3 (HPIV3) belongs to the Paramyxoviridae , causing annual worldwide epidemics of respiratory diseases, especially in newborns and infants. The core components consist of just three viral proteins: nucleoprotein (N), phosphoprotein (P), and RNA polymerase (L), playing essential roles in replication and transcription of HPIV3 as well as other paramyxoviruses. Viral genome encapsidated by N is as a template and recognized by RNA-dependent RNA polymerase complex composed of L and P. The offspring RNA also needs to assemble with N to form nucleocapsids. The N is one of the most abundant viral proteins in infected cells and chaperoned in the RNA-free form (N 0 ) by P before encapsidation. In this study, we presented the structure of unassembled HPIV3 N 0 in complex with the N-terminal portion of the P, revealing the molecular details of the N 0 and the conserved N 0 -P interaction. Combined with biological experiments, we showed that the P binds to the C-terminal domain of N 0 mainly by hydrophobic interaction and maintains the unassembled conformation of N by interfering with the formation of N-RNA oligomers, which might be a target for drug development. Based on the complex structure, we developed a method to obtain the monomeric N 0 . Furthermore, we designed a P-derived fusion peptide with 10-times higher affinity, which hijacked the N and interfered with the binding of the N to RNA significantly. Finally, we proposed a model of conformational transition of N from the unassembled state to the assembled state, which helped to further understand viral replication. IMPORTANCE Human parainfluenza virus 3 causes annual epidemics of respiratory diseases, especially in newborns and infants. For the replication of HPIV3 and other paramyxoviruses, only three viral proteins are required: phosphoprotein (P), RNA polymerase (L), and nucleoprotein (N). Here, we reported the crystal structure of the complex of N and its chaperone P. We described in details how P acts as a chaperone to maintain the unassembled conformation of N. Our analysis indicated that the interaction between P and N is conserved and mediated by hydrophobicity, which can be used as a target for drug development. We obtained a high-affinity P-derived peptide inhibitor, specifically targeted N and greatly interfered with the binding of the N to RNA, thereby inhibiting viral encapsidation and replication. In summary, our results provide new insights into the paramyxovirus genome replication and nucleocapsid assembly, and lay the basis for drug development.

scholarly journals Interaction of Human Parainfluenza Virus Type 3 Nucleoprotein with Matrix Protein Mediates Internal Viral Protein Assembly

2015 ◽  
Vol 90 (5) ◽  
pp. 2306-2315 ◽  
Author(s):  
Guangyuan Zhang ◽  
Yi Zhong ◽  
Yali Qin ◽  
Mingzhou Chen
Keyword(s):  
Virus Type ◽  
Matrix Protein ◽  
Viral Proteins ◽  
Parainfluenza Virus ◽  
M Protein ◽  
Content Type ◽  
Virion Assembly ◽  
Nucleoprotein N ◽  
Human Parainfluenza Virus ◽  
Type 3

ABSTRACTHuman parainfluenza virus type 3 (HPIV3) belongs to theParamyxoviridaefamily. Its three internal viral proteins, the nucleoprotein (N), the phosphoprotein (P), and the polymerase (L), form the ribonucleoprotein (RNP) complex, which encapsidates the viral genome and associates with the matrix protein (M) for virion assembly. We previously showed that the M protein expressed alone is sufficient to assemble and release virus-like particles (VLPs) and a mutant with the L305A point mutation in the M protein (ML305A) has a VLP formation ability similar to that of wild-type M protein. In addition, recombinant HPIV3 (rHPIV3) containing the ML305Amutation (rHPIV3-ML305A) could be successfully recovered. In the present study, we found that the titer of rHPIV3-ML305Awas at least 10-fold lower than the titer of rHPIV3. Using VLP incorporation and coimmunoprecipitation assays, we found that VLPs expressing the M protein (M-VLPs) can efficiently incorporate N and P via an N-M or P-M interaction and ML305A-VLPs had an ability to incorporate P via a P-M interaction similar to that of M-VLPs but were unable to incorporate N and no longer interacted with N. Furthermore, we found that the incorporation of P into ML305A-VLPs but not M-VLPs was inhibited in the presence of N. In addition, we provide evidence that the C-terminal region of P is involved in its interaction with both N and M and N binding to the C-terminal region of P inhibits the incorporation of P into ML305A-VLPs. Our findings provide new molecular details to support the idea that the N-M interaction and not the P-M interaction is critical for packaging N and P into infectious viral particles.IMPORTANCEHuman parainfluenza virus type 3 (HPIV3) is a nonsegmented, negative-sense, single-stranded RNA virus that belongs to theParamyxoviridaefamily and can cause lower respiratory tract infections in infants and young children as well as elderly or immunocompromised individuals. However, no effective vaccine has been developed or licensed. We used virus-like particle (VLP) incorporation and coimmunoprecipitation assays to determine how the M protein assembles internal viral proteins. We demonstrate that both nucleoprotein (N) and phosphoprotein (P) can incorporate into M-VLPs and N inhibits the M-P interaction via the binding of N to the C terminus of P. We also provide additional evidence that the N-M interaction but not the P-M interaction is critical for the regulation of HPIV3 assembly. Our studies provide a more complete characterization of HPIV3 virion assembly and substantiation that N interaction with M regulates internal viral organization.

scholarly journals Human Parainfluenza Virus Type 2 L Protein Regions Required for Interaction with Other Viral Proteins and mRNA Capping

2010 ◽  
Vol 85 (2) ◽  
pp. 725-732 ◽  
Author(s):  
M. Nishio ◽  
M. Tsurudome ◽  
D. Garcin ◽  
H. Komada ◽  
M. Ito ◽  
...  
Keyword(s):  
Virus Type ◽  
Viral Proteins ◽  
Parainfluenza Virus ◽  
L Protein ◽  
Mrna Capping ◽  
Human Parainfluenza Virus

scholarly journals Role of a Highly Conserved NH2-Terminal Domain of the Human Parainfluenza Virus Type 3 RNA Polymerase

2002 ◽  
Vol 76 (16) ◽  
pp. 8101-8109 ◽  
Author(s):  
Achut G. Malur ◽  
Suresh K. Choudhary ◽  
Bishnu P. De ◽  
Amiya K. Banerjee
Keyword(s):  
Amino Acids ◽  
Rna Polymerase ◽  
Sendai Virus ◽  
Parainfluenza Virus ◽  
L Protein ◽  
Terminal Domain ◽  
P Protein ◽  
L Proteins ◽  
Human Parainfluenza Virus ◽  
Type 3

ABSTRACT The RNA polymerase complex of human parainfluenza virus type 3 (HPIV 3), a member of the family Paramyxoviridae, is composed of two virally encoded polypeptides: a multifunctional large protein (L, 255 kDa) and a phosphoprotein (P, 90 kDa). From extensive deduced amino acid sequence analyses of the cDNA clones of a number of L proteins of nonsegmented negative-strand RNA viruses, a cluster of high-homology sequence segments have been identified within the body of the L proteins. Here, we have focused on the NH2-terminal domain of HPIV 3 L protein that is also highly conserved. Following mutational analyses within this domain, we examined the ability of the mutant L proteins to (i) transcribe an HPIV 3 minireplicon, (ii) transcribe the viral RNA in vitro using the HPIV 3 nucleocapsid RNA template, and (iii) interact with HPIV 3 P protein. Our results demonstrate that the first 15 amino acids of the NH2-terminal domain spanning a highly conserved motif is directly involved in transcription of the genome RNA and in forming a functional complex with the P protein. Substitution of eight nonconserved amino acids within this domain by the corresponding Sendai virus L protein residues yielded mutants with variable transcriptional activities. However, one mutant in which all eight amino acids were replaced with the corresponding residues of Sendai virus L protein failed to both transcribe the minireplicon and interact with HPIV 3 P and the Sendai virus P protein. The possible functional significance of the NH2-terminal domain of paramyxovirus L protein is discussed.

scholarly journals Inclusion Body Fusion of Human Parainfluenza Virus Type 3 Regulated by Acetylated α-Tubulin Enhances Viral Replication

2016 ◽  
Vol 91 (3) ◽  
Author(s):  
Shengwei Zhang ◽  
Yanliang Jiang ◽  
Qi Cheng ◽  
Yi Zhong ◽  
Yali Qin ◽  
...  
Keyword(s):  
Viral Replication ◽  
Virus Type ◽  
Inclusion Bodies ◽  
Viral Proteins ◽  
Cellular Proteins ◽  
Parainfluenza Virus ◽  
Syncytial Virus ◽  
Human Parainfluenza Virus ◽  
Type 3 ◽  
Efficient Viral Replication

ABSTRACT Viral inclusion bodies (IBs), or replication factories, are unique structures generated by viral proteins together with some cellular proteins as a platform for efficient viral replication, but little is known about the mechanism underlying IB formation and fusion. Our previous study demonstrated that the interaction between the nucleoprotein (N) and phosphoprotein (P) of human parainfluenza virus type 3 (HPIV3), an enveloped virus with great medical impact, can form IBs. In this study, we found that small IBs can fuse with each other to form large IBs that enhance viral replication. Furthermore, we found that acetylated α-tubulin interacts with the N-P complex and colocalizes with IBs of HPIV3 but does not interact with the N-P complex of human respiratory syncytial virus or vesicular stomatitis virus and does not colocalize with IBs of human respiratory syncytial virus. Most importantly, enhancement of α-tubulin acetylation using the pharmacological inhibitor trichostatin A (TSA), RNA interference (RNAi) knockdown of the deacetylase enzymes histone deacetylase 6 (HDAC6) and sirtuin 2 (SIRT2), or expression of α-tubulin acetyltransferase 1 (α-TAT1) resulted in the fusion of small IBs into large IBs and effective viral replication. In contrast, suppression of acetylation of α-tubulin by overexpressing HDAC6 and SIRT2 profoundly inhibited the fusion of small IBs and viral replication. Our findings offer previously unidentified mechanistic insights into the regulation of viral IB fusion by acetylated α-tubulin, which is critical for viral replication. IMPORTANCE Inclusion bodies (IBs) are unique structures generated by viral proteins and some cellular proteins as a platform for efficient viral replication. Human parainfluenza virus type 3 (HPIV3) is a nonsegmented single-stranded RNA virus that mainly causes lower respiratory tract disease in infants and young children. However, no vaccines or antiviral drugs for HPIV3 are available. Therefore, understanding virus-host interactions and developing new antiviral strategies are increasingly important. Acetylation on lysine (K) 40 of α-tubulin is an evolutionarily conserved modification and plays an important role in many cellular processes, but its role in viral IB dynamics has not been fully explored. To our knowledge, our findings are the first to show that acetylated α-tubulin enhances viral replication by regulating HPIV3 IB fusion.

scholarly journals Structural basis of ribosomal frameshifting during translation of the SARS-CoV-2 RNA genome

2020 ◽  
Author(s):  
Pramod R. Bhatt ◽  
Alain Scaiola ◽  
Gary Loughran ◽  
Marc Leibundgut ◽  
Annika Kratzel ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Viral Proteins ◽  
Viral Rna ◽  
Structural Basis ◽  
Rna Dependent Rna Polymerase ◽  
Ribosomal Frameshifting ◽  
Infected Cells ◽  
Rna Genome ◽  
Exit Tunnel ◽  
Viral Polyprotein

AbstractProgrammed ribosomal frameshifting is the key event during translation of the SARS-CoV-2 RNA genome allowing synthesis of the viral RNA-dependent RNA polymerase and downstream viral proteins. Here we present the cryo-EM structure of the mammalian ribosome in the process of translating viral RNA paused in a conformation primed for frameshifting. We observe that the viral RNA adopts a pseudoknot structure lodged at the mRNA entry channel of the ribosome to generate tension in the mRNA that leads to frameshifting. The nascent viral polyprotein that is being synthesized by the ribosome paused at the frameshifting site forms distinct interactions with the ribosomal polypeptide exit tunnel. We use biochemical experiments to validate our structural observations and to reveal mechanistic and regulatory features that influence the frameshifting efficiency. Finally, a compound previously shown to reduce frameshifting is able to inhibit SARS-CoV-2 replication in infected cells, establishing coronavirus frameshifting as target for antiviral intervention.

scholarly journals Structural Basis for the Inhibition of the RNA-Dependent RNA Polymerase from SARS-CoV-2 by Remdesivir

2020 ◽  
Author(s):  
Wanchao Yin ◽  
Chunyou Mao ◽  
Xiaodong Luan ◽  
Dan-Dan Shen ◽  
Qingya Shen ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Virus Disease ◽  
Complex Structure ◽  
Antiviral Drug ◽  
Viral Rna ◽  
Chain Elongation ◽  
Structural Basis ◽  
Rna Dependent Rna Polymerase ◽  
Working Mechanism ◽  
Primer Rna

The pandemic of Corona Virus Disease 2019 (COVID-19) caused by SARS-CoV-2 has become a global crisis. The replication of SARS-CoV-2 requires the viral RNA-dependent RNA polymerase (RdRp), a direct target of the antiviral drug, Remdesivir. Here we report the structure of the SARS-CoV-2 RdRp either in the apo form or in complex with a 50-base template-primer RNA and Remdesivir at a resolution range of 2.5-2.8 Å. The complex structure reveals that the partial double-stranded RNA template is inserted into the central channel of the RdRp where Remdesivir is incorporated into the first replicated base pair and terminates the chain elongation. Our structures provide critical insights into the working mechanism of viral RNA replication and a rational template for drug design to combat the viral infection.

scholarly journals Structural basis for inhibition of the RNA-dependent RNA polymerase from SARS-CoV-2 by remdesivir

Science ◽  
2020 ◽  
Vol 368 (6498) ◽  
pp. 1499-1504 ◽  
Author(s):  
Wanchao Yin ◽  
Chunyou Mao ◽  
Xiaodong Luan ◽  
Dan-Dan Shen ◽  
Qingya Shen ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Complex Structure ◽  
Antiviral Drug ◽  
Viral Rna ◽  
Chain Elongation ◽  
Structural Basis ◽  
Rna Dependent Rna Polymerase ◽  
Double Stranded Rna ◽  
Cryo Electron Microscopy ◽  
Primer Rna

The pandemic of coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has become a global crisis. Replication of SARS-CoV-2 requires the viral RNA-dependent RNA polymerase (RdRp) enzyme, a target of the antiviral drug remdesivir. Here we report the cryo–electron microscopy structure of the SARS-CoV-2 RdRp, both in the apo form at 2.8-angstrom resolution and in complex with a 50-base template-primer RNA and remdesivir at 2.5-angstrom resolution. The complex structure reveals that the partial double-stranded RNA template is inserted into the central channel of the RdRp, where remdesivir is covalently incorporated into the primer strand at the first replicated base pair, and terminates chain elongation. Our structures provide insights into the mechanism of viral RNA replication and a rational template for drug design to combat the viral infection.

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