Conserved Features of the PB2 627 Domain Impact Influenza Virus Polymerase Function and Replication

Kirui, James; Bucci, Michael D.; Poole, Daniel S.; Mehle, Andrew

doi:10.1128/jvi.00508-14

Cited by 39 publications

(32 citation statements)

References 62 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…46 Based on structural biology, the PB2 domain has a key exterior, positively charged residue at the 627 position within a flexible loop that partially wraps around an alpha helix to form what is known as a phi-loop. 53 Importantly, this residue is in the middle of a set of highly conserved, basic residues forming a net positive charge. A signature structural element is the conserved P[F/P]AAAPP motif on the N-terminal side of the 627 residue that is part of the alpha helix previously described.…”

Section: Structure and Function Of The Polymerases Of Respiratory Rnamentioning

confidence: 99%

Nucleosides for the treatment of respiratory RNA virus infections

Jordan

Stevens

Deval

2018

Antivir Chem Chemother

125

105

View full text Add to dashboard Cite

Influenza virus, respiratory syncytial virus, human metapneumovirus, parainfluenza virus, coronaviruses, and rhinoviruses are among the most common viruses causing mild seasonal colds. These RNA viruses can also cause lower respiratory tract infections leading to bronchiolitis and pneumonia. Young children, the elderly, and patients with compromised cardiac, pulmonary, or immune systems are at greatest risk for serious disease associated with these RNA virus respiratory infections. In addition, swine and avian influenza viruses, together with severe acute respiratory syndrome-associated and Middle Eastern respiratory syndrome coronaviruses, represent significant pandemic threats to the general population. In this review, we describe the current medical need resulting from respiratory infections caused by RNA viruses, which justifies drug discovery efforts to identify new therapeutic agents. The RNA polymerase of respiratory viruses represents an attractive target for nucleoside and nucleotide analogs acting as inhibitors of RNA chain synthesis. Here, we present the molecular, biochemical, and structural fundamentals of the polymerase of the four major families of RNA respiratory viruses: Orthomyxoviridae, Pneumoviridae/Paramyxoviridae, Coronaviridae, and Picornaviridae. We summarize past and current efforts to develop nucleoside and nucleotide analogs as antiviral agents against respiratory virus infections. This includes molecules with very broad antiviral spectrum such as ribavirin and T-705 (favipiravir), and others targeting more specifically one or a few virus families. Recent advances in our understanding of the structure(s) and function(s) of respiratory virus polymerases will likely support the discovery and development of novel nucleoside analogs.

show abstract

Section: Structure and Function Of The Polymerases Of Respiratory Rnamentioning

confidence: 99%

Nucleosides for the treatment of respiratory RNA virus infections

Jordan

Stevens

Deval

2018

Antivir Chem Chemother

125

105

View full text Add to dashboard Cite

show abstract

“…Viruses stably encoding a C-terminal FLAG tag on PB2 required duplication of a portion of the PB2 open reading frame to maintain a contiguous packaging signal and were engineered following the strategy of Dos Santos Afonso et al (31). WSN, WSN(PB2-FLAG), or reassortants encoding PB2-FLAG, PB1, PA, and NP derived from A/green-winged teal/OH/175/1983 (S009) or A/New York/312/2001 (NY312) were rescued in transfected HEK293T cells and amplified, and their titers were determined as described previously (32).…”

Section: Methodsmentioning

confidence: 99%

Ubiquitination Upregulates Influenza Virus Polymerase Function

Kirui

Mondal

Mehle

2016

J Virol

Self Cite

View full text Add to dashboard Cite

The influenza A virus polymerase plays an essential role in the virus life cycle, directing synthesis of viral mRNAs and genomes. It is a trimeric complex composed of subunits PA, PB1, and PB2 and associates with viral RNAs and nucleoprotein (NP) to form higher-order ribonucleoprotein (RNP) complexes. The polymerase is regulated temporally over the course of infection to ensure coordinated expression of viral genes as well as replication of the viral genome. Various host factors and processes have been implicated in regulation of the IAV polymerase function, including posttranslational modifications; however, the mechanisms are not fully understood. Here we demonstrate that ubiquitination plays an important role in stimulating polymerase activity. We show that all protein subunits in the RNP are ubiquitinated, but ubiquitination does not significantly alter protein levels. Instead, ubiquitination and an active proteasome enhance polymerase activity. Expression of ubiquitin upregulates polymerase function in a dose-dependent fashion, causing increased accumulation of viral RNA (vRNA), cRNA, and mRNA and enhanced viral gene expression during infection. Ubiquitin expression directly affects polymerase activity independent of nucleoprotein (NP) or ribonucleoprotein (RNP) assembly. Ubiquitination and the ubiquitin-proteasome pathway play key roles during multiple stages of influenza virus infection, and data presented here now demonstrate that these processes modulate viral polymerase activity independent of protein degradation. IMPORTANCEThe cellular ubiquitin-proteasome pathway impacts steps during the entire influenza virus life cycle. Ubiquitination suppresses replication by targeting viral proteins for degradation and stimulating innate antiviral signaling pathways. Ubiquitination also enhances replication by facilitating viral entry and virion disassembly. We identify here an addition proviral role of the ubiquitin-proteasome system, showing that all of the proteins in the viral replication machinery are subject to ubiquitination and this is crucial for optimal viral polymerase activity. Manipulation of the ubiquitin machinery for therapeutic benefit is therefore likely to disrupt the function of multiple viral proteins at stages throughout the course of infection.

show abstract

“…The smFRET results suggest that the population of the closed state of 627-NLS is reduced to very low levels in complex with importin corroborating the model by which the open state is the binding competent form. Experiments carried out on four importin isoforms (1, 3, 5 and 7) indicate that this mechanism is general for PB2 binding to all members of the importin  family.A recent study of residues involved in enhanced polymerase activity in 627-NLS found that mutation of the basic 650 residue, an essential constituent of the stabilizing salt bridge, resulted in efficient nuclear import but reported a lack of polymerase activity in the nucleus 38. This supports our observation that the open form mediates interaction with importin , and further supports the recent observation that the closed form is essential for function within the context of the reconstituted polymerase-RNA complex, where it has been proposed to play a crucial role in interaction with transcribed RNA in the polymerase exit tunnel 10.…”

mentioning

confidence: 98%

Large-Scale Conformational Dynamics Control H5N1 Influenza Polymerase PB2 Binding to Importin α

et al. 2015

View full text Add to dashboard Cite

Influenza A RNA polymerase complex is formed from three components, PA, PB1, and PB2. PB2 is independently imported into the nucleus prior to polymerase reconstitution. All crystallographic structures of the PB2 C-terminus (residues 536-759) reveal two globular domains, 627 and NLS, that form a tightly packed heterodimer. The molecular basis of the affinity of 627-NLS for importins remained unclear from these structures, apparently requiring large-scale conformational changes prior to importin binding. Using a combination of solution-state NMR, small-angle neutron scattering, small-angle X-ray scattering (SAXS), and Förster resonance energy transfer (FRET), we show that 627-NLS populates a temperature-dependent dynamic equilibrium between closed and open states. The closed state is stabilized by a tripartite salt bridge involving the 627-NLS interface and the linker, that becomes flexible in the open state, with 627 and NLS dislocating into a highly dynamic ensemble. Activation enthalpies and entropies associated with the rupture of this interface were derived from simultaneous analysis of temperature-dependent chemical exchange saturation transfer measurements, revealing a strong temperature dependence of both open-state population and exchange rate. Single-molecule FRET and SAXS demonstrate that only the open-form is capable of binding to importin α and that, upon binding, the 627 domain samples a dynamic conformational equilibrium in the vicinity of the C-terminus of importin α. This intrinsic large-scale conformational flexibility therefore enables 627-NLS to bind importin through conformational selection from a temperature-dependent equilibrium comprising both functional forms of the protein.

show abstract

Conserved Features of the PB2 627 Domain Impact Influenza Virus Polymerase Function and Replication

Cited by 39 publications

References 62 publications

Nucleosides for the treatment of respiratory RNA virus infections

Nucleosides for the treatment of respiratory RNA virus infections

Ubiquitination Upregulates Influenza Virus Polymerase Function

Large-Scale Conformational Dynamics Control H5N1 Influenza Polymerase PB2 Binding to Importin α

Contact Info

Product

Resources

About