Sumoylation of Influenza A Virus Nucleoprotein Is Essential for Intracellular Trafficking and Virus Growth

The nucleoprotein (NP) is a major component of the viral ribonucleoprotein (vRNP) complex. During the replication of influenza virus, the vRNP complex undergoes nuclear-cytoplasmic shuttling, during which NP serves as one of the determinants. To date, many phosphorylation sites on NP have been identified, but the biological functions of many of these phosphorylation sites remain unknown. In the present study, the functions of the phosphorylation sites S9, Y10, and Y296 were characterized. These residues are highly conserved, and their phosphorylation was essential for virus growth in cell culture and in a mouse model by regulating the activity of the viral polymerase and the nuclear-cytoplasmic shuttling of NP. The phosphorylation and dephosphorylation of S9 and Y10 controlled nuclear import of NP by affecting the binding affinity between NP and different isoforms of importin-␣. In addition, the phosphorylation of Y296 caused nuclear retention of NP by reducing the interaction between NP and CRM1. Furthermore, tyrosine phosphorylation of NP during the early stage of virus infection was ablated when Y296 was mutated to F. However, at later stages of infection, it was weakened by the Y10F mutation. Taken together, the present data indicate that the phosphorylation and dephosphorylation of NP control the shuttling of NP between the nucleus and the cytoplasm during virus replication. IMPORTANCEIt is well known that phosphorylation regulates the functions of viral proteins and the life cycle of influenza A virus. As NP is the most abundant protein in the vRNP complex of influenza A virus, several phosphorylation sites on this protein have been identified. However, the functions of these phosphorylation sites were unknown. The present study demonstrates that the phosphorylation status of these sites on NP can mediate its nuclear-cytoplasmic shuttling, which drives the trafficking of vRNP complexes in infected cells. The present data suggest that the phosphorylated residues of NP are multistep controllers of the virus life cycle and new targets for the design of anti-influenza drugs.

Section: Discussionmentioning

confidence: 52%

Phosphorylation Controls the Nuclear-Cytoplasmic Shuttling of Influenza A Virus Nucleoprotein

Zheng

Wang

et al. 2015

“…Only EV71 3C protease is SUMOylated, while coxsackievirus B5 is involved in the host cell SUMOylation system in the family Picornaviridae (38,62,70). Current understanding of viral polymerase SUMOylation is limited to RdRp of Turnip mosaic virus (71), DNA polymerase subunit UL44 of HCMV (25), nonstructural protein 5 (NS5) of dengue virus (72), and polymerase basic protein 1 (PB1) of influenza virus (30). In the currently study, we reveal that the 3D protein of EV71, an RNA-dependent RNA polymerase, is SUMOylated.…”

Section: Discussionmentioning

confidence: 99%

“…At least five influenza virus proteins are SUMO targets, and infection with influenza virus leads to a global increase in cellular SUMOylation (27). SUMOylation of nonstructural protein 1 (NS1) accelerates virus growth, M1 protein SUMOylation facilitates viral ribonucleoprotein export and the assembly of virus particles, and NP SUMOylation regulates the intracellular trafficking of NP and efficient virus production (28)(29)(30). The early lytic gene product (K-bZIP), the major transcriptional factor (K-Rta), and latencyassociated nuclear antigen 2 of Kaposi's sarcoma-associated herpesvirus are SUMOylated to achieve efficient viral replication (31)(32)(33).…”

mentioning

confidence: 99%

SUMO Modification Stabilizes Enterovirus 71 Polymerase 3D To Facilitate Viral Replication

Liu

Zheng

Shu

et al. 2016

Accumulating evidence suggests that viruses hijack cellular proteins to circumvent the host immune system. Ubiquitination and SUMOylation are extensively studied posttranslational modifications (PTMs) that play critical roles in diverse biological processes. Cross talk between ubiquitination and SUMOylation of both host and viral proteins has been reported to result in distinct functional consequences. Enterovirus 71 (EV71), an RNA virus belonging to the family Picornaviridae, is a common cause of hand, foot, and mouth disease. Little is known concerning how host PTM systems interact with enteroviruses. Here, we demonstrate that the 3D protein, an RNA-dependent RNA polymerase (RdRp) of EV71, is modified by small ubiquitin-like modifier 1 (SUMO-1) both during infection and in vitro. Residues K159 and L150/D151/L152 were responsible for 3D SUMOylation as determined by bioinformatics prediction combined with site-directed mutagenesis. Also, primer-dependent polymerase assays indicated that mutation of SUMOylation sites impaired 3D polymerase activity and virus replication. Moreover, 3D is ubiquitinated in a SUMO-dependent manner, and SUMOylation is crucial for 3D stability, which may be due to the interplay between the two PTMs. Importantly, increasing the level of SUMO-1 in EV71-infected cells augmented the SUMOylation and ubiquitination levels of 3D, leading to enhanced replication of EV71. These results together suggested that SUMO and ubiquitin cooperatively regulated EV71 infection, either by SUMO-ubiquitin hybrid chains or by ubiquitin conjugating to the exposed lysine residue through SUMOylation. Our study provides new insight into how a virus utilizes cellular pathways to facilitate its replication. IMPORTANCEInfection with enterovirus 71 (EV71) often causes neurological diseases in children, and EV71 is responsible for the majority of fatalities. Based on a better understanding of interplay between virus and host cell, antiviral drugs against enteroviruses may be developed. As a dynamic cellular process of posttranslational modification, SUMOylation regulates global cellular protein localization, interaction, stability, and enzymatic activity. However, little is known concerning how SUMOylation directly influences virus replication by targeting viral polymerase. Here, we found that EV71 polymerase 3D was SUMOylated during EV71 infection and in vitro. Moreover, the SUMOylation sites were determined, and in vitro polymerase assays indicated that mutations at SUMOylation sites could impair polymerase synthesis. Importantly, 3D is ubiquitinated in a SUMOylation-dependent manner that enhances the stability of the viral polymerase. Our findings indicate that the two modifications likely cooperatively enhance virus replication. Our study may offer a new therapeutic strategy against virus replication. S mall ubiquitin-like modifier (SUMO) modification, a member of the group of posttranslational modifications (PTMs), is important for the regulation of many cellular proteins and pathways (1). SUMO is a novel...

“…Posttranslational modifications (PTMs) of viral proteins by host enzymes have been suggested to impact this change in activity. A number of PTMs have been reported to occur on NP and subunits of the viral polymerase, including phosphorylation of PB1, PB2, PA, and NP (9), sumoylation of PB1 and NP (10,11), poly-ADP ribosylation of PB2 and PA (12), and ubiquitination of PB1 and NP (13,14). These modifications can impact multiple steps in the assembly and function of RNPs.…”

mentioning

confidence: 99%

“…These modifications can impact multiple steps in the assembly and function of RNPs. Phosphorylation and sumoylation of NP have been reported to impact its localization to the nucleus or export to the cytoplasm, respectively (11,15,16). Phosphoryla-tion of NP has also been shown to regulate its homo-oligomerization and assembly of RNP complexes (17)(18)(19).…”

mentioning

confidence: 99%

Ubiquitination Upregulates Influenza Virus Polymerase Function

Kirui

Mondal

Mehle

2016

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.