Importance of the 1+7 configuration of ribonucleoprotein complexes for influenza A virus genome packaging

Noda, Takeshi; Murakami, Shin; Nakatsu, Sumiho; Imai, Hideki; Muramoto, Yukiko; Shindo, Keiko; Sagara, Hiroshi; Kawaoka, Yoshihiro

doi:10.1038/s41467-017-02517-w

Cited by 56 publications

(44 citation statements)

References 46 publications

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“…In our previous report, 7-segmented (HA segment-deficient) IAV incorporated ribosomal RNAs into its virion (37). Thus, we examined the presence of 18S ribosomal RNA (rRNA) in purified recombinant virus, and found a greater amount of 18S rRNAs in OK-RG particles as compared to D/OK-clone particles (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…IAV and IBV incorporate eight RNPs arranged in a specific pattern “1+7”, in which seven RNPs surround the central one (40, 43, 44). Surprisingly, artificially generated 7-segmented IAV, which lacks an HA segment, incorporated rRNA as the eighth RNP instead of an HA segment (37). Interestingly, ICV and IDV both possess a 7-segmented genome, yet also incorporated 8 RNPs, although inclusion of rRNA in virions was not assessed (45).…”

Section: Discussionmentioning

confidence: 99%

“…Northern blot analysis was performed as previously described (37) with some modifications. Briefly, viral RNAs were extracted from purified viruses, plasmid-transfected, or virus-infected HRT-18G cells using ISOGEN reagents (Nippon Gene).…”

Section: Methodsmentioning

confidence: 99%

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Establishment of a Reverse Genetics System for Influenza D Virus

Ishida

Murakami

Kamiki

et al. 2019

Preprint

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250 words 15 Text 4,170 words 16 2 ABSTRACT Influenza D virus (IDV) was initially isolated in the USA in 2011. IDV is 17distributed worldwide and is one of the causative agents of bovine respiratory disease 18 complex (BRDC), which exhibits high morbidity and mortality in feedlot cattle. 19 Molecular mechanisms of IDV pathogenicity are still unknown. Reverse genetics 20 systems are vital tools not only for studying the biology of viruses, but also for use in 21 applications such as recombinant vaccine viruses. Here, we report the establishment of a 22 plasmid-based reverse genetics system for IDV. We first verified that the 3′-terminal 23 nucleotide of each 7-segmented genomic RNA contained uracil in contrary to the 24 previous report, and were then able to successfully generate recombinant IDV by 25 co-transfecting 7 plasmids containing these genomic RNAs along with 4 plasmids 26 expressing polymerase proteins and NP into HRT-18G cells. The recombinant virus had 27 a growth deficit compared to the wild-type virus, and we determined the reason for this 28 growth difference by examining the genomic RNA content of the viral particles. We 29 found that recombinant virus incorporated an unbalanced ratio of viral RNA segments 30 into particles as compared to the wild-type virus, and thus we adjusted the amount of 31 each plasmid used in transfection to obtain recombinant virus with the same replicative 32 capacity as wild-type virus. Our work here in establishing a reverse genetics system for 33 IDV will have a broad range of applications, including uses in studies focused on better 34 understanding IDV replication and pathogenicity as well as those contributing to the 35 development of BRDC countermeasures. 36 37 IMPORTANCE Bovine respiratory disease complex (BRDC) exhibits high mortality 38 and morbidity in cattle, causing economic losses worldwide. Influenza D virus (IDV) is 39 considered to be a causative agent of BRDC. Here, we developed a reverse genetics 40 3 system that allows for the generation of IDV from cloned cDNAs, and the introduction 41 of mutations into the IDV genome. This reverse genetics system will become a 42 powerful tool for use in studies related to understanding the molecular mechanisms of 43 viral replication and pathogenicity, and will also lead to the development of new 44 countermeasures against BRDC. 45 46 KEYWORD bovine respiratory disease complex, influenza D virus, mutant, 47 recombinant virus, reverse genetics, transfection 48 49 50 Influenza D virus (IDV), a member of the family Orthomyxoviridae, was first isolated 52 from pigs with respiratory illness in Oklahoma, USA, in 2011 (1, 2). Epidemiological 53 analyses revealed that cattle are the main host of the virus, due to their high 54 seroprevalence for IDV (2, 3). Further epidemiological studies revealed that IDVs 55 circulate in cattle in many countries including the USA (2-4), Mexico (5), China (6), 56 Japan (7, 8), France (9), Italy (10), Ireland (11), Luxembourg (12), and African 57 countries (13). Furthermore, serolo...

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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Establishment of a Reverse Genetics System for Influenza D Virus

Ishida

Murakami

Kamiki

et al. 2019

Preprint

Self Cite

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“…The 1 + 7 configuration may have a mechanistic function, as it is also conserved in type C and D viruses that only possess 7 vRNAs ( 36 ). Further supporting the mechanistic concept, it was recently shown that IAVs can package cellular ribosomal RNA (as a vRNP) when one of the vRNAs is made unavailable ( 37 ), possibly explaining how type C and D viruses acquire their “eighth” vRNA.…”

Section: Influenza Virusesmentioning

confidence: 89%

Influenza A Virus Cell Entry, Replication, Virion Assembly and Movement

et al. 2018

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Influenza viruses replicate within the nucleus of the host cell. This uncommon RNA virus trait provides influenza with the advantage of access to the nuclear machinery during replication. However, it also increases the complexity of the intracellular trafficking that is required for the viral components to establish a productive infection. The segmentation of the influenza genome makes these additional trafficking requirements especially challenging, as each viral RNA (vRNA) gene segment must navigate the network of cellular membrane barriers during the processes of entry and assembly. To accomplish this goal, influenza A viruses (IAVs) utilize a combination of viral and cellular mechanisms to coordinate the transport of their proteins and the eight vRNA gene segments in and out of the cell. The aim of this review is to present the current mechanistic understanding for how IAVs facilitate cell entry, replication, virion assembly, and intercellular movement, in an effort to highlight some of the unanswered questions regarding the coordination of the IAV infection process.

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“…wrapped around a protein core which is composed of the trimeric polymerase complex PA/P3, PB1, PB2, and multiple copies of nucleoprotein (NP), forming a viral ribonucleoprotein complex (vRNP). It was suggested that seven vRNPs are coordinated around the remaining one forming a "7 plus 1" pattern within the virion (5). Recent results demonstrated that the genomes of ICV and IDV also adopt this pattern, although they only encompass seven gene segments (6).…”

mentioning

confidence: 99%

The Extended C-Terminal Region of Influenza C Virus Nucleoprotein Is Important for Nuclear Import and Ribonucleoprotein Activity

Tang

Mok

et al. 2019

J Virol

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The influenza C virus (ICV) is a human-pathogenic agent, and the infections are frequently identified in children. Compared to influenza A and B viruses, the nucleoprotein of ICV (NPC) has an extended C-terminal region of which the functional significance is ill defined. We observed that the nuclear localization signals (NLSs) found on the nucleoproteins of influenza A and B virus subtypes are absent at corresponding positions on ICV. Instead, we found that a long bipartite nuclear localization signal resides at the extended C-terminal region, spanning from R513 to K549. Our experimental data determined that the KKMK motif within this region plays important roles in both nuclear import and polymerase activity. Similar to the influenza A viruses, NPC also binds to multiple human importin ␣ isoforms. Taken together, our results enhance the understanding of the virus-host interaction of the influenza C virus. IMPORTANCE As a member of the Orthomyxoviridae family, the polymerase complex of the influenza C virus structurally resembles its influenza A and influenza B virus counterparts, but the nucleoprotein differs by possessing an extra C-terminal region. We have characterized this region in view of nuclear import and interaction with the importin ␣ protein family. Our results demonstrate the functional significance of a previously uncharacterized region on Orthomyxoviridae nucleoprotein (NP). Based on this work, we propose that importin ␣ binding to influenza C virus NP is regulated by a long bipartite nuclear localization signal. Since the sequence of influenza D virus NP shares high homology to that of the influenza C virus, this work will also shed light on how influenza D virus NP functions.

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Importance of the 1+7 configuration of ribonucleoprotein complexes for influenza A virus genome packaging

Cited by 56 publications

References 46 publications

Establishment of a Reverse Genetics System for Influenza D Virus

Establishment of a Reverse Genetics System for Influenza D Virus

Influenza A Virus Cell Entry, Replication, Virion Assembly and Movement

The Extended C-Terminal Region of Influenza C Virus Nucleoprotein Is Important for Nuclear Import and Ribonucleoprotein Activity

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