Contributions of Two Nuclear Localization Signals of Influenza A Virus Nucleoprotein to Viral Replication

Abstract

The RNA genome of influenza A virus, which forms viral ribonucleoprotein complexes (vRNPs) with viral polymerase subunit proteins (PA, PB1, and PB2) and nucleoprotein (NP), is transcribed and replicated in the nucleus. NP, the major component of vRNPs, has at least two amino acid sequences that serve as nuclear localization signals (NLSs): an unconventional NLS (residues 3 to 13; NLS1) and a bipartite NLS (residues 198 to 216; NLS2). Although both NLSs are known to play a role in nuclear transport, their relative contributions to viral replication are poorly understood. We therefore investigated their contributions to NP subcellular/ subnuclear localization, viral RNA (vRNA) transcription, and viral replication. Abolishing the unconventional NLS caused NP to localize predominantly to the cytoplasm and affected its activity in vRNA transcription. However, we were able to create a virus whose NP contained amino acid substitutions in NLS1 known to abolish its nuclear localization function, although this virus was highly attenuated. These results indicate that while the unconventional NLS is not essential for viral replication, it is necessary for efficient viral mRNA synthesis. On the other hand, the bipartite NLS, whose contribution to the nuclear transport of NP is limited, was essential for vRNA transcription and NP's nucleolar accumulation. A virus with nonfunctional NLS2 could not be generated. Thus, the bipartite NLS, but not the unconventional NLS, of NP is essential for influenza A virus replication.Influenza A virus, a member of the family Orthomyxoviridae, is characterized by segmented RNA genomes of negative polarity (16). The viral genome encodes at least 11 proteins (4, 16) and consists of eight single-stranded RNA segments. These genomic RNAs are incorporated into virions as viral ribonucleoprotein complexes (vRNPs) that comprise viral RNA (vRNA), heterotrimeric viral polymerase subunit proteins (PA, PB1, and PB2), and nucleoprotein (NP). A unique property of influenza viruses among RNA viruses is that every step of vRNA synthesis take place in the nucleus by use of the nuclear machinery of the host cells (16). Therefore, newly synthesized proteins required for the vRNPs must be transported into the nuclei of the cells.The transport of proteins (larger than 50 kDa) from the cytoplasm into the nucleus is regulated by signal-mediated processes. Peptide motifs that allow the proteins to be imported through the nuclear pore complex are referred to as nuclear localization signals (NLSs). They are rich in basic amino acids and bind to NLS receptors (e.g., karyopherin family members), which are responsible for the nuclear translocation of target proteins (12,31). Of the influenza A virus proteins, NLSs have been found in three polymerase subunits, the matrix M1 protein (which associates with vRNP complexes), the nonstructural NS1 protein, and NP, the primary component of vRNPs (for a review, see reference 6).NP has at least two NLS sequences. An unconventional NLS (termed here NLS1) is located between residu...

Genome packaging in influenza A virus

“…Similar studies have now been reported for all eight segments (summarized in Fig. 4c), and in every case, the terminal coding regions, comprising sequences unique for each segment, have been found to promote more efficient packaging than the UTRs alone (Dos Santos Afonso et al, 2005;Fujii et al, 2003;Fujii et al, 2005;Gog et al, 2007;Liang et al, 2005Liang et al, , 2008Marsh et al, 2007;Muramoto et al, 2006;Ozawa et al, 2007Ozawa et al, , 2009Watanabe et al, 2003). The observation that virus formation is inefficient in the absence of the terminal regions of all eight vRNAs has also been corroborated and extended by examining the effects of removing other segments (de Wit et al, 2006;Fujii et al, 2009;Gao et al, 2008;Gao & Palese, 2009;Marsh et al, 2007).…”

“…(c) Packaging of reporter genes. The shortest sequences shown to cause efficient packaging of reporter genes are indicated by green bars Fujii et al, 2005;Liang et al, 2005;Marsh et al, 2007;Ozawa et al, 2007Ozawa et al, , 2009). The region indicated for segment 7 promotes packaging relatively poorly, and may not contain the complete packaging signal (Muramoto et al, 2006;Ozawa et al, 2009).…”

Rewiring the RNAs of influenza virus to prevent reassortment

“…A previous study showed that soon after budding, the influenza A virus ribonucleoprotein (RNP) complexes inside the virions form a specific architecture: a central segment surrounded by the seven other vRNPs (2), which suggests that influenza viral RNA (vRNA) packaging is not a random process. Moreover, segment-specific RNA packaging sequences have been identified on each segment of the influenza A/WSN/33 virus (3)(4)(5)(6)(7)(8)(9)(10)(11). A common feature of these packaging signals is that both the 3Ј and 5Ј noncoding regions (NCRs), as well as coding sequences at the two ends of each open reading frame (ORF) are important [(3-11) and Fig.…”

“…Surprisingly, except for the 12 conserved nucleotides at the 3Ј end and the 13 nucleotides at the 5Ј end of the vRNA, which are required for both polymerase recognition and binding (1), no other conserved motif has been identified in the remainder of the packaging sequences. In addition, the minimum lengths required for efficient packaging, either at the 3Ј or 5Ј ends of the vRNA, differ from one segment to another (3)(4)(5)(6)(7)(8)(9)(10)(11). Although much is known about the role of these packaging sequences in influenza RNA packaging, their mechanism has yet to be fully elucidated.…”

Critical role of segment-specific packaging signals in genetic reassortment of influenza A viruses

“…Using reverse genetics, cispackaging signals of the human H1N1 WSN and PR8 strains were found to reside at both ends of each vRNA, including the UTRs, along with up to 80 bases of adjacent coding sequences (21)(22)(23)(24)(25)(26)(27)(28). In this study, we generated reassortant viruses in vitro from avian H5N2 and human H3N2 viruses to identify incompatibilities between the two parental viruses arising at the vRNA level.…”