scholarly journals cis-Acting Packaging Signals in the Influenza Virus PB1, PB2, and PA Genomic RNA Segments

2005 ◽  
Vol 79 (16) ◽  
pp. 10348-10355 ◽  
Author(s):  
Yuying Liang ◽  
Ying Hong ◽  
Tristram G. Parslow
Keyword(s):  
Influenza Virus ◽  
Influenza A ◽  
Virus Assembly ◽  
Virus Genome ◽  
Coding Region ◽  
Coding Sequences ◽  
Viral Packaging ◽  
Cis Acting ◽  
Virus Particles ◽  
Coding Regions

ABSTRACT The influenza A virus genome consists of eight negative-sense RNA segments. The cis-acting signals that allow these viral RNA segments (vRNAs) to be packaged into influenza virus particles have not been fully elucidated, although the 5′ and 3′ untranslated regions (UTRs) of each vRNA are known to be required. Efficient packaging of the NA, HA, and NS segments also requires coding sequences immediately adjacent to the UTRs, but it is not yet known whether the same is true of other vRNAs. By assaying packaging of genetically tagged vRNA reporters during plasmid-directed influenza virus assembly in cells, we have now mapped cis-acting sequences that are sufficient for packaging of the PA, PB1, and PB2 segments. We find that each involves portions of the distal coding regions. Efficient packaging of the PA or PB1 vRNAs requires at least 40 bases of 5′ and 66 bases of 3′ coding sequences, whereas packaging of the PB2 segment requires at least 80 bases of 5′ coding region but is independent of coding sequences at the 3′ end. Interestingly, artificial reporter vRNAs carrying mismatched ends (i.e., whose 5′ and 3′ ends are derived from different vRNA segments) were poorly packaged, implying that the two ends of any given vRNA may collaborate in forming specific structures to be recognized by the viral packaging machinery.

scholarly journals Defective Assembly of Influenza A Virus due to a Mutation in the Polymerase Subunit PA

2006 ◽  
Vol 80 (1) ◽  
pp. 252-261 ◽  
Author(s):  
John F. Regan ◽  
Yuying Liang ◽  
Tristram G. Parslow
Keyword(s):  
Influenza Virus ◽  
Influenza A Virus ◽  
Influenza A ◽  
Virus Assembly ◽  
Functional Characterization ◽  
Genomic Rna ◽  
Viral Mrnas ◽  
Virus Particles ◽  
Essential Components ◽  
Viral Genomic Rna

ABSTRACT The RNA-dependent RNA polymerase of influenza A virus is composed of three subunits that together synthesize all viral mRNAs and also replicate the viral genomic RNA segments (vRNAs) through intermediates known as cRNAs. Here we describe functional characterization of 16 site-directed mutants of one polymerase subunit, termed PA. In accord with earlier studies, these mutants exhibited diverse, mainly quantitative impairments in expressing one or more classes of viral RNA, with associated infectivity defects of varying severity. One PA mutant, however, targeting residues 507 and 508, caused only modest perturbations of RNA expression yet completely eliminated the formation of plaque-forming virus. Polymerases incorporating this mutant, designated J10, proved capable of synthesizing translationally active mRNAs and of replicating diverse cRNA or vRNA templates at levels compatible with viral infectivity. Both the mutant protein and its RNA products were appropriately localized in the cytoplasm, where influenza virus assembly occurs. Nevertheless, J10 failed to generate infectious particles from cells in a plasmid-based influenza virus assembly assay, and hemagglutinating material from the supernatants of such cells contained little or no nuclease-resistant genomic RNA. These findings suggest that PA has a previously unrecognized role in assembly or release of influenza virus virions, perhaps influencing core structure or the packaging of vRNAs or other essential components into nascent influenza virus particles.

scholarly journals Analysis of the Variability in the Non-Coding Regions of Influenza A Viruses

2018 ◽  
Vol 5 (3) ◽  
pp. 76 ◽  
Author(s):  
Jessica Benkaroun ◽  
Gregory Robertson ◽  
Hugh Whitney ◽  
Andrew Lang
Keyword(s):  
Influenza Virus ◽  
Host Species ◽  
Influenza A ◽  
Wild Bird ◽  
Coding Region ◽  
Influenza A Viruses ◽  
Protein Coding ◽  
Coding Regions ◽  
Negative Sense ◽  
Specific Sequences

The genomes of influenza A viruses (IAVs) comprise eight negative-sense single-stranded RNA segments. In addition to the protein-coding region, each segment possesses 5′ and 3′ non-coding regions (NCR) that are important for transcription, replication and packaging. The NCRs contain both conserved and segment-specific sequences, and the impacts of variability in the NCRs are not completely understood. Full NCRs have been determined from some viruses, but a detailed analysis of potential variability in these regions among viruses from different host groups and locations has not been performed. To evaluate the degree of conservation in NCRs among different viruses, we sequenced the NCRs of IAVs isolated from different wild bird host groups (ducks, gulls and seabirds). We then extended our study to include NCRs available from the National Center for Biotechnology Information (NCBI) Influenza Virus Database, which allowed us to analyze a wider variety of host species and more HA and NA subtypes. We found that the amount of variability within the NCRs varies among segments, with the greatest variation found in the HA and NA and the least in the M and NS segments. Overall, variability in NCR sequences was correlated with the coding region phylogeny, suggesting vertical coevolution of the (coding sequence) CDS and NCR regions.

scholarly journals Mutational Analyses of Packaging Signals in Influenza Virus PA, PB1, and PB2 Genomic RNA Segments

2007 ◽  
Vol 82 (1) ◽  
pp. 229-236 ◽  
Author(s):  
Yuhong Liang ◽  
Taoying Huang ◽  
Hinh Ly ◽  
Tristram G. Parslow ◽  
Yuying Liang
Keyword(s):  
Protein Function ◽  
Influenza A ◽  
Virus Genome ◽  
Coding Region ◽  
Rna Packaging ◽  
Sequence Comparisons ◽  
Protein Subunits ◽  
Coding Regions ◽  
Negative Sense ◽  
Packaging Signals

ABSTRACT The influenza A virus genome consists of eight negative-sense RNA segments that must each be packaged to produce an infectious virion. We have previously mapped the minimal cis-acting regions necessary for efficient packaging of the PA, PB1, and PB2 segments, which encode the three protein subunits of the viral RNA polymerase. The packaging signals in each of these RNAs lie within two separate regions at the 3′ and 5′ termini, each encompassing the untranslated region and extending up to 80 bases into the adjacent coding sequence. In this study, we introduced scanning mutations across the coding regions in each of these RNA segments in order to finely define the packaging signals. We found that mutations producing the most severe defects were confined to a few discrete 5′ sites in the PA or PB1 coding regions but extended across the entire (80-base) 5′ coding region of PB2. In sequence comparisons among more than 580 influenza A strains from diverse hosts, these highly deleterious mutations were each found to affect one or more conserved bases, though they did not all lie within the most broadly conserved portions of the regions that we interrogated. We have introduced silent and conserved mutations to the critical packaging sites, which did not affect protein function but impaired viral replication at levels roughly similar to those of their defects in RNA packaging. Interestingly, certain mutations showed strong tendencies to revert to wild-type sequences, which implies that these putative packaging signals are critical for the influenza life cycle.

scholarly journals Importance of both the Coding and the Segment-Specific Noncoding Regions of the Influenza A Virus NS Segment for Its Efficient Incorporation into Virions

2005 ◽  
Vol 79 (6) ◽  
pp. 3766-3774 ◽  
Author(s):  
Ken Fujii ◽  
Yutaka Fujii ◽  
Takeshi Noda ◽  
Yukiko Muramoto ◽  
Tokiko Watanabe ◽  
...  
Keyword(s):  
Influenza A Virus ◽  
Influenza A ◽  
Virus Genome ◽  
Noncoding Region ◽  
Coding Region ◽  
Genome Packaging ◽  
Noncoding Regions ◽  
Coding Regions ◽  
Negative Sense ◽  
Incorporation Efficiency

ABSTRACT The genome of influenza A virus consists of eight single-strand negative-sense RNA segments, each comprised of a coding region and a noncoding region. The noncoding region of the NS segment is thought to provide the signal for packaging; however, we recently showed that the coding regions located at both ends of the hemagglutinin and neuraminidase segments were important for their incorporation into virions. In an effort to improve our understanding of the mechanism of influenza virus genome packaging, we sought to identify the regions of NS viral RNA (vRNA) that are required for its efficient incorporation into virions. Deletion analysis showed that the first 30 nucleotides of the 3′ coding region are critical for efficient NS vRNA incorporation and that deletion of the 3′ segment-specific noncoding region drastically reduces NS vRNA incorporation into virions. Furthermore, silent mutations in the first 30 nucleotides of the 3′ NS coding region reduced the incorporation efficiency of the NS segment and affected virus replication. These results suggested that segment-specific noncoding regions together with adjacent coding regions (especially at the 3′ end) form a structure that is required for efficient influenza A virus vRNA packaging.

scholarly journals Nucleotide Sequence Requirements at the 5′ End of the Influenza A Virus M RNA Segment for Efficient Virus Replication

2009 ◽  
Vol 83 (7) ◽  
pp. 3384-3388 ◽  
Author(s):  
Makoto Ozawa ◽  
Junko Maeda ◽  
Kiyoko Iwatsuki-Horimoto ◽  
Shinji Watanabe ◽  
Hideo Goto ◽  
...  
Keyword(s):  
Nucleotide Sequence ◽  
Influenza A Virus ◽  
Influenza A ◽  
Virus Genome ◽  
Viral Rna ◽  
Coding Region ◽  
Noncoding Regions ◽  
Coding Regions ◽  
Virion Incorporation ◽  
Sequence Requirements

ABSTRACT The mechanism by which the influenza A virus genome is packaged into virions is not fully understood. The coding and noncoding regions necessary for packaging of the viral RNA segments, except for the M segment, have been identified. Here, we delineate the M segment regions by incorporating a reporter viral RNA into virions and by generating viruses possessing mutations in the regions. We found that, like the other segments, the M segment coding regions are essential for virion incorporation and that the nucleotide length rather than the nucleotide sequence of the 5′ end of the coding region is important.

Evidence for specific packaging of the influenza A virus genome from conditionally defective virus particles lacking a polymerase gene

Vaccine ◽  
2006 ◽  
Vol 24 (44-46) ◽  
pp. 6647-6650 ◽  
Author(s):  
Emmie de Wit ◽  
Monique I.J. Spronken ◽  
Guus F. Rimmelzwaan ◽  
Albert D.M.E. Osterhaus ◽  
Ron A.M. Fouchier
Keyword(s):  
Influenza A Virus ◽  
Influenza A ◽  
Virus Genome ◽  
Polymerase Gene ◽  
Virus Particles ◽  
Defective Virus

scholarly journals Biochemical and genetic analysis of the role of the viral polymerase in enterovirus recombination

2016 ◽  
Vol 44 (14) ◽  
pp. 6883-6895 ◽  
Author(s):  
Andrew Woodman ◽  
Jamie J. Arnold ◽  
Craig E. Cameron ◽  
David J. Evans
Keyword(s):  
Genetic Recombination ◽  
Evolutionary Process ◽  
Virus Genome ◽  
Biochemical Assay ◽  
Strand Transfer ◽  
Turnover Rates ◽  
Coding Region ◽  
Cis Acting ◽  
Polymerase Fidelity

Abstract Genetic recombination in single-strand, positive-sense RNA viruses is a poorly understand mechanism responsible for generating extensive genetic change and novel phenotypes. By moving a critical cis-acting replication element (CRE) from the polyprotein coding region to the 3′ non-coding region we have further developed a cell-based assay (the 3′CRE-REP assay) to yield recombinants throughout the non-structural coding region of poliovirus from dually transfected cells. We have additionally developed a defined biochemical assay in which the only protein present is the poliovirus RNA dependent RNA polymerase (RdRp), which recapitulates the strand transfer events of the recombination process. We have used both assays to investigate the role of the polymerase fidelity and nucleotide turnover rates in recombination. Our results, of both poliovirus intertypic and intratypic recombination in the CRE-REP assay and using a range of polymerase variants in the biochemical assay, demonstrate that RdRp fidelity is a fundamental determinant of recombination frequency. High fidelity polymerases exhibit reduced recombination and low fidelity polymerases exhibit increased recombination in both assays. These studies provide the basis for the analysis of poliovirus recombination throughout the non-structural region of the virus genome and provide a defined biochemical assay to further dissect this important evolutionary process.

scholarly journals Hierarchy among Viral RNA (vRNA) Segments in Their Role in vRNA Incorporation into Influenza A Virions

2006 ◽  
Vol 80 (5) ◽  
pp. 2318-2325 ◽  
Author(s):  
Yukiko Muramoto ◽  
Ayato Takada ◽  
Ken Fujii ◽  
Takeshi Noda ◽  
Kiyoko Iwatsuki-Horimoto ◽  
...  
Keyword(s):  
Influenza A ◽  
Fluorescent Protein ◽  
Virus Assembly ◽  
Viral Rna ◽  
Green Fluorescent Protein Gene ◽  
Influenza A Viruses ◽  
Coding Regions ◽  
Virion Incorporation ◽  
Green Fluorescent ◽  
Efficient Viral Replication

ABSTRACT The genome of influenza A viruses comprises eight negative-strand RNA segments. Although all eight segments must be present in cells for efficient viral replication, the mechanism(s) by which these viral RNA (vRNA) segments are incorporated into virions is not fully understood. We recently found that sequences at both ends of the coding regions of the HA, NA, and NS vRNA segments of A/WSN/33 play important roles in the incorporation of these vRNAs into virions. In order to similarly identify the regions of the PB2, PB1, and PA vRNAs of this strain that are critical for their incorporation, we generated a series of mutant vRNAs that possessed the green fluorescent protein gene flanked by portions of the coding and noncoding regions of the respective segments. For all three polymerase segments, deletions at the ends of their coding regions decreased their virion incorporation efficiencies. More importantly, these regions not only affected the incorporation of the segment in which they reside, but were also important for the incorporation of other segments. This effect was most prominent with the PB2 vRNA. These findings suggest a hierarchy among vRNA segments for virion incorporation and may imply intersegment association of vRNAs during virus assembly.

scholarly journals Influenza Virus Assembly and Lipid Raft Microdomains: a Role for the Cytoplasmic Tails of the Spike Glycoproteins

2000 ◽  
Vol 74 (10) ◽  
pp. 4634-4644 ◽  
Author(s):  
Jie Zhang ◽  
Andrew Pekosz ◽  
Robert A. Lamb
Keyword(s):  
Influenza Virus ◽  
Influenza A ◽  
Virus Assembly ◽  
Intrinsic Property ◽  
Influenza Viruses ◽  
Apical Surface ◽  
Cytoplasmic Tails ◽  
Fusion Glycoprotein ◽  
Influenza C Virus ◽  
Lipid Raft Microdomains

ABSTRACT Influenza viruses encoding hemagglutinin (HA) and neuraminidase (NA) glycoproteins with deletions in one or both cytoplasmic tails (HAt− or NAt−) have a reduced association with detergent-insoluble glycolipids (DIGs). Mutations which eliminated various combinations of the three palmitoylation sites in HA exhibited reduced amounts of DIG-associated HA in virus-infected cells. The influenza virus matrix (M1) protein was also found to be associated with DIGs, but this association was decreased in cells infected with HAt− or NAt− virus. Regardless of the amount of DIG-associated protein, the HA and NA glycoproteins were targeted primarily to the apical surface of virus-infected, polarized cells. The uncoupling of DIG association and apical transport was augmented by the observation that the influenza A virus M2 protein as well as the influenza C virus HA-esterase-fusion glycoprotein were not associated with DIGs but were apically targeted. The reduced DIG association of HAt− and NAt− is an intrinsic property of the glycoproteins, as similar reductions in DIG association were observed when the proteins were expressed from cDNA. Examination of purified virions indicated reduced amounts of DIG-associated lipids in the envelope of HAt− and NAt− viruses. The data indicate that deletion of both the HA and NA cytoplasmic tails results in reduced DIG association and changes in both virus polypeptide and lipid composition.

scholarly journals Live Bivalent Vaccine for Parainfluenza and Influenza Virus Infections

2005 ◽  
Vol 79 (11) ◽  
pp. 6674-6679 ◽  
Author(s):  
Yasuko Maeda ◽  
Masato Hatta ◽  
Ayato Takada ◽  
Tokiko Watanabe ◽  
Hideo Goto ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Influenza A ◽  
Reverse Genetics ◽  
Parainfluenza Virus ◽  
Virus Infections ◽  
Coding Region ◽  
Lethal Challenge ◽  
Bivalent Vaccine ◽  
Parainfluenza Viruses ◽  
Human Parainfluenza Virus

ABSTRACT Influenza and human parainfluenza virus infections are of both medical and economical importance. Currently, inactivated vaccines provide suboptimal protection against influenza, and vaccines for human parainfluenza virus infection are not available, underscoring the need for new vaccines against these respiratory diseases. Furthermore, to reduce the burden of vaccination, the development of multivalent vaccines is highly desirable. Thus, to devise a single vaccine that would elicit immune responses against both influenza and parainfluenza viruses, we used reverse genetics to generate an influenza A virus that possesses the coding region for the hemagglutinin/neuraminidase ectodomain of parainfluenza virus instead of the influenza virus neuraminidase. The recombinant virus grew efficiently in eggs but was attenuated in mice. When intranasally immunized with the recombinant vaccine, all mice developed antibodies against both influenza and parainfluenza viruses and survived an otherwise lethal challenge with either of these viruses. This live bivalent vaccine has obvious advantages over combination vaccines, and its method of generation could, in principle, be applied in the development of a “cocktail” vaccine with efficacy against several different infectious diseases.

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