In vitro characterization of naturally occurring influenza H3NA- viruses lacking the NA gene segment: toward a new mechanism of viral resistance?

Université de Lyon, F-69000, Lyon, France.
Virology (Impact Factor: 3.28). 09/2010; 404(2):215-24. DOI: 10.1016/j.virol.2010.04.030
Source: PubMed

ABSTRACT Among a panel of 788 clinical influenza H3N2 isolates, two isolates were characterized by an oseltamivir-resistant phenotype linked to the absence of any detectable NA activity. Here, we established that the two H3NA- isolates lack any detectable full-length NA segment, and one of these could be rescued by reverse genetics in the absence of any NA segment sequence. We found that the absence of NA segment induced a moderate growth defect of the H3NA- viruses as on cultured cells. The glycoproteins density at the surface of H3NA- virions was unchanged as compared to H3N2 virions. The HA protein as well as residues 188 and 617 of the PB1 protein were shown to be strong determinants of the ability of H3NA- viruses to grow in the absence of the NA segment. The significance of these findings about naturally occurring seven-segment influenza A viruses is discussed.

1 Bookmark
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: The segmented genome of an influenza virus is encapsidated into ribonucleoprotein complexes (RNPs). Unusually among RNA viruses, influenza viruses replicate in the nucleus of an infected cell, and their RNPs must therefore recruit host factors to ensure transport across a number of cellular compartments during the course of an infection. Recent studies have shed new light on many of these processes, including the regulation of nuclear export, genome packaging, mechanisms of virion assembly and viral entry and, in particular, the identification of Rab11 on recycling endosomes as a key mediator of RNP transport and genome assembly. This review uses these recent gains in understanding to describe in detail the journey of an influenza A virus RNP from its synthesis in the nucleus through to its entry into the nucleus of a new host cell.
    Viruses 10/2013; 5(10):2424-2446. · 3.28 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Influenza viruses are highly genetically variable and escape from immunogenic pressure by antigenic changes in their surface proteins referred to as antigenic drift and antigenic shift. To assess the potential genetic plasticity under strong selection pressure, highly pathogenic avian influenza virus (HPAIV) of subtype H5N1 was passaged in embryonated chicken eggs in the presence of a neutralizing, polyclonal chicken serum for 50 times. The resulting mutant had acquired major alterations in the neuraminidase (NA)-encoding segment. Extensive deletions and rearrangements were detected in contrast to only 12 amino acid substitutions within all other segments. Interestingly, this new neuraminidase segment resulted from complex sequence shuffling and insertion of a short fragment originating from the PA segment. Characterization of that novel variant revealed a loss of the neuraminidase protein and enzymatic activity, but its replication efficiency remained comparable to the wild-type. Using reverse genetics, a recombinant virus consisting of the wild-type backbone and the shortened NA-segment could be generated, however, required the polybasic hemagglutinin cleavage site. Two independent repetitions starting with egg passage 30 in the presence of alternative chicken-derived immune sera selected mutants with similar but different large deletions within the NA segment without any neuraminidase activity, indicating a general mechanism. In chicken, these virus variants were avirulent despite the still present HPAIV polybasic hemagglutinin cleavage site. Overall, the variants reported here are the first HPAIV H5N1-viruses without functional neuraminidase which grow efficiently without any helper factor. These novel HPAIV variants may facilitate future studies shedding light on the neuraminidase in virus replication and pathogenicity.
    Journal of Virology 10/2013; · 4.65 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Influenza A viruses cause annual influenza epidemics and occasional severe pandemics. Their genome is segmented into eight fragments, which offers evolutionary advantages but complicates genomic packaging. The existence of a selective packaging mechanism, in which one copy of each viral RNA is specifically packaged into each virion, is suspected, but its molecular details remain unknown. Here, we identified a direct intermolecular interaction between two viral genomic RNA segments of an avian influenza A virus using in vitro experiments. Using silent trans-complementary mutants, we then demonstrated that this interaction takes place in infected cells and is required for optimal viral replication. Disruption of this interaction did not affect the HA titer of the mutant viruses, suggesting that the same amount of viral particles was produced. However, it nonspecifically decreased the amount of viral RNA in the viral particles, resulting in an eightfold increase in empty viral particles. Competition experiments indicated that this interaction favored copackaging of the interacting viral RNA segments. The interaction we identified involves regions not previously designated as packaging signals and is not widely conserved among influenza A virus. Combined with previous studies, our experiments indicate that viral RNA segments can promote the selective packaging of the influenza A virus genome by forming a sequence-dependent supramolecular network of interactions. The lack of conservation of these interactions might limit genetic reassortment between divergent influenza A viruses.
    Proceedings of the National Academy of Sciences 09/2013; · 9.81 Impact Factor

Full-text (2 Sources)

Available from
Jun 26, 2014