Consequences of Variation in the Influenza Virus NS1 Protein

Abstract

The NS1 protein is a major virulence factor of influenza virus. Although the protein is encoded by all natural influenza viruses, its sequence shows considerable variation suggesting that it interacts intimately with the host. Indeed this multifunctional protein has been described to bind a plethora of cellular factors. The influenza A NS1 protein from A/PR/8/34 was previously shown to enhance translation of a host expressed gene. This is likely a consequence of its ability to counter basally expressed host antiviral strategies that are activated within the cell upon transfection and transient expression of exogenous genes. This work evaluated a panel of NS1 proteins derived from different strains and subtypes of influenza, for their capacity to enhance translation. Although it is possible that all natural NS1 proteins have the capacity for this function, it was found to be often obscured by a dominant inhibitory function that was mapped to the C terminus of the protein. Only NS1 proteins that lack this second function illustrated translational enhancement without prior mutation. Modification of the C terminal domain of NS1 was able to abrogate binding to the CPSF host factor responsible for the maturation of host genes transcribed by polymerase II. This then revealed the potential of most if not all NS1 proteins to act as translational enhancers. A series of NS1 mutants were engineered to test the proposed mechanism of translational enhancement, and this work confirmed that enhancement required NS1 to be present in the cytoplasm of the transfected cell, and retain an intact dsRNA binding site. The intriguing finding that not all natural NS1 proteins bind to the CPSF host factor was investigated to ask whether CPSF interaction sometimes carried a cost to viral fitness. The hypothesis was that some NS1 proteins adopt this global mechanism for the control of interferon to compensate for high levels of PAMP produced by infection more active viral polymerase. The observations deduced from this work lead to the suggestion that NS1 participates in the regulation of interferon at the level of the polymerase complex by modulating the viral polymerase activity, in addition to its previously characterized function to counter the PRR RIG-I, and disruption of CPSF function. Combining the approaches established during this body of work, it was established that the behaviour of the NS1 protein of the newly emerged swine origin H1N1 2009 pandemic virus were unusual. Interestingly this protein was able to strongly enhance translation despite being predominantly localized to the nucleus. Importantly a reverse genetic approach demonstrated that the levels of interferon induced during infection by the pandemic H1N1 strain could be being under represented, and this may explain discrepancies in the literature between different models of pathogenicity of the pandemic virus.