Abstract
ABSTRACTAvian influenza A viruses generally do not replicate efficiently in human cells, but substitution of glutamic acid (Glu, E) for lysine (Lys, K) at residue 627 of avian influenza virus polymerase basic protein 2 (PB2) can serve to overcome host restriction and facilitate human infectivity. Although PB2 residue 627 is regarded as a species-specific signature of influenza A viruses, host restriction factors associated with PB2627E have yet to be fully investigated. We conducted immunoprecipitation, followed by differential proteomic analysis, to identify proteins associating with PB2627K (human signature) and PB2627E (avian signature) of influenza A/WSN/1933(H1N1) virus, and the results indicated that Tu elongation factor, mitochondrial (TUFM), had a higher binding affinity for PB2627E than PB2627K in transfected human cells. Stronger binding of TUFM to avian-signature PB2590G/591Q and PB2627E in the 2009 swine-origin pandemic H1N1 and 2013 avian-origin H7N9 influenza A viruses was similarly observed. Viruses carrying avian-signature PB2627E demonstrated increased replication in TUFM-deficient cells, but viral replication decreased in cells overexpressing TUFM. Interestingly, the presence of TUFM specifically inhibited the replication of PB2627E viruses, but not PB2627K viruses. In addition, enhanced levels of interaction between TUFM and PB2627E were noted in the mitochondrial fraction of infected cells. Furthermore, TUFM-dependent autophagy was reduced in TUFM-deficient cells infected with PB2627E virus; however, autophagy remained consistent in PB2627K virus-infected cells. The results suggest that TUFM acts as a host restriction factor that impedes avian-signature influenza A virus replication in human cells in a manner that correlates with autophagy.
Highlights
IMPORTANCE An understanding of the mechanisms that influenza A viruses utilize to shift host tropism and the identification of host restriction factors that can limit infection are both critical to the prevention and control of emerging viruses that cross species barriers to target new hosts
Human proteins associated with WSN PB2627K and PB2627E were collected by FLAG immunoprecipitation (FLAG-IP), separated by gradient SDS-PAGE, subjected to silver staining (Fig. 1A), and identified by matrixassisted laser desorption ionization (MALDI-TOF) mass spectrometry (MS) analysis
Avian influenza A viruses generally do not replicate efficiently in human cells, and this was observed in this study, as viral titers of rWSN PB2627E were almost 10-fold lower than those of rWSN PB2627K (Fig. 5E), suggesting that host restriction factors inhibit the viral replication of avian influenza virus in human cells
Summary
IMPORTANCE An understanding of the mechanisms that influenza A viruses utilize to shift host tropism and the identification of host restriction factors that can limit infection are both critical to the prevention and control of emerging viruses that cross species barriers to target new hosts. We further found that increased TUFM-dependent autophagy correlates with the inhibitory effect on avian-signature influenza virus replication and may serve as a key intrinsic mechanism to restrict avian influenza virus infection in humans These findings provide new insight regarding the TUFM mitochondrial protein and may have important implications for the development of novel antiviral strategies. H7N9 situation update, 24 March 2017 [http://www.fao.org/ag/againfo/programmes/en/empres/h7n9/Situation _update.html]), on the basis of confirmed cases These outbreaks underscore the need to understand how influenza A viruses cross species barriers and develop infectivity in humans. The third hypothesis posits that restriction factors selectively inhibit the avian-signature PB2627E-RNP complex in mammalian cells [17]; for example, RIG-I has greater binding affinity than NP for PB2627E, and this disrupts the viral replication machinery in human cells [18]; RIG-I knockdown failed to rescue PB2627E polymerase activity, suggesting that other restriction factors remain to be identified. TUFM is a fundamental mitochondrial protein that has been implicated in protein translation, GTPase activity, and RNA binding [22], and it has been reported to act as an NLRX1-interacting partner that enhances autophagy while
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