Recently, two new influenza A-like viruses have been discovered in bats, A/little yellow-shouldered bat/Guatemala/060/2010 (HL17NL10) and A/flat-faced bat/Peru/033/2010 (HL18NL11). The hemagglutinin (HA)-like (HL) and neuraminidase (NA)-like (NL) proteins of these viruses lack hemagglutination and neuraminidase activities, despite their sequence and structural homologies with the HA and NA proteins of conventional influenza A viruses. We have now investigated whether the NS1 proteins of the HL17NL10 and HL18NL11 viruses can functionally replace the NS1 protein of a conventional influenza A virus. For this purpose, we generated recombinant influenza A/Puerto Rico/8/1934 (PR8) H1N1 viruses containing the NS1 protein of the PR8 wild-type, HL17NL10, and HL18NL11 viruses. These viruses (r/NS1PR8, r/NS1HL17, and r/NS1HL18, respectively) were tested for replication in bat and nonbat mammalian cells and in mice. Our results demonstrate that the r/NS1HL17 and r/NS1HL18 viruses are attenuated in vitro and in vivo. However, the bat NS1 recombinant viruses showed a phenotype similar to that of the r/NS1PR8 virus in STAT1^−/− human A549 cells and mice, both in vitro and in vivo systems being unable to respond to interferon (IFN). Interestingly, multiple mouse passages of the r/NS1HL17 and r/NS1HL18 viruses resulted in selection of mutant viruses containing single amino acid mutations in the viral PB2 protein. In contrast to the parental viruses, virulence and IFN antagonism were restored in the selected PB2 mutants. Our results indicate that the NS1 protein of bat influenza A-like viruses is less efficient than the NS1 protein of its conventional influenza A virus NS1 counterpart in antagonizing the IFN response and that this deficiency can be overcome by the influenza virus PB2 protein.

IMPORTANCE Significant gaps in our understanding of the basic features of the recently discovered bat influenza A-like viruses HL17NL10 and HL18NL11 remain. The basic biology of these unique viruses displays both similarities to and differences from the basic biology of conventional influenza A viruses. Here, we show that recombinant influenza A viruses containing the NS1 protein from HL17NL10 and HL18NL11 are attenuated. This attenuation was mediated by their inability to antagonize the type I IFN response. However, this deficiency could be compensated for by single amino acid replacements in the PB2 gene. Our results unravel a functional divergence between the NS1 proteins of bat influenza A-like and conventional influenza A viruses and demonstrate an interplay between the viral PB2 and NS1 proteins to antagonize IFN.

最近，在蝙蝠中发现了两种新的类似于A型流感病毒，即A /小黄肩蝙蝠/危地马拉/ 060/2010（HL17NL10）和A /扁平蝙蝠/ Peru / 033/2010（HL18NL11）。这些病毒的血凝素（HA）样（HL）和神经氨酸酶（NA）样（NL）蛋白缺乏血凝和神经氨酸酶活性，尽管它们与常规甲型流感病毒的HA和NA蛋白具有序列和结构同源性。现在我们已经研究了HL17NL10和HL18NL11病毒的NS1蛋白是否可以在功能上替代常规A型流感病毒的NS1蛋白。为此，我们生成了重组A / Puerto Rico / 8/1934（PR8）流感H1N1病毒，其中包含PR8野生型的NS1蛋白，HL17NL10和HL18NL11病毒。这些病毒（r / NS1PR8，r / NS1HL17和r / NS1HL18，分别测试了在蝙蝠和非蝙蝠哺乳动物细胞以及小鼠中的复制。我们的结果表明，r / NS1HL17和r / NS1HL18病毒被减毒体外和体内。然而，蝙蝠NS1重组病毒显示出类似于R / NS1PR8病毒在STAT1的表型^{- / -}人A549细胞和小鼠中，都在体外和体内系统无法响应干扰素（IFN）。有趣的是，r / NS1HL17和r / NS1HL18病毒的多次小鼠传代导致选择了在病毒PB2蛋白中包含单个氨基酸突变的突变病毒。与亲本病毒相反，在选定的PB2突变体中恢复了毒性和IFN拮抗作用。我们的结果表明，蝙蝠流感样病毒的NS1蛋白在对抗IFN应答方面不如其常规甲型流感病毒NS1对应物的NS1蛋白有效，这种缺陷可以通过流感病毒PB2蛋白来克服。

重要信息在我们对最近发现的蝙蝠流感A样病毒HL17NL10和HL18NL11的基本特征的理解上，仍有很大的差距。这些独特病毒的基本生物学与常规甲型流感病毒的基本生物学既有相似之处，也有不同之处。在这里，我们显示了包含来自HL17NL10和HL18NL11的NS1蛋白的重组A型流感病毒是减毒的。这种减弱是由于它们不能拮抗I型IFN应答而介导的。但是，这种缺陷可以通过PB2基因中的单个氨基酸替代来弥补。我们的结果揭示了蝙蝠流感样病毒和常规甲型流感病毒的NS1蛋白之间的功能差异，并证明了病毒PB2和NS1蛋白之间的相互作用可拮抗IFN。