A Genetically Engineered Waterfowl Influenza Virus with a Deletion in the Stalk of the Neuraminidase Has Increased Virulence for Chickens

116

129

Negative-strand RNA viruses represent a significant class of important pathogens that cause substantial morbidity and mortality in human and animal hosts worldwide. A defining feature of these viruses is that their single-stranded RNA genomes are of opposite polarity to messenger RNA and are replicated through a positivesense intermediate. The replicative intermediate is thought to exist as a complementary ribonucleoprotein (cRNP) complex. However, isolation of such complexes from infected cells has never been accomplished. Here we report the development of an RNA-based affinity-purification strategy for the isolation of cRNPs of influenza A virus from infected cells. This technological advance enabled the structural and functional characterization of this elusive but essential component of the viral RNA replication machine. The cRNP exhibits a filamentous double-helical organization with defined termini, containing the viral RNA-dependent RNA polymerase (RdRp) at one end and a loop structure at the other end. In vitro characterization of cRNP activity yielded mechanistic insights into the workings of this RNA synthesis machine. In particular, we found that cRNPs show activity in vitro only in the presence of added RdRp. Intriguingly, a replication-inactive RdRp mutant was also able to activate cRNP-templated viral RNA synthesis. We propose a model of influenza virus genome replication that relies on the trans-activation of the cRNP-associated RdRp. The described purification strategy should be applicable to other negative-strand RNA viruses and will promote studies into their replication mechanisms.

Section: Significancesupporting

confidence: 75%

Isolation and characterization of the positive-sense replicative intermediate of a negative-strand RNA virus

York

Hengrung

Vreede

et al. 2013

116

129

“…However, the structural and functional roles of the stalk domain of NA were unknown thus far, and there was no report about the glycosylation of these canonical glycosites (19)(20)(21)(22)(23). In this study, we prove that the glycosites in the stalk domain of NA are glycosylated to regulate the activity, affinity, and specificity of NA to modulate IAV replication, suggesting that the glycans in the stalk domain of NA play an important role in the virulence and transmission of IAV.…”

Section: Discussionmentioning

confidence: 62%

Influenza A surface glycosylation and vaccine design

Lin

Tsai

et al. 2016

We have shown that glycosylation of influenza A virus (IAV) hemagglutinin (HA), especially at position N-27, is crucial for HA folding and virus survival. However, it is not known whether the glycosylation of HA and the other two major IAV surface glycoproteins, neuraminidase (NA) and M2 ion channel, is essential for the replication of IAV. Here, we show that glycosylation of HA at N-142 modulates virus infectivity and host immune response. Glycosylation of NA in the stalk region affects its structure, activity, and specificity, thereby modulating virus release and virulence, and glycosylation at the catalytic domain affects its thermostability; however, glycosylation of M2 had no effect on its function. In addition, using IAV without the stalk and catalytic domains of NA as a live attenuated vaccine was shown to confer a strong IAVspecific CD8+ T-cell response and a strong cross-strain as well as cross-subtype protection against various virus strains.influenza A virus | glycosylation | vaccine design I nfluenza A viruses (IAV) belong to the Orthomyxoviridae family and can circulate widely and cross interspecies barriers through the highly antigenic drift and shift of the 18 subtypes of HA and 11 subtypes of neuraminidase (NA) (1, 2). In addition, the posttranslational modification of the IAV surface proteins is important to circumvent host defense and support the virus life cycle (3).HA is a major surface glycoprotein of IAV and is involved in viral infection via binding to sialic acid (SA)-containing glycans on the surface of host cell (3). The other major glycoprotein of IAV, NA, is involved in the cleavage of SA on the host cell receptor to facilitate the release of viral particles to infect other cells (4). M2, the third surface protein of IAV, has ion channel activity to regulate virus penetration and uncoating (1). All surface proteins interact with M1 protein for virus assembly and release (5).The modification of HA and NA by N-glycosylation is important in the IAV life cycle (1, 6-8). Previously, we have shown that the glycosylation of HA affects its receptor binding, immune response, and structural stability, and glycosite 27 is essential for retaining the structural integrity of HA and its receptor binding (6, 9). In addition, using the monoglycosylated HA as immunogen, it showed a broader protection against various IAV subtypes compared with the fully glycosylated version (10). However, it is not clear whether the other specific glycosites and their glycan structures on HA regulate its functions. With regard to NA, the functional roles of its glycosylation are not well understood, though N-glycosylation was reported to stabilize the protein from protease digestion and may affect the enzyme activity (11,12). The aim of this study was to understand the functional effects of glycosylation on HA, NA, and M2, and how glycosylation of the surface proteins affects the life cycle of IAV. ResultsGlycosylation of HA at N-142. Because several glycosylation sites (glycosites 27, 40, 176, 303, and 497) on HA are ...

“…Passaging of the highly pathogenic HA reassortants in chickens led to few exchanges in HA (Hp-H2 poly , Hp-H4 poly ), no exchanges in NA, and two exchanges in the M2 protein (Hp-H2 poly ). The absence of mutations in the NA which originates from Hp-Wt suggests that no further adaptation was required because the stalk deletion (24), a common virulence and host range determinant in HPAIV (26)(27)(28), is already present. Those two amino acid exchanges in the M2 protein of Hp-H2 poly , D44G and R45H, are localized in the proton channel gate region (16)(17)(18), implying the need for further adaptation.…”

Section: Discussionmentioning

confidence: 99%

Avian influenza virus hemagglutinins H2, H4, H8, and H14 support a highly pathogenic phenotype

Veits

Weber²,

Stech³

et al. 2012

116

High-pathogenic avian influenza viruses (HPAIVs) evolve from lowpathogenic precursors specifying the HA serotypes H5 or H7 by acquisition of a polybasic HA cleavage site. As the reason for this serotype restriction has remained unclear, we aimed to distinguish between compatibility of a polybasic cleavage site with H5/H7 HA only and unique predisposition of these two serotypes for insertion mutations. To this end, we introduced a polybasic cleavage site into the HA of several low-pathogenic avian strains with serotypes H1, H2, H3, H4, H6, H8, H10, H11, H14, or H15, and rescued HA reassortants after cotransfection with the genes from either a low-pathogenic H9N2 or high-pathogenic H5N1 strain. Oculonasal inoculation with those reassortants resulted in varying pathogenicity in chicken. Recombinants containing the engineered H2, H4, H8, or H14 in the HPAIV background were lethal and exhibited i.v. pathogenicity indices of 2.79, 2.37, 2.85, and 2.61, respectively, equivalent to naturally occurring H5 or H7 HPAIV. Moreover, the H2, H4, and H8 reassortants were transmitted to some contact chickens. The H2 reassortant gained two mutations in the M2 proton channel gate region, which is affected in some HPAIVs of various origins. Taken together, in the presence of a polybasic HA cleavage site, non-H5/H7 HA can support a highly pathogenic phenotype in the appropriate viral background, indicating requirement for further adaptation. Therefore, the restriction of natural HPAIV to serotypes H5 and H7 is likely a result of their unique predisposition for acquisition of a polybasic HA cleavage site.