Featured Article - October 2008
Short description: Proc. Natl Acad. Sci. USA 105, 13093-13098 (2008)
Influenza A viruses cause both seasonal flu outbreaks and the severe pandemics that result in high death rates. The H5N1 viruses, often referred to as bird flu, are type A influenza viruses, as was the virus responsible for the flu pandemic in 1918 that caused approximately 50 million deaths.
In infected cells, double-stranded RNA produced by the virus usually triggers the production of interferon-β, a cytokine with antiproliferative and antiviral activities. But influenza A avoids this response by producing the virulence factor NS1A, which inhibits the production of interferon-β.
The amino-terminal domain of NS1A binds to double-stranded RNA, but previous studies have shown no role for this fragment in inhibiting interferon-β messenger RNA production. The remainder of the protein, residues 85–215, termed the effector domain, binds to the 30 kDa host protein cleavage and polyadenylation specificity factor (CPSF30). This factor is required for all 3′ end processing of pre-mRNAs, including interferon-β mRNA.
A collaboration between the Northeast Structural Genomics Consortium (NESG) and the laboratories of Robert Krug at the University of Texas at Austin and Kalyan Das and Eddy Arnold at Rutgers University resulted in the 1.95 Å resolution X-ray crystallographic structure of NS1A of human influenza A/Udorn/72 (Ud) virus in a complex with CPSF30. The complex was formed by the second and third zinc finger motif (F2F3) domain of CPSF30 and the effector domain of NS1A. The F2F3 domain was chosen because it had been previously shown to bind to Ud NS1A and because expression of F2F3 in virus-infected cells leads to inhibition of Ud virus replication and increased interferon-β production.
The complex formed is tetrameric, with two F2F3 molecules wrapped around two NS1A molecules, whose effector domains interact in a head-to-head conformation. The structure revealed a binding pocket for CPSF30 that is composed mainly of hydrophobic residues on NS1A.
The authors mutated three individual residues within the pocket: Gly184, Trp187 and Gln121. Substitutions of Gly184 and Trp187 with arginine abolished binding, as did mutation of Gln121 to alanine.
The researchers then performed an in-depth analysis of the Gly184Arg mutation but first they confirmed using circular dichroism and NMR that the substitution did not disrupt the overall fold.
Then they generated a mutant virus that produced an NS1A protein with the Gly184Arg mutation and examined its effect on the processing of interferon-β pre-mRNA. Substantial levels of unprocessed interferon-β mRNA were found in cells infected with the wild-type virus, whereas roughly one fifth of the unprocessed mRNA accumulated in cells infected with the mutated virus. In contrast, they found that the amount of mature interferon-β mRNA was five times that found in wild-type-infected cells. They concluded that processing of interferon-β pre-mRNA is much more efficient in cells infected with the Gly184Arg virus, thereby explaining why replication of the Gly184Arg virus was attenuated.
The amino acids in the CPSF30-binding pocket are highly conserved among human influenza A viruses, including H5N1 viruses isolated from humans and the pandemic 1918 strain. Thus, this site looks like a good target for antiviral drug development.
The structure also showed that the interaction site between NS1A and CPSF30 stretches beyond the pocket. Two amino acids outside of the pocket, Phe103 and Met106, are highly conserved and help to stabilize the complex through hydrophobic interactions. These residues are conserved in more than 99% of NS1A proteins, but a few of the proteins have other residues at this site.
Influenza A viruses H5N1, HK97 (the 1997 bird flu) and PR8 encode NS1A proteins with different amino acids at positions 103 and 106. In HK97, binding is stabilized in infected cells by the interaction of the viral polymerase with the NS1A:CPSF30 complex. All H5N1 viruses isolated since 2003 have the consensus Phe103 and Met106.
PR8 uses a different approach from HK97. At position 103, it has a hydrophilic residue that almost eliminates binding; instead it works by suppressing the activation of the transcription factor IRF-3 by an as yet unknown mechanism that does not involve the NS1A protein. Only five out of approximately 2,800 influenza A viruses isolated from humans have a hydrophilic residue at position 103, and the last such virus appeared in 1976. All influenza A viruses currently circulating in humans have the consensus Phe103 and Met106, demonstrating the importance of the CPSF30 binding site for the replication of influenza A viruses in humans.
Consequently, the structure of the NS1A:CPSF30 complex identifies the CPSF30- binding pocket on NS1A as a potential target site for the development of small-molecule antiviral drugs.
Kalyan Das, Li-Chung Ma, Rong Xiao, Brian Radvansky, James Aramini et al. Structural basis for suppression of a host antiviral response by influenza A virus.
Proc. Natl Acad. Sci. USA 105, 13093-13098 (2008). doi:10.1073/pnas.0805213105