PMID- 24155389
OWN - NLM
STAT- MEDLINE
DCOM- 20140408
LR  - 20211021
IS  - 1098-5514 (Electronic)
IS  - 0022-538X (Print)
IS  - 0022-538X (Linking)
VI  - 88
IP  - 1
DP  - 2014 Jan
TI  - Adaptive mutations in the nuclear export protein of human-derived H5N1 strains 
      facilitate a polymerase activity-enhancing conformation.
PG  - 263-71
LID - 10.1128/JVI.01495-13 [doi]
AB  - The nuclear export protein (NEP) (NS2) of the highly pathogenic human-derived 
      H5N1 strain A/Thailand/1(KAN-1)/2004 with the adaptive mutation M16I greatly 
      enhances the polymerase activity in human cells in a concentration-dependent 
      manner. While low NEP levels enhance the polymerase activity, high levels are 
      inhibitory. To gain insights into the underlying mechanism, we analyzed the 
      effect of NEP deletion mutants on polymerase activity after reconstitution in 
      human cells. This revealed that the polymerase-enhancing function of NEP resides 
      in the C-terminal moiety and that removal of the last three amino acids 
      completely abrogates this activity. Moreover, compared to full-length NEP, the 
      C-terminal moiety alone exhibited significantly higher activity and seemed to be 
      deregulated, since even the highest concentration did not result in an inhibition 
      of polymerase activity. To determine transient interactions between the N- and 
      C-terminal domains in cis, we fused both ends of NEP to a split click beetle 
      luciferase and performed fragment complementation assays. With decreasing 
      temperature, increased luciferase activity was observed, suggesting that 
      intramolecular binding between the C- and N-terminal domains is preferentially 
      stabilized at low temperatures. This stabilizing effect was significantly reduced 
      with the adaptive mutation M16I or a combination of adaptive mutations (M16I, 
      Y41C, and E75G), which further increased polymerase activity also at 34 degrees C. We 
      therefore propose a model in which the N-terminal moiety of NEP exerts an 
      inhibitory function by back-folding to the C-terminal domain. In this model, 
      adaptive mutations in NEP decrease binding between the C- and N-terminal domains, 
      thereby allowing the protein to "open up" and become active already at a low 
      temperature.
FAU - Reuther, Peter
AU  - Reuther P
AD  - Institute for Virology, University Medical Center Freiburg, Freiburg, Germany.
FAU - Giese, Sebastian
AU  - Giese S
FAU - Gotz, Veronika
AU  - Gotz V
FAU - Kilb, Normann
AU  - Kilb N
FAU - Manz, Benjamin
AU  - Manz B
FAU - Brunotte, Linda
AU  - Brunotte L
FAU - Schwemmle, Martin
AU  - Schwemmle M
LA  - eng
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
DEP - 20131023
PL  - United States
TA  - J Virol
JT  - Journal of virology
JID - 0113724
RN  - 0 (Viral Nonstructural Proteins)
SB  - IM
MH  - Cell Nucleus/*metabolism
MH  - HEK293 Cells
MH  - Humans
MH  - Influenza A Virus, H5N1 Subtype/genetics/*metabolism/physiology
MH  - *Mutation
MH  - Protein Conformation
MH  - Protein Folding
MH  - Protein Transport
MH  - Temperature
MH  - Viral Nonstructural Proteins/chemistry/*genetics
MH  - Virus Replication
PMC - PMC3911765
EDAT- 2013/10/25 06:00
MHDA- 2014/04/09 06:00
PMCR- 2014/07/01
CRDT- 2013/10/25 06:00
PHST- 2013/10/25 06:00 [entrez]
PHST- 2013/10/25 06:00 [pubmed]
PHST- 2014/04/09 06:00 [medline]
PHST- 2014/07/01 00:00 [pmc-release]
AID - JVI.01495-13 [pii]
AID - 01495-13 [pii]
AID - 10.1128/JVI.01495-13 [doi]
PST - ppublish
SO  - J Virol. 2014 Jan;88(1):263-71. doi: 10.1128/JVI.01495-13. Epub 2013 Oct 23.