PMID- 25926641
OWN - NLM
STAT- MEDLINE
DCOM- 20150831
LR  - 20181113
IS  - 1098-5514 (Electronic)
IS  - 0022-538X (Print)
IS  - 0022-538X (Linking)
VI  - 89
IP  - 14
DP  - 2015 Jul
TI  - A Combined Genetic-Proteomic Approach Identifies Residues within Dengue Virus 
      NS4B Critical for Interaction with NS3 and Viral Replication.
PG  - 7170-86
LID - 10.1128/JVI.00867-15 [doi]
AB  - Dengue virus (DENV) infection causes the most prevalent arthropod-borne viral 
      disease worldwide. Approved vaccines are not available, and targets suitable for 
      the development of antiviral drugs are lacking. One possible drug target is 
      nonstructural protein 4B (NS4B), because it is absolutely required for virus 
      replication; however, its exact role in the DENV replication cycle is largely 
      unknown. With the aim of mapping NS4B determinants critical for DENV replication, 
      we performed a reverse genetic screening of 33 NS4B mutants in the context of an 
      infectious DENV genome. While the majority of these mutations were lethal, for 
      several of them, we were able to select for second-site pseudoreversions, most 
      often residing in NS4B and restoring replication competence. To identify all 
      viral NS4B interaction partners, we engineered a fully viable DENV genome 
      encoding an affinity-tagged NS4B. Mass spectrometry-based analysis of the NS4B 
      complex isolated from infected cells identified the NS3 protease/helicase as a 
      major interaction partner of NS4B. By combining the genetic complementation map 
      of NS4B with a replication-independent expression system, we identified the NS4B 
      cytosolic loop-more precisely, amino acid residue Q134-as a critical determinant 
      for NS4B-NS3 interaction. An alanine substitution at this site completely 
      abrogated the interaction and DENV RNA replication, and both were restored by 
      pseudoreversions A69S and A137V. This strict correlation between the degree of 
      NS4B-NS3 interaction and DENV replication provides strong evidence that this 
      viral protein complex plays a pivotal role during the DENV replication cycle, 
      hence representing a promising target for novel antiviral strategies. IMPORTANCE: 
      With no approved therapy or vaccine against dengue virus infection, the viral 
      nonstructural protein 4B (NS4B) represents a possible drug target, because it is 
      indispensable for virus replication. However, little is known about its precise 
      structure and function. Here, we established the first comprehensive genetic 
      interaction map of NS4B, identifying amino acid residues that are essential for 
      virus replication, as well as second-site mutations compensating for their 
      defects. Additionally, we determined the NS4B viral interactome in infected cells 
      and identified the NS3 protease/helicase as a major interaction partner of NS4B. 
      We mapped residues in the cytosolic loop of NS4B as critical determinants for 
      interaction with NS3, as well as RNA replication. The strong correlation between 
      NS3-NS4B interaction and RNA replication provides strong evidence that this 
      complex plays a pivotal role in the viral replication cycle, hence representing a 
      promising antiviral drug target.
CI  - Copyright (c) 2015, American Society for Microbiology. All Rights Reserved.
FAU - Chatel-Chaix, Laurent
AU  - Chatel-Chaix L
AD  - Department of Infectious Diseases, Molecular Virology, University of Heidelberg, 
      Heidelberg, Germany.
FAU - Fischl, Wolfgang
AU  - Fischl W
AD  - Department of Infectious Diseases, Molecular Virology, University of Heidelberg, 
      Heidelberg, Germany.
FAU - Scaturro, Pietro
AU  - Scaturro P
AD  - Department of Infectious Diseases, Molecular Virology, University of Heidelberg, 
      Heidelberg, Germany.
FAU - Cortese, Mirko
AU  - Cortese M
AD  - Department of Infectious Diseases, Molecular Virology, University of Heidelberg, 
      Heidelberg, Germany.
FAU - Kallis, Stephanie
AU  - Kallis S
AD  - Department of Infectious Diseases, Molecular Virology, University of Heidelberg, 
      Heidelberg, Germany.
FAU - Bartenschlager, Marie
AU  - Bartenschlager M
AD  - Department of Infectious Diseases, Molecular Virology, University of Heidelberg, 
      Heidelberg, Germany.
FAU - Fischer, Bernd
AU  - Fischer B
AD  - Computational Genome Biology, German Cancer Research Center, Heidelberg, Germany.
FAU - Bartenschlager, Ralf
AU  - Bartenschlager R
AD  - Department of Infectious Diseases, Molecular Virology, University of Heidelberg, 
      Heidelberg, Germany ralf_bartenschlager@med.uni-heidelberg.de.
LA  - eng
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
DEP - 20150429
PL  - United States
TA  - J Virol
JT  - Journal of virology
JID - 0113724
RN  - 0 (Mutant Proteins)
RN  - 0 (NS3 protein, flavivirus)
RN  - 0 (NS4B protein, flavivirus)
RN  - 0 (Viral Nonstructural Proteins)
RN  - EC 3.4.21.- (Serine Endopeptidases)
RN  - EC 3.6.4.13 (RNA Helicases)
SB  - IM
MH  - Amino Acid Substitution
MH  - Cell Line
MH  - DNA Mutational Analysis
MH  - Dengue Virus/genetics/*physiology
MH  - Genetic Complementation Test
MH  - Hepatocytes/virology
MH  - Humans
MH  - Mass Spectrometry
MH  - Microbial Viability
MH  - Mutant Proteins/genetics/metabolism
MH  - Protein Binding
MH  - *Protein Interaction Mapping
MH  - RNA Helicases/metabolism
MH  - Reverse Genetics
MH  - Serine Endopeptidases/metabolism
MH  - Suppression, Genetic
MH  - Viral Nonstructural Proteins/genetics/*metabolism
MH  - *Virus Replication
PMC - PMC4473547
EDAT- 2015/05/01 06:00
MHDA- 2015/09/01 06:00
PMCR- 2016/01/15
CRDT- 2015/05/01 06:00
PHST- 2015/03/31 00:00 [received]
PHST- 2015/04/23 00:00 [accepted]
PHST- 2015/05/01 06:00 [entrez]
PHST- 2015/05/01 06:00 [pubmed]
PHST- 2015/09/01 06:00 [medline]
PHST- 2016/01/15 00:00 [pmc-release]
AID - JVI.00867-15 [pii]
AID - 00867-15 [pii]
AID - 10.1128/JVI.00867-15 [doi]
PST - ppublish
SO  - J Virol. 2015 Jul;89(14):7170-86. doi: 10.1128/JVI.00867-15. Epub 2015 Apr 29.