PMID- 25878113
OWN - NLM
STAT- MEDLINE
DCOM- 20150824
LR  - 20181113
IS  - 1098-5514 (Electronic)
IS  - 0022-538X (Print)
IS  - 0022-538X (Linking)
VI  - 89
IP  - 13
DP  - 2015 Jul
TI  - Mutation of Putative N-Glycosylation Sites on Dengue Virus NS4B Decreases RNA 
      Replication.
PG  - 6746-60
LID - 10.1128/JVI.00423-15 [doi]
AB  - Dengue virus (DENV) nonstructural protein 4B (NS4B) is an endoplasmic reticulum 
      (ER) membrane-associated protein, and mutagenesis studies have revealed its 
      significance in viral genome replication. In this work, we demonstrated that NS4B 
      is an N-glycosylated protein in virus-infected cells as well as in recombinant 
      protein expression. NS4B is N glycosylated at residues 58 and 62 and exists in 
      two forms, glycosylated and unglycosylated. We manipulated full-length infectious 
      RNA clones and subgenomic replicons to generate N58Q, N62Q, and N58QN62Q mutants. 
      Each of the single mutants had distinct effects, but the N58QN62Q mutation 
      resulted in dramatic reduction of viral production efficiency without affecting 
      secretion or infectivity of the virion in mammalian and mosquito C6/36 hosts. 
      Real-time quantitative PCR (qPCR), subgenomic replicon, and trans-complementation 
      assays indicated that the N58QN62Q mutation affected RNA replication possibly by 
      the loss of glycans. In addition, four intragenic mutations (S59Y, S59F, T66A, 
      and A137T) were obtained from mammalian and/or mosquito C6/36 cell culture 
      systems. All of these second-site mutations compensated for the replication 
      defect of the N58QN62Q mutant without creating novel glycosylation sites. In vivo 
      protein stability analyses revealed that the N58QN62Q mutation alone or plus a 
      compensatory mutation did not affect the stability of NS4B. Overall, our findings 
      indicated that mutation of putative N-glycosylation sites affected the biological 
      function of NS4B in the viral replication complex. IMPORTANCE: This is the first 
      report to identify and reveal the biological significance of dengue virus (DENV) 
      nonstructural protein 4B (NS4B) posttranslation N-glycosylation to the virus life 
      cycle. The study demonstrated that NS4B is N glycosylated in virus-infected cells 
      and in recombinant protein expression. NS4B is modified by glycans at Asn-58 and 
      Asn-62. Functional characterization implied that DENV NS4B utilizes the 
      glycosylation machinery in both mammalian and mosquito hosts. Four intragenic 
      mutations were found to compensate for replication and subsequent viral 
      production deficiencies without creating novel N-glycosylation sites or 
      modulating the stabilities of the protein, suggesting that glycans may be 
      involved in maintaining the NS4B protein conformation. NS4B glycans may be 
      necessary elements of the viral life cycle, but compensatory mutations can 
      circumvent their requirement. This novel finding may have broader implications in 
      flaviviral biology as the most likely glycan at Asn-62 of NS4B is conserved in 
      DENV serotypes and in some related flaviviruses.
CI  - Copyright (c) 2015, American Society for Microbiology. All Rights Reserved.
FAU - Naik, Nenavath Gopal
AU  - Naik NG
AD  - Molecular Cell Biology, Taiwan International Graduate Program, Graduate Institute 
      of Life Sciences, National Defense Medical Centre and Academia Sinica, Taipei, 
      Taiwan Institute of Molecular Biology, Academia Sinica, Taipei, Taiwan.
FAU - Wu, Huey-Nan
AU  - Wu HN
AD  - Institute of Molecular Biology, Academia Sinica, Taipei, Taiwan 
      hnwu@gate.sinica.edu.tw.
LA  - eng
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
DEP - 20150415
PL  - United States
TA  - J Virol
JT  - Journal of virology
JID - 0113724
RN  - 0 (Mutant Proteins)
RN  - 0 (NS4B protein, flavivirus)
RN  - 0 (RNA, Viral)
RN  - 0 (Viral Nonstructural Proteins)
SB  - IM
MH  - Animals
MH  - Cell Line
MH  - Dengue Virus/genetics/*physiology
MH  - Genetic Complementation Test
MH  - Glycosylation
MH  - Humans
MH  - Mutant Proteins/genetics/metabolism
MH  - *Mutation, Missense
MH  - RNA, Viral/*metabolism
MH  - Real-Time Polymerase Chain Reaction
MH  - Viral Nonstructural Proteins/genetics/*metabolism
MH  - *Virus Replication
PMC - PMC4468497
EDAT- 2015/04/17 06:00
MHDA- 2015/08/25 06:00
PMCR- 2016/01/01
CRDT- 2015/04/17 06:00
PHST- 2015/02/16 00:00 [received]
PHST- 2015/04/12 00:00 [accepted]
PHST- 2015/04/17 06:00 [entrez]
PHST- 2015/04/17 06:00 [pubmed]
PHST- 2015/08/25 06:00 [medline]
PHST- 2016/01/01 00:00 [pmc-release]
AID - JVI.00423-15 [pii]
AID - 00423-15 [pii]
AID - 10.1128/JVI.00423-15 [doi]
PST - ppublish
SO  - J Virol. 2015 Jul;89(13):6746-60. doi: 10.1128/JVI.00423-15. Epub 2015 Apr 15.