PMID- 12663761
OWN - NLM
STAT- MEDLINE
DCOM- 20030505
LR  - 20190508
IS  - 0022-538X (Print)
IS  - 1098-5514 (Electronic)
IS  - 0022-538X (Linking)
VI  - 77
IP  - 8
DP  - 2003 Apr
TI  - Homo-oligomerization of the porcine reproductive and respiratory syndrome virus 
      nucleocapsid protein and the role of disulfide linkages.
PG  - 4546-57
AB  - As a step toward understanding the assembly pathway of the porcine reproductive 
      and respiratory syndrome virus (PRRSV), the oligomeric properties of the 
      nucleocapsid (N) protein were investigated. In this study, we have demonstrated 
      that under nonreducing conditions the N protein forms disulfide-linked 
      homodimers. However, inclusion of an alkylating agent (N-ethylmaleimide [NEM]) 
      prevented disulfide bond formation, suggesting that these intermolecular 
      disulfide linkages were formed as a result of spurious oxidation during cell 
      lysis. In contrast, N protein homodimers isolated from extracellular virions were 
      shown to have formed NEM-resistant intermolecular disulfide linkages, the 
      function of which is probably to impart stability to the virion. Pulse-chase 
      analysis revealed that N protein homodimers become specifically disulfide linked 
      within the virus-infected cell, albeit at the later stages of infection, 
      conceivably when the virus particle buds into the oxidizing environment of the 
      endoplasmic reticulum. Moreover, NEM-resistant disulfide linkages were shown to 
      occur only during productive PRRSV infection, since expression of recombinant N 
      protein did not result in the formation of NEM-resistant disulfide-linked 
      homodimers. Mutational analysis indicated that of the three conserved cysteine 
      residues in the N protein, only the cysteine at position 23 was involved in the 
      formation of disulfide linkages. The N protein dimer was shown to be stable both 
      in the presence and absence of intermolecular disulfide linkages, indicating that 
      noncovalent interactions also play a role in dimerization. Non-disulfide-mediated 
      N protein interactions were subsequently demonstrated both in vitro by the 
      glutathione S-transferase (GST) pull-down assay and in vivo by the mammalian 
      two-hybrid assay. Using a series of N protein deletion mutants fused to GST, 
      amino acids 30 to 37 were shown to be essential for N-N interactions. 
      Furthermore, since RNase A treatment markedly decreased N protein-binding 
      affinity, it appears that at least in vitro, RNA may be involved in bridging N-N 
      interactions. In cross-linking experiments, the N protein was shown to assemble 
      into higher-order structures, including dimers, trimers, tetramers, and 
      pentamers. Together, these findings demonstrate that the N protein possesses 
      self-associative properties, and these likely provide the basis for PRRSV 
      nucleocapsid assembly.
FAU - Wootton, Sarah K
AU  - Wootton SK
AD  - Department of Pathobiology, Ontario Veterinary College, University of Guelph, 
      Guelph, Ontario, Canada N1G 2W1.
FAU - Yoo, Dongwan
AU  - Yoo D
LA  - eng
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
PL  - United States
TA  - J Virol
JT  - Journal of virology
JID - 0113724
RN  - 0 (Disulfides)
RN  - 0 (Nucleocapsid Proteins)
RN  - O3C74ACM9V (Ethylmaleimide)
SB  - IM
MH  - Animals
MH  - Cell Line
MH  - Dimerization
MH  - Disulfides/chemistry/*metabolism
MH  - Ethylmaleimide/pharmacology
MH  - HeLa Cells
MH  - Humans
MH  - Nucleocapsid Proteins/chemistry/genetics/*metabolism
MH  - Porcine Reproductive and Respiratory Syndrome/virology
MH  - Porcine respiratory and reproductive syndrome virus/genetics/*metabolism
MH  - Swine
MH  - Virion/metabolism
PMC - PMC152152
EDAT- 2003/03/29 05:00
MHDA- 2003/05/06 05:00
PMCR- 2003/04/01
CRDT- 2003/03/29 05:00
PHST- 2003/03/29 05:00 [pubmed]
PHST- 2003/05/06 05:00 [medline]
PHST- 2003/03/29 05:00 [entrez]
PHST- 2003/04/01 00:00 [pmc-release]
AID - 2197 [pii]
AID - 10.1128/jvi.77.8.4546-4557.2003 [doi]
PST - ppublish
SO  - J Virol. 2003 Apr;77(8):4546-57. doi: 10.1128/jvi.77.8.4546-4557.2003.