PMID- 15582652
OWN - NLM
STAT- MEDLINE
DCOM- 20050208
LR  - 20061115
IS  - 0042-6822 (Print)
IS  - 0042-6822 (Linking)
VI  - 331
IP  - 1
DP  - 2005 Jan 5
TI  - A DNA-launched reverse genetics system for porcine reproductive and respiratory 
      syndrome virus reveals that homodimerization of the nucleocapsid protein is 
      essential for virus infectivity.
PG  - 47-62
AB  - Reverse genetic systems were developed for a highly virulent 'atypical' porcine 
      reproductive and respiratory syndrome virus (PRRSV). The full-length genome of 
      15395 nucleotides was assembled as a single cDNA clone and placed under either 
      the prokaryotic T7 or eukaryotic CMV promoter. Transfection of cells with the RNA 
      transcripts or the DNA clone induced cytopathic effects and produced infectious 
      progeny. The reconstituted virus was stable and grew to the titer of the parental 
      virus in cells. Upon infection, pigs produced clinical signs and lung pathology 
      typical for PRRSV and induced viremia and specific antibodies. Previously, we 
      showed that the PRRSV nucleocapsid (N) protein forms homodimers via both 
      noncovalent and covalent interactions and that cysteine at position 23 is 
      responsible for the covalent interaction. The functional significance of 
      cysteines of N for PRRSV infectivity was assessed using the infectious cDNA 
      clone. Each cysteine of N at positions 23, 75, and 90 was replaced with serine 
      and the individual mutation was incorporated into the cDNA clone such that three 
      independent cysteine mutants were constructed. When transfected, the wild type 
      and C75S clones induced cytopathic effects and produced infectious virus with 
      indistinguishable plaque morphology. In contrast, the C23S mutation completely 
      abolished infectivity of the clone, indicating that C23-mediated N protein 
      homodimerization plays a critical role in PRRSV infectivity. Unexpectedly, the 
      C90S mutation also appeared to be lethal for virus infectivity. Genome 
      replication and mRNA transcription were both positive for the 
      replication-defective C23S and C90S mutants. The data suggest that, in addition 
      to homodimerization, the PRRSV N protein may also undergo heterodimerization with 
      another structural protein using cysteine 90 and that the N protein 
      heterodimerization is essential for PRRSV infectivity.
FAU - Lee, Changhee
AU  - Lee C
AD  - Department of Pathobiology, Ontario Veterinary College, University of Guelph, 
      Guelph, Ontario, Canada N1G 2W1.
FAU - Calvert, Jay G
AU  - Calvert JG
FAU - Welch, Siao-Kun W
AU  - Welch SK
FAU - Yoo, Dongwan
AU  - Yoo D
LA  - eng
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
PL  - United States
TA  - Virology
JT  - Virology
JID - 0110674
RN  - 0 (Antibodies, Viral)
RN  - 0 (DNA, Complementary)
RN  - 0 (Nucleocapsid Proteins)
RN  - 0 (RNA, Viral)
SB  - IM
MH  - Animals
MH  - Antibodies, Viral/blood
MH  - Base Sequence
MH  - Cell Line
MH  - Cloning, Molecular
MH  - DNA, Complementary
MH  - Humans
MH  - Molecular Sequence Data
MH  - Mutagenesis, Site-Directed
MH  - Mutation
MH  - Nucleocapsid Proteins/*chemistry/physiology
MH  - Porcine Reproductive and Respiratory Syndrome/virology
MH  - Porcine respiratory and reproductive syndrome 
      virus/*chemistry/genetics/immunology/pathogenicity
MH  - RNA, Viral/chemistry
MH  - Recombination, Genetic
MH  - Swine
MH  - Viremia/blood
MH  - Virulence
MH  - Virus Replication/genetics/physiology
EDAT- 2004/12/08 09:00
MHDA- 2005/02/09 09:00
CRDT- 2004/12/08 09:00
PHST- 2004/05/19 00:00 [received]
PHST- 2004/08/25 00:00 [revised]
PHST- 2004/10/06 00:00 [accepted]
PHST- 2004/12/08 09:00 [pubmed]
PHST- 2005/02/09 09:00 [medline]
PHST- 2004/12/08 09:00 [entrez]
AID - S0042-6822(04)00680-4 [pii]
AID - 10.1016/j.virol.2004.10.026 [doi]
PST - ppublish
SO  - Virology. 2005 Jan 5;331(1):47-62. doi: 10.1016/j.virol.2004.10.026.