PMID- 17522233
OWN - NLM
STAT- MEDLINE
DCOM- 20071017
LR  - 20201209
IS  - 0022-538X (Print)
IS  - 1098-5514 (Electronic)
IS  - 0022-538X (Linking)
VI  - 81
IP  - 18
DP  - 2007 Sep
TI  - Identification of nonessential regions of the nsp2 replicase protein of porcine 
      reproductive and respiratory syndrome virus strain VR-2332 for replication in 
      cell culture.
PG  - 9878-90
AB  - The nonstructural protein 2 (nsp2) of porcine reproductive and respiratory 
      syndrome virus (PRRSV) is a multidomain protein and has been shown to undergo 
      remarkable genetic variation, primarily in its middle region, while exhibiting 
      high conservation in the N-terminal putative protease domain and the C-terminal 
      predicted transmembrane region. A reverse genetics system of PRRSV North American 
      prototype VR-2332 was developed to explore the importance of different regions of 
      nsp2 for viral replication. A series of mutants with in-frame deletions in the 
      nsp2 coding region were engineered, and infectious viruses were subsequently 
      recovered from transfected cells and further characterized. The results 
      demonstrated that the cysteine protease domain (PL2), the PL2 downstream flanking 
      sequence (amino acids [aa] 181 to 323), and the putative transmembrane domain 
      were critical for replication. In contrast, the segment of nsp2 preceding the PL2 
      domain (aa 13 to 35) was dispensable for viral replication, and the nsp2 middle 
      hypervariable region (aa 324 to 813) tolerated 100-aa or 200-aa deletions but 
      could not be removed as a whole; the largest deletion was about 400 aa 
      (nsp2Delta324-726). Characterization of the mutants demonstrated that those with 
      small deletions possessed growth kinetics and RNA expression profiles similar to 
      those of the parental virus, while the nsp2Delta324-726 mutant displayed 
      decreased cytolytic activity on MARC-145 cells and did not develop visible 
      plaques. Finally, the utilization of the genetic flexibility of nsp2 to express 
      foreign genes was examined by inserting the gene encoding green fluorescent 
      protein (GFP) in frame into one nsp2 deletion mutant construct. The recombinant 
      virus was viable but impaired and unstable and gradually gained parental growth 
      kinetics by the loss of most of the GFP gene.
FAU - Han, Jun
AU  - Han J
AD  - Department of Veterinary and Biomedical Sciences, University of Minnesota, Saint 
      Paul, MN 55108, USA.
FAU - Liu, Gongping
AU  - Liu G
FAU - Wang, Yue
AU  - Wang Y
FAU - Faaberg, Kay S
AU  - Faaberg KS
LA  - eng
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
DEP - 20070523
PL  - United States
TA  - J Virol
JT  - Journal of virology
JID - 0113724
RN  - 0 (Membrane Proteins)
RN  - 0 (RNA, Viral)
RN  - 0 (Viral Nonstructural Proteins)
RN  - EC 2.7.7.48 (RNA-Dependent RNA Polymerase)
RN  - EC 3.4.22.- (Cysteine Endopeptidases)
RN  - EC 3.4.22.- (nsP2 proteinase)
SB  - IM
MH  - Amino Acid Sequence
MH  - Animals
MH  - Cell Line
MH  - Chlorocebus aethiops
MH  - Cysteine Endopeptidases/genetics/*metabolism
MH  - Gene Expression Regulation, Viral/genetics
MH  - Kinetics
MH  - Membrane Proteins/genetics/*metabolism
MH  - Porcine Reproductive and Respiratory Syndrome/genetics/metabolism
MH  - Porcine respiratory and reproductive syndrome virus/genetics/*metabolism
MH  - Protein Structure, Tertiary/genetics
MH  - RNA, Viral/biosynthesis/genetics
MH  - RNA-Dependent RNA Polymerase/genetics/*metabolism
MH  - Sequence Deletion
MH  - Swine
MH  - Viral Nonstructural Proteins/genetics/*metabolism
MH  - Virus Replication/*physiology
PMC - PMC2045381
EDAT- 2007/05/25 09:00
MHDA- 2007/10/18 09:00
PMCR- 2008/01/01
CRDT- 2007/05/25 09:00
PHST- 2007/05/25 09:00 [pubmed]
PHST- 2007/10/18 09:00 [medline]
PHST- 2007/05/25 09:00 [entrez]
PHST- 2008/01/01 00:00 [pmc-release]
AID - JVI.00562-07 [pii]
AID - 0562-07 [pii]
AID - 10.1128/JVI.00562-07 [doi]
PST - ppublish
SO  - J Virol. 2007 Sep;81(18):9878-90. doi: 10.1128/JVI.00562-07. Epub 2007 May 23.