TY - JOUR
T1 - Purification and functional characterization of a biologically active full-length feline immunodeficiency virus (FIV) pr50gag
AU - Krishnan, Anjana
AU - Pillai, Vineeta N.
AU - Chameettachal, Akhil
AU - Ali, Lizna Mohamed
AU - Pitchai, Fathima Nuzra Nagoor
AU - Tariq, Saeed
AU - Mustafa, Farah
AU - Marquet, Roland
AU - Rizvi, Tahir A.
N1 - Publisher Copyright:
© 2019 by the authors. Licensee MDPI, Basel, Switzerland.
PY - 2019/8
Y1 - 2019/8
N2 - The feline immunodeficiency virus (FIV) full-length Pr50Gag precursor is a key player in the assembly of new viral particles. It is also a critical component of the efficient selection and packaging of two copies of genomic RNA (gRNA) into the newly formed virus particles from a wide pool of cellular and spliced viral RNA. To understand the molecular mechanisms involved during FIV gRNA packaging, we expressed the His6-tagged and untagged recombinant FIV Pr50Gag protein both in eukaryotic and prokaryotic cells. The recombinant Pr50Gag-His6-tag fusion protein was purified from soluble fractions of prokaryotic cultures using immobilized metal affinity chromatography (IMAC). This purified protein was able to assemble in vitro into virus-like particles (VLPs), indicating that it preserved its ability to oligomerize/multimerize. Furthermore, VLPs formed in eukaryotic cells by the FIV full-length Pr50Gag both in the presence and absence of His6-tag could package FIV sub-genomic RNA to similar levels, suggesting that the biological activity of the recombinant full-length Pr50Gag fusion protein was retained in the presence of His6-tag at the carboxy terminus. Successful expression and purification of a biologically active, recombinant full-length Pr50Gag-His6-tag fusion protein will allow study of the intricate RNA-protein interactions involved during FIV gRNA encapsidation.
AB - The feline immunodeficiency virus (FIV) full-length Pr50Gag precursor is a key player in the assembly of new viral particles. It is also a critical component of the efficient selection and packaging of two copies of genomic RNA (gRNA) into the newly formed virus particles from a wide pool of cellular and spliced viral RNA. To understand the molecular mechanisms involved during FIV gRNA packaging, we expressed the His6-tagged and untagged recombinant FIV Pr50Gag protein both in eukaryotic and prokaryotic cells. The recombinant Pr50Gag-His6-tag fusion protein was purified from soluble fractions of prokaryotic cultures using immobilized metal affinity chromatography (IMAC). This purified protein was able to assemble in vitro into virus-like particles (VLPs), indicating that it preserved its ability to oligomerize/multimerize. Furthermore, VLPs formed in eukaryotic cells by the FIV full-length Pr50Gag both in the presence and absence of His6-tag could package FIV sub-genomic RNA to similar levels, suggesting that the biological activity of the recombinant full-length Pr50Gag fusion protein was retained in the presence of His6-tag at the carboxy terminus. Successful expression and purification of a biologically active, recombinant full-length Pr50Gag-His6-tag fusion protein will allow study of the intricate RNA-protein interactions involved during FIV gRNA encapsidation.
KW - Feline immunodeficiency virus (FIV)
KW - Gag protein purification
KW - His-tag fusion protein
KW - Pr50 protein expression
KW - Retroviral RNA packaging
KW - Viral assembly
UR - http://www.scopus.com/inward/record.url?scp=85070100689&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=85070100689&partnerID=8YFLogxK
U2 - 10.3390/v11080689
DO - 10.3390/v11080689
M3 - Article
C2 - 31357656
AN - SCOPUS:85070100689
SN - 1999-4915
VL - 11
JO - Viruses
JF - Viruses
IS - 8
M1 - 689
ER -