Construction and characterization of a full-length infectious simian T-cell lymphotropic virus type 3 molecular clone.
Together with their simian T-cell lymphotropic virus (STLV) equivalent, human T-cell lymphotropic virus type 1 (HTLV-1), HTLV-2, and HTLV-3 form the primate T-cell lymphotropic virus (PTLV) group. Over the years, understanding the biology and pathogenesis of HTLV-1 and HTLV-2 has been widely improved by the creation of molecular clones. In contrast, so far, PTLV-3 experimental studies have been restricted to the overexpression of the tax gene using reporter assays. We have therefore decided to construct an STLV-3 molecular clone. We generated a full-length STLV-3 proviral clone (8,891 bp) by PCR amplification of overlapping fragments. This STLV-3 molecular clone was then transfected into 293T cells. Reverse transcriptase PCR experiments followed by sequence analysis of the amplified products allowed us to establish that both gag and tax/rex mRNAs were transcribed. Western blotting further demonstrated the presence of the STLV-3 p24gag protein in the cell culture supernatant from transfected cells. Transient transfection of 293T cells and of 293T-long terminal repeat-green fluorescent protein cells with the STLV-3 clone promoted syncytium formation, a hallmark of PTLV Env expression, as well as the appearance of fluorescent cells, also demonstrating that the Tax3 protein was expressed. Virus particles were visible by electron microscopy. These particles are infectious, as demonstrated by our cell-free-infection experiments with purified virions. All together, our data demonstrate that the STLV-3 molecular clone is functional and infectious. This clone will give us a unique opportunity to study in vitro the different pX transcripts and the putative presence of antisense transcripts and to evaluate the PTLV-3 pathogenicity in vivo.
Chevalier
Sébastien Alain
SA
Unité d'Epidémiologie et Physiopathologie des Virus Oncogènes, CNRS URA 3015, Département de Virologie, Institut Pasteur, 28 rue du Docteur Roux, 75724 Paris cedex 15, France.
Walic
Marine
M
Calattini
Sara
S
Mallet
Adeline
A
Prévost
Marie-Christine
MC
Gessain
Antoine
A
Mahieux
Renaud
R
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D003001:Cloning, Molecular; D015967:Gene Expression Regulation, Viral; D049452:Green Fluorescent Proteins / Q000378:metabolism; D000882:Haplorhini; D006801:Humans; D008957:Models, Genetic; D008969:Molecular Sequence Data; D016376:Oligonucleotides, Antisense / Q000737:chemistry; D030282:Primate T-lymphotropic virus 3 / Q000235:genetics* / Q000502:physiology*; D012367:RNA, Viral / Q000378:metabolism; D014759:Viral Envelope Proteins / Q000737:chemistry
D016376:Oligonucleotides, Antisense; D012367:RNA, Viral; D014759:Viral Envelope Proteins; D049452:Green Fluorescent Proteins