Construction and biological characterization of infectious molecular clones of HIV-1 subtypes B and E (CRF01_AE) generated by the polymerase chain reaction.
We previously described the use of extended polymerase chain reaction (PCR) to amplify contiguous 9.2-kilobase (kb) single-long terminal repeat (LTR) proviral sequences from HIV-1 genetic subtypes A through G. We now extend these findings by describing a novel vector system to recover infectious molecular clones from long PCR amplicons. Directional ligation of 9.2-kb proviral amplicons into a recovery vector reconstitutes missing LTR sequences, providing candidate molecular clones for infectivity screening. We show that a previously characterized infectious molecular clone of HIV-1 retains its biological properties upon recovery with this strategy. Three additional infectious molecular clones generated, from primary isolates of subtype B (HIV-1(WR27)) and circulating recombinant form 01_AE (subtype E) (HIV-1(CM235)) by subtype-specific LTR reconstitution, displayed biological properties reflecting their cognate parental isolates. This represents the first report of infectious molecular clones from circulating recombinant form 01_AE (subtype E).
Salminen
M O
MO
The Henry M. Jackson Foundation for the Advancement of Military Medicine, Rockville, Maryland 20850, USA.
Ehrenberg
P K
PK
Mascola
J R
JR
Dayhoff
D E
DE
Merling
R
R
Blake
B
B
Louder
M
M
Hegerich
S
S
Polonis
V R
VR
Birx
D L
DL
Robb
M L
ML
McCutchan
F E
FE
Michael
N L
NL
D002478:Cells, Cultured; D003001:Cloning, Molecular; D004279:DNA, Viral / Q000032:analysis; D016655:HIV Core Protein p24 / Q000032:analysis; D016325:HIV Long Terminal Repeat / Q000235:genetics; D015497:HIV-1 / Q000235:genetics* / Q000254:growth & development / Q000276:immunology / Q000502:physiology; D006801:Humans; D007700:Kinetics; D016133:Polymerase Chain Reaction / Q000379:methods; D011533:Proviruses / Q000235:genetics* / Q000502:physiology; D014779:Virus Replication
D004279:DNA, Viral; D016655:HIV Core Protein p24