73. An inhibitory effect of a sequence just upstream of the central polypurine tract of HIV-1 on RNA production of an HIV-1-based vector
Authors: Ryuta Sakuma, Aikichi Iwamoto, Yoshihiro Kitamura
Journal: Molecular Therapy (2004)

The central polypurine tract (cPPT) with downstream central termination sequence (CTS) (about 100nt apart) is common in all lentivirus genomes. In reverse transcription of human immunodeficiency virus type 1 (HIV-1), plus-strand DNA synthesis is initiated at cPPT and 3'PPT. The latter stops at CTS. Thus, the final product of reverse transcription is a linear DNA molecule with a stable plus strand overlap, the central DNA flap. Reportedly, flap-negative HIV-1 was unable to replicate and defective for nuclear import of preintegration complex. In contrast, many flap-negative HIV-1 vectors could transduce many kind of cells. To confirm these observations and to develop high-titer HIV-1 vectors, we tested an effect of insertion of cPPT/CTS on vector titers. We chose the HXN vector as a parental vector, which carried a TK-promoter-driven neomycin-resistant gene (neor) between the 5'- and 3'-LTRs. We inserted 282nt or 178nt fragment containing cPPT/CTS of the HIV-1LAI just upstream of the TK promoter in the HXN DNA in the sense or anti-sense orientation, generating HXN-282S, HXN-282R, HXN-178S, or HXN-178R DNA, respectively. We produced vector particles by cotransfecting 293T cells with one of the above vector DNAs together with expression plasmids for Gag-Pol (pCMV-GP) and vesicular somatitis viral envelope protein (pMD.G, a gift from Dr. Trono). We obtained about 2 times more G418-resistant colonies using HXN-178S or HXN-178R than the parental HXN vector normalized to one nanogram of p24, after two weeks of G418-selection. In contrast, the number of G418 resistant colonies with HXN-282S or HXN-282R was 5-10 times less than that with HXN. The two cPPT/CTS fragments differed in that the longer had 104 (=282-178) nt additional sequence at 5' end. We named it as the dZ sequence. In order to know whether the dZ sequence had made HXN-282S vector particles less infectious, we constructed pHXN-dZ, pHXN DNA with dZ but without cPPT/CTS by inserting dZ into pHXN. Surprisingly, RT-PCR revealed that little vector RNA was present in the culture supernatant even though abundant p24 was present. Similarly, northern blot analysis showed that little vector RNA was present in transfected Huh7 cells. We concluded that dZ completely decreased an intracellular amount of vector RNA. This conclusion simply predicted that insertion of the longer cPPT/CTS (282nt) — a fusion of the inhibitory dZ and the enhancing shorter cPPT (178nt) — to HXN would have resulted in production of vector particles with a medium amount of vector RNA per unit amount of p24 capsid protein. In fact, RT-PCR revealed that the RNA/p24 ratio of HXN-282S was about 8 times lower than that of HXN-178S and about 100 times higher than that of HXN-dZ as if a tug-of-war took place between the negative and positive sequence elements. dZ was likely to decrease an amount of intracellular RNA molecules by rendering them instable rather than by suppressing transcription per se because insertion of dZ upstream of luciferase gene did not affect luciferase activity very much.