In the present study we examined the effect of Vpu inactivation on Gag-Env interaction particle release

The clones were screened using Southern Blot analysis with a lentiviral specific probe and clones with a single integration were further investigated. Whereas almost no survival of undifferentiated cells was obtained for differentiated single integrant clones OT #4 and OT# 11, more than 1% of Oct-3/4 positive cells were still observed after GCV treatment of NT clones. This incomplete elimination might be due to an epigenetic silencing of the transgene. Another explanation for the presence of Oct-3/4 positive cells after GCV treatment and differentiation might be contaminations by a few untransduced ES cells. To avoid this, the single integrant clones can be further subcloned to achieve pure single integrant ES cell clones but this is more time consuming. Furthermore, in contrast to the generation of single clones, the usage of mixed ES cell populations is much simpler to handle since the additional process of clone selection and screening by Southern blot is not necessary. Irrespectively whether the TK vector is not functional or not present, a pre-selection strategy can be employed to obtain pure ES cell populations that express TK. Therefore, we incorporated hygromycin resistance gene as a pre-selection tool in the LVs to select for transduced ES cells. And indeed, treatment of STPH-transduced ES cells with hygromycin and GCV abolished undifferentiated cells in vitro. Importantly, no teratoma formation in the in vivo studies could be observed when mice were injected with these pre-selected transduced ES cells and treated with GCV. This could be a promising application of LVs and the TK/GCV system in future clinical approaches. The GCV treatment has to be applied directly after ES cell injection to prevent tumor formation caused by pluripotent cells as it is not able to eliminate or stop the growth of established tumors. This in accordance with previously published results. In addition, we tried to raise copy numbers of STPHtransduced ES cells to decline the untransduced cell population. Even with higher average copy numbers, several ES cells seemed to be not transduced because undifferentiated cells survived the GCV treatment. In conclusion, complete elimination was only achieved by hygromycin pre-selection. The functional interaction of Vpu with Gag through a tetratricopeptide repeat protein, UBP has been shown and its role as an intermediate between Vpu and Gag was proposed to play an important role in virus assembly. A recent study demonstrated that Vpu enhances virion release by preventing endosomal accumulation of Gag. In addition to its effect on Gag trafficking, Vpu slows down the progress of Env glycoproteins along biosynthetic pathways, from ER and Golgi to plasma membrane. On the other hand, Gag MA has been shown to have predominant effect on Envelope function. Previous studies demonstrated that specific amino acid substitutions in the Gag MA region abrogate incorporation of HIV-1 Env glycoproteins into virus particles and redirects Echinatin particle assembly to intracellular sites. Several studies suggest that the cytoplasmic domain of Env gp41 and the MA domain of Gag, together with various cellular cofactors, play a central role in HIV1 assembly and specific alterations in the MA region of Gag affects Env incorporation onto virion particles, association with lipid rafts and thereby virion infectivity. While MA was shown to modulate Env assembly on mature virions, absence of Vpu has been shown to modulate Gag trafficking, assembly and infectivity besides modulation of particle release. Taken together, available information suggests interplay between Gag, Env and Vpu proteins and their Ginsenoside-Ro cross-talk seems to be important for assembly and production of infectious virus particles.

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