Fig. 4.

Viral target for H2 mediated resistance. (A) Comparison between different WT HIV and retroviral vectors gene products (arrow) restricted by H2 cells. Amplified dashed box represents the common features: the U3 deleted 3′LTR (dU3LTR), containing the poly(A) signal and a GT rich region, both defining the transcript end. RRE, Rev-responsive element; SFFV, spleen focus forming virus; WPRE, Woodchuck posttranscriptional regulatory element. (B) HIV-puro and a modified HIV-puro with SV40 poly(A) or a BGH poly(A) sequence replacing dU3LTR were transiently transfected into TE or H2 cell lines. Two days after transfection, puromycin was added to the medium, and, 5–8 days later, resistant colonies were counted after Giemsa staining. Results are shown as the ratio between the numbers of TE versus H2 resistant colonies and are representative of 3 independent experiments. Error bars represent standard error. (C) In vitro polyadenylation reaction. Poly(A) addition. The ³²P-labeled precleaved RNA substrate was incubated in nuclear extracts from HeLa-CD4-pBabe-HAZ, HeLa-CD4-H2, or HeLa cells and the recombinant eIF3f-GST, N91-eIF3f-GST, and GST proteins, with ATP for 30 min at 30 °C. The RNA products were isolated and resolved on a denaturing 10% polyacrylamide gel. Lanes 10 and 11 are controls of polyadenylation. Precleaved HIV-1 input and HeLa nuclear extracts with 3′dATP or ATP respectively. (D) Poly(A) site cleavage. The ³²P-labeled uncleaved 3′LTR HIV RNA substrate was incubated with nuclear extracts of TE671 or HeLa cells expressing empty vector control (HAZ) or N91-eIF3f (H2) for 30 min at 30 °C. The RNA products were isolated and resolved on a denaturing 10% polyacrylamide gel. The 3′LTR RNA substrate (HIVwt) or a substrate with a core poly(A) hexamer deletion (HIVΔHex) were incubated with HeLa HAZ extracts for 5′cleavage product control.