FIG. 6.
Overexpression of HA-Nuc(212) in producer cells reduces Mo-MuLV release. 293T cells were transiently transfected with 8 μg of HA-p11, HA-Nuc(212), or nucleolin-Myc expression plasmid, together with 2 μg of plasmid expressing Mo-MuLV. The HA- and Myc-tagged proteins, but not Mo-MuLV, were expressed from plasmids containing the SV40 origin of replication. Levels of viral protein expression were determined 2 days later. (A) Quantitative RT assay (see Materials and Methods) of unpurified virions in supernatants of transfected cells. The HA- or Myc-tagged proteins that were coexpressed with Mo-MuLV are indicated below the columns. RT activity is represented in arbitrary pixel units, quantitated by a PhosphorImager, divided by the reaction time in minutes. Mock, transfection without adding plasmid DNA. (B) Cell extracts and purified virions from the experiment described for panel A were analyzed by the Western blot procedure as described for Fig. 2A. The membrane was probed with polyclonal antibodies against capsid. Positions of migration of size marker bands (right) and of full-length Pr65gag and capsid (left) are shown. (C) Exogenous RT assay of unpurified virions from supernatants of transfected cells. The HA- or Myc-tagged proteins indicated at the top were coexpressed with Mo-MuLV. MoZipWT virus that lacks RT was used as a mock control.