A cell-line-specific defect in the intracellular transport and release of assembled retroviral capsids

Retrovirus assembly involves a complex series of events in which a large nu mber of proteins must be targeted to a point on the plasma membrane where i mmature viruses bud from the cell. Gag polyproteins of most retroviruses as semble an immature capsid on the cytoplasmic side of the plasma membrane du ring the budding process (C-type assembly), but a few assemble immature cap sids deep in the cytoplasm and are then transported to the plasma membrane (B- or D-type assembly), where they are enveloped. With both assembly pheno types, Gag polyproteins must be transported to the site of viral budding in either a relatively unassembled form (C type) or a completely assembled fo rm (B and D types). The molecular nature of this transport process and the host cell factors that are involved have remained obscure. During the devel opment of a recombinant baculovirus/insect cell system for the expression o f both C-type and D-type Gag polyproteins, we discovered an insect cell lin e (High Five),vith two distinct defects that resulted in the reduced releas e of virus-like particles. The first of these was a pronounced defect in th e transport of D-type but not C-type Gag polyproteins to the plasma membran e. High Five cells expressing wild-type Mason-Pfizer monkey virus (M-PMV) G ag precursors accumulate assembled immature capsids in large cytoplasmic ag gregates similar to a transport-defective mutant (MA-A18V), In contrast, a larger fraction of the Gag molecules encoded by the M-PMV C-type morphogene sis mutant (MA-R55W) and those of human immunodeficiency virus were transpo rted to the plasma membrane for assembly and budding of virions. When pulse -labeled Gag precursors from High Five cells were fractionated on velocity gradients, they sedimented more rapidly, indicating that they are sequester ed in a higher-molecular-mass complex. Compared to Sf9 insect cells, the Hi gh Five cells also demonstrate a defect in the release of C-type virus part icles. These findings support the hypothesis that host cell factors are imp ortant in the process of Gag transport and in the release of enveloped vira l particles.