No abstract

Packaging of retroviral genomic RNA during virion assembly is thought to be mediated by specific interactions between the gag polyprotein and RNA sequences (often termed the or E region) near the 5' end of the genome. For many retroviruses, including human immunodeficiency virus type 1 (HIV-1), the portions of the gag protein and the RNA that are required for this interaction remain poorly defined. We have used an RNA gel mobility shift assay to measure the in vitro binding of purified glutathione S-transferase-HIV-1 gag fusion proteins to RNA riboprobes. Both the complete gag polyprotein and the nucleocapsid (NC) protein alone were found to bind specifically to an HIV-1 riboprobe. Either Cys-His box of NC could be removed without eliminating specific binding to the riboprobe, but portions of gag containing only the MA and CA proteins without NC did not bind to RNA. There were at least two binding sites in HIV-1 genomic RNA that bound to the gag polyprotein: one entirely 5' to gag and one entirely within gag. The HIV-1 NC protein bound to riboprobes containing other retroviral t sequences almost as well as to the HIV-1 W riboprobe.

No abstract

The retroviral nucleocapsid (NC) protein is necessary for the specific encapsidation of the viral genomic RNA by the assembling virion. However, it is unclear whether NC contains the determinants for the specific recognition of the viral RNA or instead contributes nonspecific RNA contacts to strengthen a specific contact made elsewhere in the Gag polyprotein. To discriminate between these two possibilities, we have swapped the NC domains of the human immunodeficiency virus type 1 (HIV-1) and Moloney murine leukemia virus (M-MuLV), generating an HIV-1 mutant containing the M-MuLV NC domain and an M-MuLV mutant containing the HIV-1 NC domain. These mutants, as well as several others, were characterized for their abilities to encapsidate HIV-1, M-MuLV, and nonviral RNAs and to preferentially package genomic viral RNAs over spliced viral RNAs. We found that the M-MuLV NC domain mediates the specific packaging of RNAs containing the M-MuLV ⌿ packaging element, while the HIV-1 NC domain confers an ability to package the unspliced HIV-1 RNA over spliced HIV-1 RNAs. In addition, we found that the HIV-1 mutant containing the M-MuLV NC domain exhibited a 20-fold greater ability than wild-type HIV-1 to package a nonviral RNA. These results help confirm the notion that the NC domain specifically recognizes the retroviral genomic RNA during RNA encapsidation.