Retroviral nucleocapsid domains mediate the specific recognition of genomic viral RNAs by chimeric Gag polyproteins during RNA packaging in vivo.
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 psi 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.
Berkowitz
R D
RD
Department of Microbiology, Columbia University, New York, New York 10032, USA.
Ohagen
A
A
Höglund
S
S
Goff
S P
SP
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002213:Capsid / Q000378:metabolism*; D002460:Cell Line; D002522:Chlorocebus aethiops; D017931:DNA Primers; D015683:Gene Products, gag / Q000378:metabolism*; D005814:Genes, Viral; D016325:HIV Long Terminal Repeat; D015497:HIV-1 / Q000235:genetics / Q000378:metabolism*; D007668:Kidney; D008969:Molecular Sequence Data; D008979:Moloney murine leukemia virus / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D016133:Polymerase Chain Reaction; D012326:RNA Splicing; D012367:RNA, Viral / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015183:Restriction Mapping; D014162:Transfection; D014758:Viral Core Proteins / Q000378:metabolism*
D017931:DNA Primers; D015683:Gene Products, gag; D012367:RNA, Viral; D011993:Recombinant Fusion Proteins; D014758:Viral Core Proteins