Insertion of a classical nuclear import signal into the matrix domain of the Rous sarcoma virus Gag protein interferes with virus replication.
The Rous sarcoma virus Gag protein undergoes transient nuclear trafficking during virus assembly. Nuclear import is mediated by a nuclear targeting sequence within the MA domain. To gain insight into the role of nuclear transport in replication, we investigated whether addition of a "classical " nuclear localization signal (NLS) in Gag would affect virus assembly or infectivity. A bipartite NLS derived from nucleoplasmin was inserted into a region of the MA domain of Gag that is dispensable for budding and infectivity. Gag proteins bearing the nucleoplasmin NLS insertion displayed an assembly defect. Mutant virus particles (RC.V8.NLS) were not infectious, although they were indistinguishable from wild-type virions in Gag, Gag-Pol, Env, and genomic RNA incorporation and Gag protein processing. Unexpectedly, postinfection viral DNA synthesis was also normal, as similar amounts of two-long-terminal-repeat junction molecules were detected for RC.V8.NLS and wild type, suggesting that the replication block occurred after nuclear entry of proviral DNA. Phenotypically revertant viruses arose after continued passage in culture, and sequence analysis revealed that the nucleoplasmin NLS coding sequence was deleted from the gag gene. To determine whether the nuclear targeting activity of the nucleoplasmin sequence was responsible for the infectivity defect, two critical basic amino acids in the NLS were altered. This virus (RC.V8.KR/AA) had restored infectivity, and the MA.KR/AA protein showed reduced nuclear localization, comparable to the wild-type MA protein. These data demonstrate that addition of a second NLS, which might direct MA and/or Gag into the nucleus by an alternate import pathway, is not compatible with productive virus infection.
Garbitt
Rachel A
RA
Department of Microbiology, Pennsylvania State University College of Medicine, Hershey, PA 17033, USA.
Bone
Karen R
KR
Parent
Leslie J
LJ
D021581:Active Transport, Cell Nucleus; D000595:Amino Acid Sequence; D000818:Animals; D001358:Avian Sarcoma Viruses / Q000235:genetics / Q000472:pathogenicity / Q000502:physiology*; D002467:Cell Nucleus / Q000378:metabolism*; D002478:Cells, Cultured; D015967:Gene Expression Regulation, Viral; D015683:Gene Products, gag / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D019913:Nuclear Localization Signals / Q000737:chemistry / Q000235:genetics*; D011784:Quail; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013347:Subcellular Fractions / Q000378:metabolism; D014763:Viral Matrix Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D019065:Virus Assembly; D014779:Virus Replication
D015683:Gene Products, gag; D019913:Nuclear Localization Signals; D011993:Recombinant Fusion Proteins; D014763:Viral Matrix Proteins; D049452:Green Fluorescent Proteins