Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2004 Dec;78(24):13534-42.
doi: 10.1128/JVI.78.24.13534-13542.2004.

Insertion of a classical nuclear import signal into the matrix domain of the Rous sarcoma virus Gag protein interferes with virus replication

Rachel A Garbitt  1 , Karen R Bone, Leslie J Parent
Affiliations

Insertion of a classical nuclear import signal into the matrix domain of the Rous sarcoma virus Gag protein interferes with virus replication

Rachel A Garbitt et al. J Virol. 2004 Dec.

Abstract

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.

PubMed Disclaimer

Figures

FIG. 1.
FIG. 1.
Schematic diagram of wild-type and mutant MA proteins. The RSV Gag polyprotein is depicted at the top, with the MA, p2, p10, CA, NC, and PR domains indicated. Gag.NLS contains a bipartite NLS derived from nucleoplasmin inserted between positions 86 and 100 of the MA domain. Gag.NLS.KR/AA contains substitutions of K to A and R to A as shown.
FIG. 2.
FIG. 2.
Subcellular localization of wild-type and mutant MA-GFP and Gag-GFP fusion proteins. (A) Live QT6 cells expressing wild-type or mutant MA-GFP fusion proteins were examined by fluorescence confocal microscopy 18 to 24 h after transfection. (B) QT6 cells transfected with plasmid DNAs expressing wild-type or mutant Gag-GFP fusion proteins were either untreated (−LMB) or treated (+LMB) with 20 nM LMB for 2 h and examined by confocal microscopy.
FIG. 3.
FIG. 3.
Budding efficiency of NLS mutant viruses in avian cells. (A) QT6 cells were transiently transfected with the indicated wild-type or mutant plasmids, metabolically labeled for either 5 min (left panel) or 2.5 h (right panel), and lysed, and viral proteins were immunoprecipitated from lysates and growth media by using polyclonal RSV antiserum. (B) The amount of Gag polyprotein (Pr76) isolated from cell lysates after the 5-min pulse and the amount of CA released into the medium after the 2.5-h labeling period were quantified by phosphorimager analysis. Budding efficiency was calculated by comparing the amount of CA released into the medium to the amount of Pr76 produced in cells. Budding efficiency for the wild type was assigned a value of 100, and mutant budding values were compared to the wild-type value. Each bar represents the average of eight independent experiments. The budding efficiency of RC.V8.NLS was statistically different from that of wild type (RC.V8) (P < 0.0001).
FIG. 4.
FIG. 4.
Infectivity of the RC.V8.NLS viral mutant. QT6 cells were transfected with either the wild-type, RC.V8.NLS, or RC.V8.KR/AA proviral constructs and passaged in culture every 3 days for 21 days. Medium was collected prior to each passage, pelleted through a 25% sucrose cushion, and frozen. At the end of the 3-week period, RT assays on all of the stored samples were performed in triplicate and average values were determined for each.
FIG. 5.
FIG. 5.
Isolation and sequencing of viral revertants. (A and B) QT6 cells were transiently transfected with proviral constructs expressing the wild-type or mutant sequences and passaged every 3 days for periods ranging from 21 to 49 days, and media were analyzed as described in the legend for Fig. 4. (C) Following transfection of QT6 cells, equivalent amounts of virus particles, as determined by RT activity, were added to fresh QT6 cells, which were then passaged and analyzed as described for panels A and B. (D) Genomic DNA was extracted from the transfected cells (from panels A and B) or infected cells (from panel C) and subjected to PCR amplification of the gag gene, and the PCR products were analyzed by dideoxy sequencing. The predicted amino acid sequence of the NLS mutant and three sequences of phenotypically revertant viruses isolated from independent experiments are shown. The bold letters represent the nucleoplasmin NLS sequence, and the dashed lines indicate amino acids that were deleted from the revertant viruses.
FIG. 6.
FIG. 6.
Quantitative viral RNA packaging. Virus-associated RNA was extracted from virus particles, which were collected from the media of transiently transfected QT6 cells. (A) RPAs were performed with a probe which spans the 3′ env splice site to distinguish between spliced (183-bp) and unspliced (263-bp) viral RNA. As a control, 5 μg of yeast RNA was added to the reaction mixture to indicate that an adequate concentration of RNase A/T1 was used (lane 7). Undigested probe is shown as a 316-bp band (lane 8). A twofold dilution of each viral RNA was included to demonstrate that the RPA was in the linear range (lanes 2, 4, and 6). (B) Viral RNA packaging efficiency for the wild type (RC.V8) was assigned a value of 1.0, and the ratios for each mutant are shown relative to the wild-type ratio. Each bar represents the average of at least three independent experiments.
FIG. 7.
FIG. 7.
Viral DNA synthesis postinfection. QT6 cells were infected with the indicated virus, low-molecular-weight DNA was isolated 18 h later, and PCR analysis was performed using primers specific for 2-LTR circle junctions (A) and quail mitochondrial DNA (B). Decreasing amounts of DNA in serial twofold dilutions were used as the template for each PCR, as indicated by the black triangles. Wild-type virus (RC.V8) was heat inactivated at 65°C (RC.V8 HI) for 20 min prior to infection, and Hirt supernatant DNA was used undiluted as a negative control for the 2-LTR circle PCR primers and as a positive control for the quail mitochondrial DNA PCR primers.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Cairns, T. M., and R. C. Craven. 2001. Viral DNA synthesis defects in assembly-competent Rous sarcoma virus CA mutants. J. Virol. 75:242-250. - PMC - PubMed
    1. Callahan, E. M., and J. W. Wills. 2000. Repositioning basic residues in the M domain of the Rous sarcoma virus Gag protein. J. Virol. 74:11222-11229. - PMC - PubMed
    1. Callahan, E. M., and J. W. Wills. 2003. Link between genome packaging and rate of budding for Rous sarcoma virus. J. Virol. 77:9388-9398. - PMC - PubMed
    1. Conti, E., and E. Izaurralde. 2001. Nucleocytoplasmic transport enters the atomic age. Curr. Opin. Cell Biol. 13:310-319. - PubMed
    1. Craven, R. C., A. E. Leure-duPree, R. A. Weldon, Jr., and J. W. Wills. 1995. Genetic analysis of the major homology region of the Rous sarcoma virus Gag protein. J. Virol. 69:4213-4227. - PMC - PubMed

Publication types

MeSH terms

LinkOut - more resources