JVI Accepts, published online ahead of print on 8 February 2012 J. Virol. doi:10.1128/JVI.07199-11 Copyright © 2012, American Society for Microbiology. All Rights Reserved.

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CRM1-dependent trafficking of retroviral Gag proteins revisited

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Mariju F. Baluyot1*, Sarah A. Grosse2*, Terri D. Lyddon3, Sanath Kumar Janaka3, Marc

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C. Johnson3#

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1New

York Medical College, Valhalla, NY 2Rosalind Franklin University of Medicine

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and Science, Chicago, IL 3Molecular Microbiology and Immunology, Christopher S.

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Bond Life Science Center, University of Missouri, Columbia, MO 65211

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*The

following authors contributed equally to this work

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#Corresponding

author:

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Department of Molecular Microbiology and Immunology,

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Christopher S. Bond Life Science Center,

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University of Missouri, Columbia, MO 65211

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Phone: 573-882-1519

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Email: [email protected]

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Running title: Lack of Crm1 dependent Gag trafficking

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Abstract

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We analyzed the nuclear trafficking ability of Gag proteins from six retroviral

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genera. Contrary to a previous report, Human immunodeficiency virus (HIV-1) Gag

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showed no propensity to cycle through the nucleus. The only Gag protein that

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displayed CRM1-dependent nuclear cycling was that of Rous sarcoma virus (RSV).

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Surprisingly, this cycling could be eliminated without compromising infectivity by

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replacing the RSV Gag N-terminal matrix (MA) domain with HIV MA.

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The retroviral structural protein, Gag, from several viral genera has been reported

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to be present in minor amounts in the nucleus of infected cells (3, 11, 14, 17).

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These viruses include RSV, human immunodeficiency virus (HIV-1), murine

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leukemia virus (MLV) and prototypic foamy virus (PFV). For RSV, the mechanism of

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this trafficking has been carefully studied. RSV Gag contains two nuclear localization

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sequences (NLSs), one in the matrix (MA) domain and one in the nucleocapsid (NC)

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domain of Gag. These NLSs bind to the nuclear import receptors importin-11 and

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importin-alpha, respectively (1, 5). RSV Gag also contains a nuclear export sequence

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(NES) in the p10 domain that interacts with the CRM1 nuclear export machinery

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(14, 16). At steady state very little RSV Gag is located in the nucleus, but treatment

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of cells with the CRM1 inhibitor leptomycin B (LMB) results in a striking

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accumulation of Gag in the nucleus within minutes of treatment (14). It was been

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proposed that the nuclear trafficking of RSV Gag is required for proper packaging of

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the genomic RNA (4, 5, 12). In support of this hypothesis, RSV Gag mutants that do

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not traffic through the nucleus fail to properly package their RNA genome and are

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non-infectious (2, 4, 15, 16). However, another report suggests that RSV Gag does

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not play a role in the export of unspliced genomic RNA (9).

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For HIV-1, according to a single report (3), the MA domain contains both an NLS

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and an NES sequence. A HIV-1 Gag mutant know as M4 that contains two point

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mutations in MA (K18A, R22G) was reported to be severely replication deficient,

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apparently due to loss of genomic RNA packaging. According to the data in that

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study, wildtype HIV-1 Gag was exclusively cytoplasmic, but HIV-1 M4 Gag

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accumulated in the nucleus. Further, a green fluorescent protein (GFP)-tagged HIV-

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1 MA was also expressed exclusively in the cytoplasm, but accumulated in the

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nucleus upon LMB treatment (3).

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For PFV it was recently reported that cell division was required for nuclear Gag

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accumulation, that PFV Gag does not have a functional NLS, and that the nuclear

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trafficking of PFV Gag was not essential for infectivity (10). The report of MLV Gag

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nuclear accumulations did not determine the mechanism of transport (11).

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Because it appeared from these publications that nuclear trafficking of retroviral

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Gag proteins might be a common feature among retroviruses, we chose to assess the

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nuclear trafficking characteristics of fluorescently tagged Gag proteins from each of

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the major retroviral genera (Fig 1A). These viruses included an alpharetrovirus

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(RSV)(18), a betaretrovirus (Mason-Pfizer monkey virus (MPMV)), a

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gammaretrovirus (MLV), a deltaretrovirus (HTLV-1), a lentivirus (HIV-1)(6), and a

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spumaretrovirus (human foamy virus (HFV) GagGFP). Plasmid constructs

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expressing each of these tagged Gag proteins were transfected into 293FT cells

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(Invitrogen) using Fugene6 and analyzed by western blotting to confirm

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appropriate protein expression using an antibody against GFP (Fig 1B). Next, the

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transfections were repeated in glass bottom dishes (Mattek) and cells were imaged

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the next day by epifluorescence. None of the cells other than a portion of the HFV

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Gag expressing cells displayed significant nuclear accumulation of Gag. To

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determine if any of the Gag proteins were being exported from the nucleus by the

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CRM1 pathway, we treated the cells with LMB (10 ng/ml) and imaged the cells 30

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minutes later (Fig 2). As expected (14), RSV Gag was found to be predominantly in

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the nucleus after treatment. None of the other retroviral Gag proteins demonstrated

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increased nuclear accumulation after LMB treatment, although MPMV Gag began to

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lose nuclear exclusion in some cells (Fig. 2). By four hours post treatment the

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majority of MPMV Gag expressing cells had lost nuclear exclusion, but even after 24

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hours there was no accumulation in the nucleus like was observed with RSV Gag.

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Each transfection was also repeated in the HeLa derived cell line TZM-bl cells, with

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identical results (not shown). It should be noted that this assay would only show

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nuclear accumulation if Gag contain both a nuclear import sequence and a CRM-1

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dependent export sequence.

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The absence of HIV Gag accumulation after LMB treatment was unexpected, given

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the apparently unambiguous data in the single relevant publication. To further

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probe the CRM1-dependent trafficking of HIV-1 Gag, we generated an HIV-1 MA-GFP

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expression construct, as well as HIV- GagGFP and MAGFP constructs with the M4

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mutations in MA exactly as previously described (3). Surprisingly, we saw no

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nuclear accumulation of any of these proteins with or without LMB treatment (Fig.

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2).

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Because RSV MA contains an NLS, but in our experiments HIV MA does not, we

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tested whether an RSV Gag chimera that contained HIV MA in place of RSV MA

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(H/RSV) would continue to traffic through the nucleus. This construct was

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generated in the context of RSV GagGFP and tested as above (Fig 3A,B). At steady

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state H/RSV GagGFP was found predominantly in the cytoplasm, and LMB treatment

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did not lead to significant nuclear accumulation.

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To test if this chimeric protein is capable of generating infectious particles, the

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chimeric gene was introduced into an RSV provirus. Because the major splice donor

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for RSV is located in MA, we were not able to generate a chimera capable for

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producing both Gag and Env. Therefore, an Env-defective provirus was used and a

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functional viral glycoprotein was provided in trans from a separate plasmid. To

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monitor infectivity, we used a two-color flow cytometry system described

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previously (7). For this, we engineered a plasmid that contained a GFP cassette

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under the control of a CMV promoter within the RSV provirus in place of Env, and a

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red fluorescent protein cassette (TdTomato) also under CMV control in the plasmid

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backbone (Fig 3C). Cells transfected with the provirus express both red and green.

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However, if infectious particles are generated, the newly infected cells express green

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only, because only the GFP expression cassette is incorporated into the viral

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genome. The chicken fibroblast cell line DF1 was transfected with this provirus

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alone, or in combination with a VSV-G expression vector, and the cells were

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collected for analysis two to three days later. Infectivity was measured as the ratio

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of transfected (red and green) cells to infected (green only) cells (Fig 3C, Table 1).

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Because unlike RSV Gag the H/RSV Gag chimera does not cycle through the nucleus

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in a CRM-dependent fashion, we did not expect it to be capable of generating

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infectious particles. To our surprise, the H/RSV chimera was still fully capable of

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generating infectious particles, with the infectivity reduced only a few-fold

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compared with wildtype. This is the first known example of an RSV Gag protein that

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does not display significant CRM1-dependent nuclear trafficking, but remains

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capable of producing infectious particles.

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If nuclear trafficking of the chimeric RSV Gag is not required for infectivity, then the

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CRM1-dependent NES in p10 should be dispensable in this context. Unfortunately,

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the NES in RSV p10 overlaps an important structural domain that is required for

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proper assembly (8, 13, 15). Because both functions have been probed extensively,

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we were able to predict which amino acids are required for each function. The

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position most likely to abolish the NES function without completely destroying

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structural function is the Leucine 21 amino acid residues before the p10/CA

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cleavage site. When this amino acid was mutated to Alanine (L219A) the NES

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activity was abolished (15), but mutation of the same amino acid to Methionine did

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not abolish viral assembly in an in vitro assembly assay (8). We therefore

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introduced this L219A mutation into the RSV and H/RSV GagGFP constructs and

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into the proviral construct (Fig. 3D,E). As expected, the wildtype RSV GagGFP was

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cytoplasmic at steady state, but GagGFP L219A was exclusively nuclear. In contrast

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H/RSV GagGFP was predominantly cytoplasmic both with and without the L219A

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mutation. Although there was no nuclear accumulation of H/RSV GagGFP L219A, a

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fraction of the cells did not display complete nuclear exclusion. It is not clear

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whether this was a result of cell division or very weak nuclear import.

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Next we tested the NES deficient mutants for infectivity (Fig. 3C, Table 1). As

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expected, the L219A mutation dramatically reduced wildtype RSV infectivity

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(average 33-fold drop, Table 1). The effect on the H/RSV chimera was less dramatic,

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reducing infectivity by 6.5 fold on average. It is not possible to know with certainty

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if this drop was due to an effect on the NES or to changes in the overlapping

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structural domain. This experiment was repeated three times and the H/RSV

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chimera on average was 2.5-fold less infections than wildtype RSV when the NES

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was intact, but 2.5 more infection than wildtype RSV when the NES was inactivated

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(Table 1). Because the H/RSV chimera and the L219A mutation undoubtedly cause

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addition changes to the viral lifecycle, besides affecting the NLS and NES functions,

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we cannot conclude that the nuclear trafficking of Gag does not contribute to

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genome packaging efficiency. However, because the non-trafficking Gag proteins

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generate infectious particles, we can conclude that bulk CRM1-dependent nuclear

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trafficking of Gag is not absolutely required for genome packaging and infectivity.

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Since the imaging assay used to analyze Gag trafficking is qualitative, it is not

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possible to exclude that a small percentage of Gag molecules traffic through the

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nucleus. Nor can it be excluded that there is trafficking that does not utilize the

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CRM1 export pathway. However, our data provide no evidence that nuclear

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trafficking is a critical step in the production of infectious viral particles for any of

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the retroviruses tested.

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Acknowledgements.

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We thank John Wills for the RSV GagGFP plasmid, Gisela Heideker and David Derse

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for the HTLV plasmid, Alan Rein for an MLV plasmid, Marilyn Resh for the HIV-1

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plasmid, and Maxine Linial for the HFV plasmid. This work was supported by U.S.

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Public Health Service grant AI73098.

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1.

2. 3.

4. 5.

6.

Butterfield-Gerson, K. L., L. Z. Scheifele, E. P. Ryan, A. K. Hopper, and L. J. Parent. 2006. Importin-beta family members mediate alpharetrovirus gag nuclear entry via interactions with matrix and nucleocapsid. Journal of Virology 80:1798-1806. 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. Dupont, S., N. Sharova, C. DeHoratius, C. M. Virbasius, X. Zhu, A. G. Bukrinskaya, M. Stevenson, and M. R. Green. 1999. A novel nuclear export activity in HIV-1 matrix protein required for viral replication. Nature 402:681-685. Garbitt-Hirst, R., S. P. Kenney, and L. J. Parent. 2009. Genetic evidence for a connection between Rous sarcoma virus gag nuclear trafficking and genomic RNA packaging. J Virol 83:6790-6797. Gudleski, N., J. M. Flanagan, E. P. Ryan, M. C. Bewley, and L. J. Parent. 2010. Directionality of nucleocytoplasmic transport of the retroviral gag protein depends on sequential binding of karyopherins and viral RNA. Proc Natl Acad Sci U S A 107:9358-9363. Hermida-Matsumoto, L., and M. D. Resh. 2000. Localization of human immunodeficiency virus type 1 Gag and Env at the plasma membrane by confocal imaging. J Virol 74:8670-8679. 8

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Jorgenson, R. L., V. M. Vogt, and M. C. Johnson. 2009. Foreign glycoproteins can be actively recruited to virus assembly sites during pseudotyping. J Virol 83:4060-4067. Joshi, S. M., and V. M. Vogt. 2000. Role of the Rous sarcoma virus p10 domain in shape determination of gag virus-like particles assembled in vitro and within Escherichia coli. J Virol 74:10260-10268. LeBlanc, J. J., S. Uddowla, B. Abraham, S. Clatterbuck, and K. L. Beemon. 2007. Tap and Dbp5, but not Gag, are involved in DR-mediated nuclear export of unspliced Rous sarcoma virus RNA. Virology 363:376-386. Mullers, E., K. Stirnnagel, S. Kaulfuss, and D. Lindemann. 2011. Prototype foamy virus gag nuclear localization: a novel pathway among retroviruses. J Virol 85:9276-9285. Nash, M. A., M. K. Meyer, G. L. Decker, and R. B. Arlinghaus. 1993. A subset of Pr65gag is nucleus associated in murine leukemia virus-infected cells. J Virol 67:1350-1356. Parent, L. J. 2011. New insights into the nuclear localization of retroviral Gag proteins. Nucleus 2:92-97. Phillips, J. M., P. S. Murray, D. Murray, and V. M. Vogt. 2008. A molecular switch required for retrovirus assembly participates in the hexagonal immature lattice. EMBO Journal 27:1411-1420. Scheifele, L. Z., R. A. Garbitt, J. D. Rhoads, and L. J. Parent. 2002. Nuclear entry and CRM1-dependent nuclear export of the Rous sarcoma virus Gag polyprotein. Proc Natl Acad Sci U S A 99:3944-3949. Scheifele, L. Z., S. P. Kenney, T. M. Cairns, R. C. Craven, and L. J. Parent. 2007. Overlapping roles of the Rous sarcoma virus Gag p10 domain in nuclear export and virion core morphology. J Virol 81:10718-10728. Scheifele, L. Z., E. P. Ryan, and L. J. Parent. 2005. Detailed mapping of the nuclear export signal in the Rous sarcoma virus Gag protein. J Virol 79:87328741. Schliephake, A. W., and A. Rethwilm. 1994. Nuclear localization of foamy virus Gag precursor protein. J Virol 68:4946-4954. Spidel, J. L., R. C. Craven, C. B. Wilson, A. Patnaik, H. Wang, L. M. Mansky, and J. W. Wills. 2004. Lysines close to the Rous sarcoma virus late domain critical for budding. J Virol 78:10606-10616.

LEGENDS

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FIGURE 1. Fluorescently tagged retroviral Gag constructs. A. RSV construct

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(RSV.3h.GFP) was obtained from John Wills, HTLV-1 construct (mCMVREM-MAYFP)

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from David Derse, HIV-1 construct from Marilyn Resh and HFV (pcHFV/gfp) 9

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construct from Maxine Linial. MLV GagCFP was obtained from Alan Rein and

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subcloned into a GFP vector. All other clones were generated using standard

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cloning procedures. Amino acid sequence at junctions are shown. Bold letters

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represents Gag sequence, underlined letters represent GFP/YFP sequence. B.

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Vectors were transfected into 293FT cells and a western blot was performed on the

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cell lysates using an antibody against GFP.

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FIGURE 2. LMB sensitivity of retroviral Gag proteins. A. 293FT cells were

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transfected with Gag constructs illustrated in Fig 1. Cells were imaged the next day.

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LMB treated cells received 10 ng/ml LMB for 30 minutes prior to imaging. B.

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Distribution of cellular phenotypes. Y-axis is the percentage of cells in each

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treatment with the various distributions. Cells were scored as nuclear if there was a

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noticeable accumulation of protein in the nucleus. Cells were scored as cytoplasmic

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if the protein was enriched in the cytoplasm or plasma membrane. Cells were

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scored as nuclear and cytoplasmic if the protein was equally distributed between

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the nucleus and cytoplasm.

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Figure 3. An HIV/RSV Gag Chimera does not require the CRM1 export sequence. A.

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Amino acid sequence at the junction sequence is shown. LMB treatment was

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performed as in Fig 2. B. Distribution of cells from (A), as described in Fig 2.

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C. Illustration of the RSV reporter construct. DF1 cells were transfected with 1600

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ng of provirus along with 400 ng of filler DNA or 400 ng of VSV-G. Cells were

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collected 2-3 days post transfection and analyzed on a MoFloXDP flow cytometer.

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Percentage of cells transfected (expressing TdTomato and GFP) is shown in the

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upper right, percentage of cells infected (expressing GFP only) is shown in the lower

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right hand corner. D. Cells were transfected and imaged 6-8 hours later to avoid

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possible nuclear translocation during cell division. E. Distribution of cells from (D).

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TABLE 1. Proviral infectivity. Infected Cells/Transfected cell Experiment 1*

Experiment 2

Experiment 3

RSV

77.4

6.62

16.65

RSV L219A

1.8

0.22

0.60

H/RSV

40.0

2.96

5.52

H/RSV L219A

6.5

0.66

0.61

*The

raw data from experiment 1 are shown in Fig. 3.

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