fThe mouse mammary tumor virus (MMTV) Gag protein directs the assembly in the cytoplasm of immature viral capsids, which subsequently bud from the plasma membranes of infected cells. MMTV Gag localizes to discrete cytoplasmic foci in mouse mammary epithelial cells, consistent with the formation of cytosolic capsids. Unexpectedly, we also observed an accumulation of Gag in the nucleoli of infected cells derived from mammary gland tumors. To detect Gag-interacting proteins that might influence its subcellular localization, a yeast two-hybrid screen was performed. Ribosomal protein L9 (RPL9 or L9), an essential component of the large ribosomal subunit and a putative tumor suppressor, was identified as a Gag binding partner. Overexpression of L9 in cells expressing the MMTV(C3H) provirus resulted in specific, robust accumulation of Gag in nucleoli. Förster resonance energy transfer (FRET) and coimmunoprecipitation analyses demonstrated that Gag and L9 interact within the nucleolus, and the CA domain was the major site of interaction. In addition, the isolated NC domain of Gag localized to the nucleolus, suggesting that it contains a nucleolar localization signal (NoLS). To determine whether L9 plays a role in virus assembly, small interfering RNA (siRNA)-mediated knockdown was performed. Although Gag expression was not reduced with L9 knockdown, virus production was significantly impaired. Thus, our data support the hypothesis that efficient MMTV particle assembly is dependent upon the interaction of Gag and L9 in the nucleoli of infected cells. Since its discovery as a milk-transmitted agent in the 1930s, the oncogenic retrovirus mouse mammary tumor virus (MMTV) has served as an important model in breast cancer research and immunology (1). However, little is known about the molecular mechanisms that govern MMTV assembly. The 9-kb MMTV RNA genome consists of the common retroviral elements gag, pro, pol, and env, as well as dut (dUTPase) (2), sag (superantigen) (3), and rem (regulator of export of MMTV mRNA) (4, 5). Like all retroviruses, MMTV uses full-length viral RNA to transcribe the viral structural proteins Gag, Gag-Pro, and Gag-Pro-Pol. The Gag protein directs assembly of complete, immature viral capsids in the cytoplasm, which are subsequently transported to the plasma membrane for release by budding.Unlike acutely transforming retroviruses like Rous sarcoma virus (RSV), MMTV does not carry an oncogene and instead induces tumors primarily by integrating near cellular oncogenes and disrupting their regulation. In addition, the MMTV Gag and Env proteins also promote tumorigenesis independently of the proviral integration site (6, 7). Moreover, differences in pathogenesis between the highly tumorigenic MMTV(C3H) strain and the tumor-attenuated MMTV hybrid provirus (HP) strain map to the CA and NC regions of the Gag protein (6), which led us to hypothesize that the Gag proteins from the C3H and HP strains might differentially interact with cellular proteins to promote malignant transformation.The eukaryotic ribos...

Packaging of genomic RNA (gRNA) by retroviruses is essential for infectivity, yet the subcellular site of the initial interaction between the Gag polyprotein and gRNA remains poorly defined. Because retroviral particles are released from the plasma membrane, it was previously thought that Gag proteins initially bound to gRNA in the cytoplasm or at the plasma membrane. However, the Gag protein of the avian retrovirus Rous sarcoma virus (RSV) undergoes active nuclear trafficking, which is required for efficient gRNA encapsidation (L. Z. Scheifele, R. A. Garbitt, J. D. Rhoads, and L. J. Parent, Proc Natl Acad Sci U S A 99:3944–3949, 2002, https://doi.org/10.1073/pnas.062652199; R. Garbitt-Hirst, S. P. Kenney, and L. J. Parent, J Virol 83:6790–6797, 2009, https://doi.org/10.1128/JVI.00101-09). These results raise the intriguing possibility that the primary contact between Gag and gRNA might occur in the nucleus. To examine this possibility, we created a RSV proviral construct that includes 24 tandem repeats of MS2 RNA stem-loops, making it possible to track RSV viral RNA (vRNA) in live cells in which a fluorophore-conjugated MS2 coat protein is coexpressed. Using confocal microscopy, we observed that both wild-type Gag and a nuclear export mutant (Gag.L219A) colocalized with vRNA in the nucleus. In live-cell time-lapse images, the wild-type Gag protein trafficked together with vRNA as a single ribonucleoprotein (RNP) complex in the nucleoplasm near the nuclear periphery, appearing to traverse the nuclear envelope into the cytoplasm. Furthermore, biophysical imaging methods suggest that Gag and the unspliced vRNA physically interact in the nucleus. Taken together, these data suggest that RSV Gag binds unspliced vRNA to export it from the nucleus, possibly for packaging into virions as the viral genome. IMPORTANCE Retroviruses cause severe diseases in animals and humans, including cancer and acquired immunodeficiency syndromes. To propagate infection, retroviruses assemble new virus particles that contain viral proteins and unspliced vRNA to use as gRNA. Despite the critical requirement for gRNA packaging, the molecular mechanisms governing the identification and selection of gRNA by the Gag protein remain poorly understood. In this report, we demonstrate that the Rous sarcoma virus (RSV) Gag protein colocalizes with unspliced vRNA in the nucleus in the interchromatin space. Using live-cell confocal imaging, RSV Gag and unspliced vRNA were observed to move together from inside the nucleus across the nuclear envelope, suggesting that the Gag-gRNA complex initially forms in the nucleus and undergoes nuclear export into the cytoplasm as a viral ribonucleoprotein (vRNP) complex.

Retroviruses are positive-sense, single-stranded RNA viruses that reverse transcribe their RNA genomes into double-stranded DNA for integration into the host cell chromosome. The integrated provirus is used as a template for the transcription of viral RNA. The full-length viral RNA can be used for the translation of the Gag and Gag-Pol structural proteins or as the genomic RNA (gRNA) for encapsidation into new virions by the Gag protein. The mechanism by which Gag selectively incorporates unspliced gRNA into virus particles is poorly understood. Although Gag was previously thought to localize exclusively to the cytoplasm and plasma membrane where particles are released, we found that the Gag protein of Rous sarcoma virus, an alpharetrovirus, undergoes transient nuclear trafficking. When the nuclear export signal of RSV Gag is mutated (Gag.L219A), the protein accumulates in discrete subnuclear foci reminiscent of nuclear bodies such as splicing speckles, paraspeckles, and PML bodies. In this report, we observed that RSV Gag.L219A foci appeared to be tethered in the nucleus, partially co-localizing with the splicing speckle components SC35 and SF2. Overexpression of SC35 increased the number of Gag.L219A nucleoplasmic foci, suggesting that SC35 may facilitate the formation of Gag foci. We previously reported that RSV Gag nuclear trafficking is required for efficient gRNA packaging. Together with the data presented herein, our findings raise the intriguing hypothesis that RSV Gag may co-opt splicing factors to localize near transcription sites. Because splicing occurs co-transcriptionally, we speculate that this mechanism could allow Gag to associate with unspliced viral RNA shortly after its transcription initiation in the nucleus, before the viral RNA can be spliced or exported from the nucleus as an mRNA template.

Retroviruses package their full-length, dimeric genomic RNA (gRNA) via specific interactions between the Gag polyprotein and a “Ψ” packaging signal located in the gRNA 5′-UTR. Rous sarcoma virus (RSV) gRNA has a contiguous, well-defined Ψ element, that directs the packaging of heterologous RNAs efficiently. The simplicity of RSV Ψ makes it an informative model to examine the mechanism of retroviral gRNA packaging, which is incompletely understood. Little is known about the structure of dimerization initiation sites or specific Gag interaction sites of RSV gRNA. Using selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE), we probed the secondary structure of the entire RSV 5′-leader RNA for the first time. We identified a putative bipartite dimerization initiation signal (DIS), and mutation of both sites was required to significantly reduce dimerization in vitro. These mutations failed to reduce viral replication, suggesting that in vitro dimerization results do not strictly correlate with in vivo infectivity, possibly due to additional RNA interactions that maintain the dimers in cells. UV crosslinking-coupled SHAPE (XL-SHAPE) was next used to determine Gag-induced RNA conformational changes, revealing G218 as a critical Gag contact site. Overall, our results suggest that disruption of either of the DIS sequences does not reduce virus replication and reveal specific sites of Gag–RNA interactions.

Retroviral Gag polyproteins orchestrate the assembly and release of nascent virus particles from the plasma membranes of infected cells. Although it was traditionally thought that Gag proteins trafficked directly from the cytosol to the plasma membrane, we discovered that the oncogenic avian alpharetrovirus Rous sarcoma virus (RSV) Gag protein undergoes transient nucleocytoplasmic transport as an intrinsic step in virus assembly. Using a genetic approach in yeast, we identified three karyopherins that engage the two independent nuclear localization signals (NLS) in Gag. The primary NLS is in the nucleocapsid (NC) domain of Gag and binds directly to importin-α, which recruits importin-β to mediate nuclear entry. The second NLS, which resides in the matrix (MA) domain, is dependent on importin-11 and transportin-3 (TNPO3), known as MTR10p and Kap120p in yeast, although it is not clear whether these import factors are independent or additive. The functionality of importin α/β and importin-11 has been verified in avian cells, whereas the role of TNPO3 has not been studied. In this report, we demonstrate that TNPO3 mediates nuclear entry of Gag and directly binds to Gag. To our surprise, this interaction did not require the cargo-binding domain of TNPO3, which typically mediates nuclear entry for other binding partners of TNPO3 including SR-domain containing splicing factors and tRNAs that re-enter the nucleus. These results suggest that RSV hijacks the host nuclear import pathway using a unique mechanism, potentially allowing other cargo to bind TNPO3 simultaneously. IMPORTANCE RSV Gag nuclear entry is facilitated using three distinct host import factors that interact with nuclear localization signals in the Gag MA and NC domains. Here we show that the MA region is required for nuclear import of Gag through the TNPO3 pathway. Gag nuclear entry does not require the cargo binding domain of TNPO3. Understanding the molecular basis for TNPO3-mediated nuclear trafficking of the RSV Gag protein may lead to a deeper appreciation for whether different import factors play distinct roles in retrovirus replication.

No abstract

11]. The NLS in the Gag MA domain also contributes to nuclear transport, although it interacts with 43 two different host importins [9]. A nuclear export signal (NES) in the Gag p10 domain functions 44 Viruses 2020, 12, x FOR PEER REVIEW 2 of 14 through its interaction with CRM1/RanGTP export complex. Mutations of the p10 domain or 45 treatment with leptomycin B, a CRM1 inhibitor, result in accumulation of Gag in the nucleus with 46 formation of numerous nucleoplasmic and nucleolar foci [11][12][13][14]. These foci are dependent on the 47 presence of the NC domain and its nucleic-acid binding function [11,14,15]. 48The RSV Gag NC domain contains two Cys-His domains, or zinc knuckles, that bind RNA and 49 are required for specific gRNA packaging [16][17][18][19]. The basic residues in the Gag NC domain play 50 numerous roles in virus assembly, including promoting Gag-Gag interactions leading to dimer and 51 oligomer formation, nonspecific and specific nucleic acid binding, and gRNA encapsidation [20][21][22][23]. 52The RSV NC domain contains sixteen basic residues, however only eight are required to promote 53 Gag-Gag interactions and virus particle assembly [21]. Lee et al. performed several studies examining 54 basic residues in NC [21,22]. When the RKR residues immediately following the second zinc knuckle 55 were deleted, they found little evidence for binding of NC to the MΨ RNA using a yeast three-hybrid 56 assay [22]. In their follow up work, they observed that the RKR deletion mutant was still able to 57 undergo Gag-Gag interactions [21], suggesting importance of these residues in MΨ binding, but not 58 in Gag-Gag interactions. 59To further examine roles the C-terminal basic residues in NC play in Gag subcellular 60 localization, virus budding, gRNA packaging, and infectivity, we examined deletions and 61 substitutions of basic residues derived from heterologous viral proteins with roles similar to NC. For 62 this purpose, we chose the highly basic region (BR) of the herpes simplex type 1 (HSV-1) ICP27 63 protein, which contains an RGG Box RNA binding domain [24]. ICP27 localizes to nuclear foci and 64 nucleoli [25-27] interacts with splicing components in nuclear speckles, [28] and binds intronless 65 HSV-1 RNAs for nuclear export [26]. As a second viral RNA binding protein, we chose the HIV-1 Rev 66 protein, which also localizes to nucleoli, contains an RNA binding domain enriched in basic residues, 67 and binds to the Rev-response element to facilitate export of unspliced viral RNA from the nucleus 68 [29-31].69 2. Materials and Methods 70 2.1. Expression vectors, plasmids, and cells 71 The Prague C RSV Gag expression vector containing YFP (pGag-YFP) fluorophore was 72 previously described [14]. RSV NC was expressed from a pEYFP-N1 containing vector (Clontech) 73 described previously [9]. Proviral constructs were created by site-directed mutagenesis in the NC 74 domain of pCMV.GagPol (kind gift of Rebecca Craven, Penn State College of Medicine). To create 75 the pRS.V8.Gag.D61-73, pRS.V8.Gag.ICP27, and...

The Rous sarcoma virus Gag polyprotein transiently traffics through the nucleus, which is required for efficient incorporation of the viral genomic RNA (gRNA) into virus particles. Packaging of gRNA is mediated by two zinc knuckles and basic residues located in the nucleocapsid (NC) domain in Gag. To further examine the role of basic residues located downstream of the zinc knuckles in gRNA encapsidation, we used a gain-of-function approach. We replaced a basic residue cluster essential for gRNA packaging with heterologous basic residue motif (BR) with RNA-binding activity from either the HIV-1 Rev protein (Rev BR) or the HSV ICP27 protein (ICP27 BR). Compared to wild-type Gag, the mutant ICP27 BR and Rev BR Gag proteins were much more strongly localized to the nucleus and released significantly lower levels of virus particles. Surprisingly, both the ICP27 BR and Rev BR mutants packaged normal levels of gRNA per virus particle when examined in the context of a proviral vector, yet both mutants were noninfectious. These results support the hypothesis that basic residues located in the C-terminal region of NC are required for selective gRNA packaging, potentially by binding non-specifically to RNA via electrostatic interactions.