scholarly journals Lysines Close to the Rous Sarcoma Virus Late Domain Critical for Budding

2004 ◽  
Vol 78 (19) ◽  
pp. 10606-10616 ◽  
Author(s):  
Jared L. Spidel ◽  
Rebecca C. Craven ◽  
Carol B. Wilson ◽  
Akash Patnaik ◽  
Huating Wang ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Rous Sarcoma Virus ◽  
Leukemia Virus ◽  
Cell Leukemia Virus Type ◽  
Gag Protein ◽  
Rous Sarcoma ◽  
Human T Cell ◽  
The Matrix ◽  
T Cell Leukemia Virus ◽  
Sarcoma Virus

ABSTRACT The release of retroviruses from the plasma membrane requires host factors that are believed to be recruited to the site of budding by the late (L) domain of the virus-encoded Gag protein. The L domain of Rous sarcoma virus (RSV) has been shown to interact with a ubiquitin (Ub) ligase, and budding of this virus is dependent on Ub. RSV is similar to other retroviruses in that it contains ∼100 molecules of Ub, but it is unique in that none of these molecules has been found to be conjugated to Gag. If transient ubiquitination of RSV Gag is required for budding, then replacement of the target lysine(s) with arginine should prevent the addition of Ub and reduce budding. Based on known sites of ubiquitination in other viruses, the important lysines would likely reside near the L domain. In RSV, there are five lysines located just upstream of the L domain in a region of the matrix (MA) protein that is dispensable for membrane binding, and replacement of these with arginine (mutant 1-5KR) reduced budding 80 to 90%. The block to budding was found to be on the plasma membrane; however, the few virions that were released had normal size, morphology, and infectivity. Budding was restored when any one of the residues was changed back to lysine or when lysines were inserted in novel positions, either within this region of MA or within the downstream p10 sequence. Moreover, the 1-5KR mutant could be rescued into particles by coexpression of budding-competent Gag molecules. These data argue that the phenotype of mutant 1-5KR is not due to a conformational defect. Consistent with the idea that efficient budding requires a specific role for lysines, human T-cell leukemia virus type 1, which does not bud well compared to RSV and lacks lysines close to its L domain, was found to be released at a higher level upon introduction of lysines near its L domain. This report strongly supports the hypothesis that ubiquitination of the RSV Gag protein (and perhaps those of other retroviruses) is needed for efficient budding.

scholarly journals Visualizing Association of the Retroviral Gag Protein with Unspliced Viral RNA in the Nucleus

mBio ◽  
2020 ◽  
Vol 11 (2) ◽  
Author(s):  
Rebecca J. Kaddis Maldonado ◽  
Breanna Rice ◽  
Eunice C. Chen ◽  
Kevin M. Tuffy ◽  
Estelle F. Chiari ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Nuclear Envelope ◽  
Rous Sarcoma Virus ◽  
Nuclear Export ◽  
Live Cell ◽  
Viral Rna ◽  
Wild Type ◽  
Gag Protein ◽  
Rous Sarcoma ◽  
Sarcoma Virus

ABSTRACT 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.

scholarly journals The PPPY Motif of Human T-Cell Leukemia Virus Type 1 Gag Protein Is Required Early in the Budding Process

2002 ◽  
Vol 76 (19) ◽  
pp. 10024-10029 ◽  
Author(s):  
Isabelle Le Blanc ◽  
Marie-Christine Prévost ◽  
Marie-Christine Dokhélar ◽  
Arielle R. Rosenberg
Keyword(s):  
T Cell ◽  
Virus Type ◽  
Leukemia Virus ◽  
T Cell Leukemia ◽  
Cell Leukemia ◽  
Cell Leukemia Virus Type ◽  
Gag Protein ◽  
Human T Cell ◽  
T Cell Leukemia Virus

ABSTRACT Domains required late in the virus budding process (L domains) have been identified in the Gag proteins of a number of retroviruses. Here we show that the human T-cell leukemia virus type 1 candidate L domain motif PPPY is indeed required for virus production. Strikingly, however, mutation of this motif arrested virus particles at an earlier stage in the budding process than was seen for mutation of the L domain motifs thus far described for retroviruses. In view of the exchangeability of such domains, we propose that the retrovirus budding process may involve a continuum from bud formation to membrane fission.

scholarly journals Alterations in the MA and NC Domains Modulate Phosphoinositide-Dependent Plasma Membrane Localization of the Rous Sarcoma Virus Gag Protein

2013 ◽  
Vol 87 (6) ◽  
pp. 3609-3615 ◽  
Author(s):  
S. Nadaraia-Hoke ◽  
D. V. Bann ◽  
T. L. Lochmann ◽  
N. Gudleski-O'Regan ◽  
L. J. Parent
Keyword(s):  
Plasma Membrane ◽  
Rous Sarcoma Virus ◽  
Membrane Localization ◽  
Gag Protein ◽  
Plasma Membrane Localization ◽  
Rous Sarcoma ◽  
Sarcoma Virus

scholarly journals Hydrodynamic and Membrane Binding Properties of Purified Rous Sarcoma Virus Gag Protein

2015 ◽  
Vol 89 (20) ◽  
pp. 10371-10382 ◽  
Author(s):  
Robert A. Dick ◽  
Siddhartha A. K. Datta ◽  
Hirsh Nanda ◽  
Xianyang Fang ◽  
Yi Wen ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Rous Sarcoma Virus ◽  
Acyl Chain ◽  
Membrane Binding ◽  
Membrane Association ◽  
Gag Protein ◽  
Rous Sarcoma ◽  
Sarcoma Virus ◽  
Hiv 1

ABSTRACTPreviously, no retroviral Gag protein has been highly purified in milligram quantities and in a biologically relevant and active form. We have purified Rous sarcoma virus (RSV) Gag protein and in parallel several truncation mutants of Gag and have studied their biophysical properties and membrane interactionsin vitro. RSV Gag is unusual in that it is not naturally myristoylated. From its ability to assemble into virus-like particlesin vitro, we infer that RSV Gag is biologically active. By size exclusion chromatography and small-angle X-ray scattering, Gag in solution appears extended and flexible, in contrast to previous reports on unmyristoylated HIV-1 Gag, which is compact. However, by neutron reflectometry measurements of RSV Gag bound to a supported bilayer, the protein appears to adopt a more compact, folded-over conformation. At physiological ionic strength, purified Gag binds strongly to liposomes containing acidic lipids. This interaction is stimulated by physiological levels of phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P2] and by cholesterol. However, unlike HIV-1 Gag, RSV Gag shows no sensitivity to acyl chain saturation. In contrast with full-length RSV Gag, the purified MA domain of Gag binds to liposomes only weakly. Similarly, both an N-terminally truncated version of Gag that is missing the MA domain and a C-terminally truncated version that is missing the NC domain bind only weakly. These results imply that NC contributes to membrane interactionin vitro, either by directly contacting acidic lipids or by promoting Gag multimerization.IMPORTANCERetroviruses like HIV assemble at and bud from the plasma membrane of cells. Assembly requires the interaction between thousands of Gag molecules to form a lattice. Previous work indicated that lattice formation at the plasma membrane is influenced by the conformation of monomeric HIV. We have extended this work to the more tractable RSV Gag. Our results show that RSV Gag is highly flexible and can adopt a folded-over conformation on a lipid bilayer, implicating both the N and C termini in membrane binding. In addition, binding of Gag to membranes is diminished when either terminal domain is truncated. RSV Gag membrane association is significantly less sensitive than HIV Gag membrane association to lipid acyl chain saturation. These findings shed light on Gag assembly and membrane binding, critical steps in the viral life cycle and an untapped target for antiretroviral drugs.

scholarly journals Intracellular Distribution of Human T-Cell Leukemia Virus Type 1 Gag Proteins Is Independent of Interaction with Intracellular Membranes

2002 ◽  
Vol 76 (2) ◽  
pp. 905-911 ◽  
Author(s):  
Isabelle Le Blanc ◽  
Vincent Blot ◽  
Isabelle Bouchaert ◽  
Jean Salamero ◽  
Bruno Goud ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
T Cell ◽  
Virus Type ◽  
Leukemia Virus ◽  
Cell Leukemia ◽  
Cell Leukemia Virus Type ◽  
Gag Proteins ◽  
Human T Cell ◽  
T Cell Leukemia Virus

ABSTRACT Retrovirus Gag proteins are synthesized on free ribosomes, and are sufficient to govern the assembly and release of virus particles. Like type C retroviruses, human T-cell leukemia virus type 1 (HTLV-1) assembles and buds at the plasma membrane. After immunofluorescence staining, HTLV-1 Gag proteins appear as punctuated intracellular clusters, which suggests that they are associated either with intracellular membranes or with the plasma membrane. However, colocalization experiments using a panel of markers demonstrated that Gag proteins were not associated with the membranes involved in the secretory or endocytosis pathway. Small amounts of Gag proteins were detected at the plasma membrane and colocalized with the envelope glycoproteins. Moreover, Gag proteins were excluded from streptolysin-O permeabilized cells and in this respect behaved like cytoplasmic proteins. This suggests that the trafficking of HTLV-1 Gag proteins through the cytoplasm of the host cell is independent of any cell membrane system.

scholarly journals Structural basis for human T-cell leukemia virus type 1 Gag targeting to the plasma membrane for assembly

2021 ◽  
Author(s):  
Dominik Herrmann ◽  
Lynne W Zhou ◽  
Heather M. Hanson ◽  
Nora A Willkomm ◽  
Louis M. Mansky ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
T Cell ◽  
Virus Type ◽  
Leukemia Virus ◽  
T Cell Leukemia ◽  
Cell Leukemia ◽  
Cell Leukemia Virus Type ◽  
Human T Cell ◽  
T Cell Leukemia Virus

During the late phase of retroviral replication, the virally encoded Gag polyprotein is targeted to the plasma membrane (PM) for assembly. GagPM binding is mediated by the N-terminal matrix (MA) domain of Gag. For many retroviruses, Gag binding to the PM was found to be dependent on phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2]. However, it was shown that for human T-cell leukemia virus type 1 (HTLV-1), Gag binding to membranes is less dependent on PI(4,5)P2, suggesting that other factors may modulate Gag assembly. To elucidate the mechanism by which HTLV-1 Gag binds to the PM, we employed NMR techniques to solve the structure of unmyristoylated MA (myr(−)MA) and to characterize its interactions with lipids and liposomes. The MA structure consists of four α-helices and unstructured N- and C-termini. We show that myr(−)MA binds to PI(4,5)P2 via the polar head and that myr(−)MA binding to inositol phosphates (IPs) is significantly enhanced by increasing the number of phosphate groups on the inositol ring, indicating that the MAIP binding is governed by chargecharge interactions. The IP binding site was mapped to a well-defined basic patch formed by lysine and arginine residues. Using a sensitive NMR-based liposome binding assay, we show that myr(−)MA binding to membranes is significantly enhanced by phosphatidylserine (PS). Confocal microscopy data show that Gag is localized to the inner leaflet of the PM of infected cells, while the Gag G2A mutant, lacking myristoylation, is diffuse and cytoplasmic. These findings advance our understanding of a key mechanism in retroviral assembly.

scholarly journals Multiple Functions for the Basic Amino Acids of the Human T-Cell Leukemia Virus Type 1 Matrix Protein in Viral Transmission

1999 ◽  
Vol 73 (3) ◽  
pp. 1860-1867 ◽  
Author(s):  
Isabelle Le Blanc ◽  
Arielle R. Rosenberg ◽  
Marie-Christine Dokhélar
Keyword(s):  
Amino Acids ◽  
Matrix Protein ◽  
Leukemia Virus ◽  
Cell Leukemia Virus Type ◽  
Particle Release ◽  
Basic Amino Acids ◽  
Human T Cell ◽  
Cell Transmission ◽  
The Matrix ◽  
T Cell Leukemia Virus

ABSTRACT We studied the involvement of the human T-cell leukemia virus type 1 (HTLV-1) Gag matrix protein in the cell-to-cell transmission of the virus using missense mutations of the basic amino acids. These basic amino acids are clustered at the N terminus of the protein in other retroviruses and are responsible for targeting the Gag proteins to the plasma membrane. In the HTLV–bovine leukemia virus genus of retroviruses, the basic amino acids are distributed throughout the matrix protein sequence. The HTLV-1 matrix protein contains 11 such residues. A wild-type phenotype was obtained only for mutant viruses with mutations at one of two positions in the matrix protein. The phenotypes of the other nine mutant viruses showed that the basic amino acids are involved at various steps of the replication cycle, including some after membrane targeting. Most of these nine mutations allowed normal synthesis, transport, and cleavage of the Gag precursor, but particle release was greatly affected for seven of them. In addition, four mutated proteins with correct particle release and envelope glycoprotein incorporation did not however permit cell-to-cell transmission of HTLV-1. Thus, particle release, although required, is not sufficient for the cell-to-cell transmission of HTLV-1, and the basic residues of the matrix protein are involved in steps that occur after viral particle release.

scholarly journals Both the PPPY and PTAP Motifs Are Involved in Human T-Cell Leukemia Virus Type 1 Particle Release

2004 ◽  
Vol 78 (3) ◽  
pp. 1503-1512 ◽  
Author(s):  
Huating Wang ◽  
Nicholas J. Machesky ◽  
Louis M. Mansky
Keyword(s):  
Plasma Membrane ◽  
Leukemia Virus ◽  
Cell Leukemia ◽  
Cell Leukemia Virus Type ◽  
Particle Release ◽  
Virus Particles ◽  
Human T Cell ◽  
T Cell Leukemia Virus ◽  
Ptap Motif

ABSTRACT In retroviruses, the late (L) domain has been defined as a conserved motif in the Gag polyprotein precursor that, when mutated, leads to the emergence of virus particles that fail to pinch off from the plasma membrane. These domains have been observed to contain the PPXY, PTAP, or YXXL motifs. The deltaretroviruses, which include bovine leukemia virus (BLV) and human T-cell leukemia virus type 1 (HTLV-1) and HTLV-2, have a conserved PPPY motif in the C-terminal region of the matrix (MA) domain of Gag, while HTLV-1 also encodes a PTAP motif in MA. In this study, we analyzed the roles of the PPPY and PTAP motifs in the C terminus of MA in HTLV-1 particle release. Mutation of either motif (i.e., PPPY changed to APPY or PTAP changed to PTRP) reduced budding efficiencies. Particle buds and electron-dense regions of plasma membrane were observed by electron microscopy. When the locations of PPPY and PTAP were switched, particle release was eliminated. Intriguingly, the replacement of the PTAP motif with either the PPPY or YPDL motifs did not influence the release of virus particles, but the replacement of the PPPY motif with either PTAP or YPDL eliminated particle production. This indicates that the role that PPPY plays in HTLV-1 budding cannot be replaced with either PTAP or YPDL. A similar observation was made with the BLV PPPY motif. Finally, HTLV-1 particle release was found to be sensitive to proteasome inhibitors, implicating a role for ubiquitin in HTLV-1 budding. In summary, our observations indicate that (i) the PPPY motif plays a crucial role in virus budding and (ii) the PTAP motif plays a more subtle role in HTLV-1 particle release. Each of these motifs may play an important role in virus release from specific cell types and therefore be important in efficient virus spread and transmission.

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