Preassembled capsids of type D retroviruses contain a signal sufficient for targeting specifically to the plasma membrane

Rhee, Sehun; Hui, Huxiong; Hunter, Eric

doi:10.1128/jvi.64.8.3844-3852.1990

Cited by 69 publications

(39 citation statements)

References 54 publications

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“…While the absence of Env did not result in the complete inhibition of transport that we observed at 20 °C, we did observe an approximately 7‐fold reduction in the rate of Gag maturation during the second and third hours of a pulse‐chase experiment, which is the time of maximum Gag release and maturation for wild‐type virus. This observation differs from a previous report from this laboratory by Rhee et al (13) that described apparently normal Gag polyprotein processing and viral release kinetics for a provirus expressing a truncated Env that is retained in the ER. While the basis for this discrepancy is not fully understood, in the initial report, pulse‐chase experiments were performed 72 h after calcium phosphate transfection, in contrast to 16 h following Fugene 6 transfection for the experiments described here.…”

Section: Discussioncontrasting

confidence: 99%

M‐PMV Capsid Transport Is Mediated by Env/Gag Interactions at the Pericentriolar Recycling Endosome

Sfakianos

Hunter

2003

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Cytoplasmic transport of Gag molecules to the site of budding is an important but poorly understand process in retroviral assembly. Our previous studies of MasonPfizer monkey virus showed that, for this retrovirus, Gag is assembled into capsids at a pericentriolar region and that Env is necessary for efficient transport out of the site. An Env requirement for cytoplasmic transport implicates vesicular trafficking in this process even though the capsids remain cytoplasmic and do not bud into intracellular compartments in the cells studied to date. We show here that the secretory pathway of the cell is not directly involved in Gag transport since the latter was not inhibited by BFA, nor did Gag colocalize with markers of the ER, Golgi, or TGN. Instead, colocalization was observed between Gag and endocytosed transferrin and with Rab11, suggesting that pericentriolar recycling endosomes play a critical role in this process. Mutants of Rab11 that inhibit efflux of transferrin from the recycling endosome also inhibited Gag transport. Our studies show that Env colocalizes with Gag at the pericentriolar assembly site, and provide evidence that Env must travel through this compartment in order to initiate export of the capsids from the site of assembly. Thus, for the first time, endocytic trafficking of a retroviral Env glycoprotein is linked to the efficient cytoplasmic transport of Gag. Mason-Pfizer monkey virus (M-PMV), the prototypic D-type retrovirus, is characterized by assembly of spherical immature capsids in the cytoplasm (1-3). The cytoplasmic assembly of M-PMV is directed by an 18-amino acid sequence within the MA domain, termed the cytoplasmic targeting/retention signal (CTRS). Despite the presence of the bipartite plasma membrane targeting signal found in retroviral Gag molecules that assemble at the periphery of the cell, the CTRS acts in a dominant manner. Our previous studies (4) suggest that the CTRS functions in a cotranslational manner to interact with the dynein/dynactin molecular motor complex and transport nascent Gag molecules, along with the polysome, to the pericentriolar region of the cell.This region of the cell provides an advantageous location for translation and assembly of M-PMV capsids. The region is host to many chaperonins, including TRiC, which has been demonstrated to participate in M-PMV capsid assembly (5). Additionally, the region serves as the microtubule-organizing center (MTOC) that is responsible for anchoring many of the cell's organelles. Thus, the pericentriolar assembly site of this virus provides an optimal location for protein folding as well as for interactions with cytoskeletal and vesicular components of the cell. This is important because in the accompanying paper we show that efficient transport of assembled M-PMV capsids from the pericentriolar region requires coexpression of the M-PMV Env protein and functional vesicular trafficking. In the absence of Env or under conditions of a low temperature (20°C) block to vesicular transport (6,7) Gag accumulated at the assemb...

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Section: Discussioncontrasting

confidence: 99%

M‐PMV Capsid Transport Is Mediated by Env/Gag Interactions at the Pericentriolar Recycling Endosome

Sfakianos

Hunter

2003

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“…The cytoplasmic targeting/retention signal or CTRS sequence of the M‐PMV Gag polyprotein was previously demonstrated to be responsible for the intracytoplasmic assembly of retroviruses exhibiting B/D‐type morphogenesis (15,27). Here we show that the CTRS is responsible for dynein‐mediated targeting/retention of nascent Gag polyproteins at the centriolar region of the cell, and that assembly of immature M‐PMV capsids occurs at this location.…”

Section: Discussionmentioning

confidence: 99%

The M‐PMV Cytoplasmic Targeting‐Retention Signal Directs Nascent Gag Polypeptides to a Pericentriolar Region of the Cell

Sfakianos¹,

LaCasse²,

Hunter³

2003

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Intracytoplasmic protein targeting in mammalian cells is critical for organelle function as well as virus assembly, but the signals that mediate it are poorly defined. We show here that Mason‐Pfizer monkey virus specifically targets Gag precursor proteins to the pericentriolar region of the cytoplasm in a microtubule dependent process through interactions between a short peptide signal, known as the cytoplasmic targeting‐retention signal, and the dynein/dynactin motor complex. The Gag molecules are concentrated in pericentriolar microdomains, where they assemble to form immature capsids. Depletion of Gag from this region by cycloheximide treatment, coupled with the presence of ribosomal clusters that are in close vicinity to the assembling capsids, suggests that the dominant N‐terminal cytoplasmic targeting‐retention signal functions in a cotranslational manner. Transport of the capsids out of the pericentriolar assembly site requires the env‐gene product, and a functional vesicular transport system. A single point mutation that renders the cytoplasmic targeting‐retention signal defective abrogates pericentriolar targeting of Gag molecules. Thus the previously defined cytoplasmic targeting‐retention signal appears to act as a cotranslational intracellular targeting signal that concentrates Gag proteins at the centriole for assembly of capsids.

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“…: 46 proteins of flaviviruses (2,24,34,63) and coronaviruses (58) have been demonstrated to form subviral lipoprotein particles. Retroviruses represent yet a third type of viral budding mechanism, and in this case the viral core protein precursor Gag is capable of self-assembling into enveloped virus-like particles (9,12,48,53,62), thereby indicating that binding of a cytoplasmic core protein into the membrane and its oligomerization into a core structure provide the driving force for retrovirus budding. Recently, it was reported that the core components of the rhabdovirus rabies virus was capable of assembling into extracellular enveloped particles in the absence of the viral spike proteins (36).…”

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confidence: 99%

Targeting of Moloney murine leukemia virus gag precursor to the site of virus budding.

Suomalainen

Hultenby

Garoff

1996

The Journal of Cell Biology

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Abstract. Retrovirus Moloney murine leukemia virus(M-MuLV) matures by budding at the cell surface. Central to the budding process is the myristoylated viral core protein precursor Gag which, even in the absence of all other viral components, is capable of associating with the cytoplasmic leaflet of the plasma membrane and assembling into extracellular virus-like particles. In this paper we have used heterologous, Semliki Forest virus-driven, expression of M-MuLV Gag to study the mechanism by which this protein is targeted to the cell surface. In pulse-chase experiments, BFA, monensin, and 20°C block did not affect incorporation of Gag into extracellular particles thereby indicating that the secretory pathway is not involved in targeting of Gag to the cell surface. Subcellular fractionation studies demonstrated that newly synthesized Gag became rapidly and efficiently associated with membranes which had a density similar to that of plasma membrane-derived vesicles. Protease-protection studies confirmed that the Gag-containing membranes were of plasma membrane origin, since in crude cell homogenates, the bulk of newly synthesized Gag was protease-resistant as expected of a protein that binds to the cytoplasmic leaflet of the plasma membrane. Taken together these data indicate that targeting of M-MuLV Gag to the cell surface proceeds via direct insertion of the protein to the cytoplasmic side of the plasma membrane. Furthermore, since the membrane insertion reaction is highly efficient and specific, this suggests that the reaction is dependent on as-yet-unidentified cellular factors.

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Preassembled capsids of type D retroviruses contain a signal sufficient for targeting specifically to the plasma membrane

Cited by 69 publications

References 54 publications

M‐PMV Capsid Transport Is Mediated by Env/Gag Interactions at the Pericentriolar Recycling Endosome

M‐PMV Capsid Transport Is Mediated by Env/Gag Interactions at the Pericentriolar Recycling Endosome

The M‐PMV Cytoplasmic Targeting‐Retention Signal Directs Nascent Gag Polypeptides to a Pericentriolar Region of the Cell

Targeting of Moloney murine leukemia virus gag precursor to the site of virus budding.

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