Chimeras of receptors for gibbon ape leukemia virus/feline leukemia virus B and amphotropic murine leukemia virus reveal different modes of receptor recognition by retrovirus

Pedersen, Lene; Johann, S V; Zeijl, Marja van; Pedersen, Finn Skou; O’Hara, Brian J.

doi:10.1128/jvi.69.4.2401-2405.1995

Cited by 54 publications

(68 citation statements)

References 19 publications

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“…CHO K1 cells expressing human PiT2 were permissive for infection by both vector pseudotypes (Fig. 4B,C) in agreement with PiT2¢s well-described receptor function for AM-MLV and 10A1 MLV [26,28,36]. All mutants supported infection with both viruses at wild-type receptor levels or higher (Fig.…”

Section: Pi Transporter Pit Family Signature Sequencessupporting

confidence: 79%

Evolutionary and experimental analyses of inorganic phosphate transporter PiT family reveals two related signature sequences harboring highly conserved aspartic acids critical for sodium‐dependent phosphate transport function of human PiT2

Bøttger

Pedersen

2005

The FEBS Journal

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The mammalian members of the inorganic phosphate (P i ) transporter (PiT) family, the type III sodium-dependent phosphate (NaP i ) transporters PiT1 and PiT2, have been assigned housekeeping P i transport functions and are suggested to be involved in chondroblastic and osteoblastic mineralization and ectopic calcification. The PiT family members are conserved throughout all kingdoms and use either sodium (Na + ) or proton (H + ) gradients to transport P i . Sequence logo analyses revealed that independent of their cation dependency these proteins harbor conserved signature sequences in their N-and C-terminal ends with the common core consensus sequence GANDVANA. With the exception of 10 proteins from extremophiles all 109 proteins analyzed carry an aspartic acid in one or both of the signature sequences. We changed either of the highly conserved aspartates, Asp28 and Asp506, in the N-and C-terminal signature sequences, respectively, of human PiT2 to asparagine and analyzed P i uptake function in Xenopus laevis oocytes. Both mutant proteins were expressed at the cell surface of the oocytes but exhibited knocked out NaP i transport function. Human PiT2 is also a retroviral receptor and we have previously shown that this function can be exploited as a control for proper processing and folding of mutant proteins. Both mutant transporters displayed wild-type receptor functions implying that their overall architecture is undisturbed. Thus the presence of an aspartic acid in either of the PiT family signature sequences is critical for the Na + -dependent P i transport function of human PiT2. The conservation of the aspartates among proteins using either Na + -or H + -gradients for P i transport suggests that they are involved in H + -dependent P i transport as well. Current results favor a membrane topology model in which the N-and C-terminal PiT family signature sequences are positioned in intra-and extracellular loops, respectively, suggesting that they are involved in related functions on either side of the membrane. The present data are in agreement with a possible role of the signature sequences in translocation of cations.Abbreviations 10A1, an MLV isolate related to AM-MLV; AM-MLV, amphotropic MLV; CHO, Chinese hamster ovary; FeLV-B, feline leukemia virus subgroup B; GALV, gibbon ape leukemia virus; MLV, murine leukemia virus; NaP i , sodium-dependent phosphate; PiT, inorganic phosphate transporter; RADAR, rapid automatic detection and alignment of repeats.

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Section: Pi Transporter Pit Family Signature Sequencessupporting

confidence: 79%

Evolutionary and experimental analyses of inorganic phosphate transporter PiT family reveals two related signature sequences harboring highly conserved aspartic acids critical for sodium‐dependent phosphate transport function of human PiT2

Bøttger

Pedersen

2005

The FEBS Journal

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“…␤-Galactosidase assay of infected cells. The ␤-galactosidase (␤-Gal) assay was performed as described previously (37). The cells were fixed with 0.05% glutaraldehyde, washed with phosphate-buffered saline (PBS), and overlaid with X-Gal (5-bromo-4-chloro-3-indolyl-␤-D-galactopyranoside) containing (1 mg/ml) staining solution (5 mM potassium ferricyanate, 5 mM potassium ferrocyanate, 2 mM MgCl 2 ).…”

Section: Methodsmentioning

confidence: 99%

Matrix Fibronectin Binds Gammaretrovirus and Assists in Entry: New Light on Viral Infections

Beer¹,

Pedersen

2007

J Virol

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A major entry route for the gammaretrovirus amphotropic murine leukemia virus (A-MLV) into NIH 3T3 fibroblasts is via caveola-dependent endocytosis. However, during the infection time, few viral particles can be observed intracellularly. Analyzing the dynamics of the A-MLV infection process by using total internal reflection fluorescence microscopy, we show that the majority of viruses are extracellular and bound to the fibronectin matrix. Moreover, the amounts of bound virus and of fibronectin correlated. Using confocal microscopy, nanoparticles targeted to fibronectin by a III1C-fibronectin fragment or anti-fibronectin antibody were detected intracellularly in NIH 3T3 cells; unconjugated nanoparticles neither bound to cells nor were detectable intracellularly. Furthermore, A-MLV colocalized intracellularly with the fibronectin-targeted nanoparticles, suggesting that they were taken up by the same cellular pathway. Both A-MLV entry and fibronectin turnover depend on caveolar endocytosis, and we found that inhibiting viral binding to the extracellular NIH 3T3 fibronectin-matrix dramatically reduced A-MLV infection, indeed, showing an active role of fibronectin in infection. We suggest that binding to the cellular fibronectin matrix provides a new mechanism by which viruses can enter cells.Knowledge of the role of cellular factors in retroviral entry increases our understanding of how viruses can exploit cellular features to enter host cells. Gammaretroviruses, like other enveloped viruses, are dependent on specific cellular receptors for fusion of the viral membrane with the cellular membrane; e.g., amphotropic murine leukemia virus (A-MLV) depends on the presence of the ubiquitously expressed sodium-dependent phosphate transporter Pit2 (17,29,48,50). MLV-based retroviral vectors including vectors carrying A-MLV envelope proteins are widely used in gene therapy protocols, and although retroviruses and retroviral vectors can infect a variety of dividing cell types, the efficiencies vary greatly among different cell types despite the fact that these cells all express Pit2 (48). Specifically, efficient transduction of hematopoietic cells can only be achieved when infection occurs in the presence of chymotryptic fibronectin (FN) fragments like 30/35 FN, recombinant chimeric FN fragments like CH-296 (RetroNectin) (13, 32), or shed FN (sFN) derived from NIH 3T3-based packaging cell lines (21 and C. S. Søndergaard, C. Haldrup, C. Beer, D. B. Kohn, and L. Pedersen, submitted for publication). It has been suggested that increased infection is due to concomitant binding of vectors and cells to the fibronectin fragments and that this increases the likelihood that a vector and a cell will interact compared to the situation where both vectors and cells would be in suspension (13, 31). In agreement with this hypothesis, we could recently show that gammaretroviral vectors bind to sFN from NIH 3T3 cultures (Søndergaard et al., submitted).However, the role of naturally occurring FN in viral entry is largely unknown.FN is a compo...

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“…Virus titer. The titers of A-MLV and 10A1 MLV pseudotypes were determined on D17 cells as previously described (39); titers of VSV pseudotypes were determined in a similar set-up on NIH 3T3 cells. The titers obtained were ca.…”

Section: Methodsmentioning

confidence: 99%

“…Transient cotransfections and infection assays. Transient transfection and infection assays were performed essentially as described elsewhere (6,39). CHO K1 cells were seeded in 60-mm dishes at 2 ϫ 10 3 cells/cm 2 .…”

Section: Methodsmentioning

confidence: 99%

“…The next day, the cells were cotransfected by calcium phosphate DNA precipitation. Each precipitate (1 ml) contained 5 g of pOJ74 (Wyeth-Ayerst Research, Pearl River, NY) encoding human Pit2 (39) or pcDNA3 (empty vector), 2.5 g of C-or N-terminally green fluorescent protein (GFP)-tagged caveolin-1 expression plasmid in pEGFP-derived vectors from Clontech (kind gifts from Ari Helenius) (41) and 7.5 g of pUC19 plasmid as a carrier. Aliquots of 200 l precipitate were used per 60-mm dish, and three independent precipitates (three dishes) were made for each combination.…”

Section: Methodsmentioning

confidence: 99%

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Caveola-Dependent Endocytic Entry of Amphotropic Murine Leukemia Virus

Beer

Andersen²,

Rojek

et al. 2005

J Virol

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Early results suggested that the amphotropic murine leukemia virus (A-MLV) does not enter cells via endocytosis through clathrin-coated pits and this gammaretrovirus has therefore been anticipated to fuse directly with the plasma membrane. However, here we present data implicating a caveola-mediated endocytic entry route for A-MLV via its receptor Pit2. Caveolae belong to the cholesterol-rich microdomains characterized by resistance to nonionic detergents such as Triton X-100. Extraction of murine fibroblastic NIH 3T3 cells in cold Triton X-100 showed the presence of the A-MLV receptor Pit2 in detergent-insoluble microdomains. Using coimmunoprecipitation of cell extracts, we were able to demonstrate direct association of Pit2 with caveolin-1, the structural protein of caveolae. Other investigations revealed that A-MLV infection in contrast to vesicular stomatitis virus infection is a slow process (t 1 ⁄2 Ϸ5 h), which is dependent on plasma membrane cholesterol but independent of NH 4 Cl treatment of cells; NH 4 Cl impairs entry via clathrin-coated pits.

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Chimeras of receptors for gibbon ape leukemia virus/feline leukemia virus B and amphotropic murine leukemia virus reveal different modes of receptor recognition by retrovirus

Cited by 54 publications

References 19 publications

Evolutionary and experimental analyses of inorganic phosphate transporter PiT family reveals two related signature sequences harboring highly conserved aspartic acids critical for sodium‐dependent phosphate transport function of human PiT2

Evolutionary and experimental analyses of inorganic phosphate transporter PiT family reveals two related signature sequences harboring highly conserved aspartic acids critical for sodium‐dependent phosphate transport function of human PiT2

Matrix Fibronectin Binds Gammaretrovirus and Assists in Entry: New Light on Viral Infections

Caveola-Dependent Endocytic Entry of Amphotropic Murine Leukemia Virus

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