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Journal of Virology, December 2001, p. 11464-11473, Vol. 75, No. 23
0022-538X/01/$04.00+0   DOI: 10.1128/JVI.75.23.11464-11473.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Functional Murine Leukemia Virus Vectors Pseudotyped with the Visna Virus Envelope Show Expanded Visna Virus Cell Tropism

Linda Bruett and Janice E. Clements*

Division of Comparative Medicine, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21287

Received 20 March 2001/Accepted 18 August 2001


    ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

Pseudotype virus vectors serve as a powerful tool for the study of virus receptor usage and entry. We describe the development of murine leukemia virus (MuLV) particles pseudotyped with the visna virus envelope glycoprotein and encoding a green fluorescent protein reporter as a tool to study the expression of the visna virus receptor. Functional MuLV/visna virus pseudotypes were obtained when the cytoplasmic tail of the visna virus envelope TM protein was truncated to 3, 7, or 11 amino acids in length. MuLV/visna virus particles were used to transduce a panel of cell types from various organisms, including sheep, goat, human, hamster, mouse, monkey, and quail. The majority of the cells examined were susceptible to MuLV/visna pseudotype viruses, supporting the notion that the visna virus cellular receptor is a widely expressed protein found in many species. Of 16 different cell types tested, only mouse embryo fibroblast NIH 3T3 cells, hamster ovary CHO cells, and the human promonocyte cell line U937 cells were not susceptible to transduction by the pseudotyped virus. The production of functional MuLV/visna virus pseudotypes has provided a sensitive, biologically relevant system to study visna virus cell entry and envelope-receptor interactions.


    INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

Visna virus is a member of the Lentivirus family of retroviruses. The genetic and structural organization of visna virus is very similar to that of other members of this virus family, including the human immunodeficiency virus (HIV) and the simian immunodeficiency virus (SIV). The pathobiology of visna virus in sheep includes interstitial pneumonia, encephalitis, and arthritis (30-32). Reports from our laboratory and others have studied visna virus cell tropism, focusing on cells of sheep and goat origin, demonstrating an ability of visna virus to enter a variety of cell types in vitro with a restricted tropism for replication in vivo to cells of the monocyte lineage (1, 5, 10, 12, 17, 19, 37, 46). Unlike the primate lentiviruses HIV and SIV, the mechanism for entry of visna virus into its host cells is still unknown. Putative receptors for visna virus entry have been suggested, but to date, the identity of the cellular receptor for visna virus has not been elucidated (4, 6, 13, 15).

Studies on visna virus cell entry have been limited by a lack of available tools to clearly study the initial interaction of the envelope glycoprotein with its conjugate cellular receptor. Most studies on visna virus cell entry to date have utilized infectious visna virus to challenge cells and have measured viral output, observed cell-cell fusion, or detected viral RNA or DNA by PCR, each detectable phenotype being dependent on virus integration, replication, and/or production. In addition to the aforementioned methods, coculture assays have been used to study visna virus receptor distribution (24). In a coculture assay the envelope is expressed on one cell type which is grown together with a test cell line to look for cell-cell fusion and multinucleated cells as an indicator of receptor expression.

Many advances in the understanding of retrovirus cellular entry have been made by the utilization of virus pseudotype systems (2, 16, 34, 35, 40, 41). Pseudotyping refers to the incorporation of an envelope glycoprotein onto a viral core whose proteins and machinery are derived from a different virus than that of the envelope. The resulting pseudotyped particles should show a cell tropism indicative of the virus from which the envelope glycoprotein was derived, but the machinery and cellular requirements for packaged gene expression will be that of the virus which makes up the core and genome of the particle. Pseudotypes provide an ideal system for the separation of the entry step of viral infection from subsequent events in replication.

Murine leukemia virus (MuLV) is commonly used as a vector for pseudotype production, as it is able to incorporate heterologous envelope proteins onto its core in place of the native envelope protein. In many instances, including vesicular stomatitis virus (VSV) (7), Ebola virus (43), SIV (22), feline leukemia virus subgroup B (33), LaCrosse and Hantaan virus (25), and feline endogenous virus RD114 (11), pseudotypes of MuLV with the foreign viral glycoprotein are obtained at a reasonably high titer. Some viral envelope glycoproteins, however, do not incorporate into the MuLV core, and pseudotype formation is restricted unless modifications to the cytoplasmic tail of the envelope are made (21, 27, 39).

This report describes the development of functional MuLV/visna virus pseudotypes. MuLV/visna virus pseudotype production was dependent on envelope construct expression and on cytoplasmic tail length. Optimized MuLV/visna virus pseudotypes were used to facilitate increased understanding of visna virus entry into host cells. Replication of visna virus is limited to sheep and goat cells in vitro; however, these pseudotype experiments demonstrate that the observed limited tropism is not a block at the level of cell entry. Our data demonstrate that the visna virus receptor is widely expressed in cells of sheep and goat derivation, as well as cells from various other species, including quail, monkey, hamster, and human.


    MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

Cell lines. Sheep choroid plexus (SCP) cells and goat synovial membrane (GSM) cells were obtained as previously described (30-32). SCP and GSM cells were maintained in minimum essential medium with Earle's salts (EMEM) (GIBCO BRL, Gaithersburg, Md.) supplemented with 10% fetal bovine serum (FBS) and gentamicin (50 µg/ml) reagent solution (GIBCO BRL). Human embryonic kidney 293T cells (American Type Culture Collection [ATCC], Manassas, Va.) were maintained in Dulbecco's modified Eagle's medium (DMEM) (GIBCO BRL) containing 10% FBS and supplemented with, 2 mM L-glutamine, 2 mM Na pyruvate, 10 mM HEPES, and penicillin (90 U/ml)-streptomycin (90 µg/ml) (GIBCO BRL). Human endothelial cells (iBMEC) were obtained from Shahin Rafii (8). Quail QT6 cells, African green monkey fibroblast COS-7 cells, HeLa cells, and murine embryonic fibroblast NIH 3T3 cells were each obtained from the ATCC. Murine macrophage-like RAW264.2 cells were provided by Stephanie Vogel (Uniformed Services University of the Health Sciences, Bethesda, Md.), Syrian golden baby hamster kidney cells (BHK-21) were provided by Carolyn Machamer (Johns Hopkins School of Medicine, Baltimore, Md.), and Chinese hamster ovary cells (CHO) were provided by Diane Griffin (Johns Hopkins School of Public Health, Baltimore, Md.). QT6, COS-7, HeLa, NIH 3T3, RAW264.2, and iBMEC cells were all grown in DMEM supplemented with 10% FBS. BHK-21 cells were grown in DMEM with 5% FBS. CHO cells were maintained in F-12 nutrient mixture (Ham) medium with 10% FBS (GIBCO BRL).

Nonadherent cells, U937, THP-1, and Jurkat cells were each obtained from the ATCC. CEM(E) and CEMx174 cells were provided by James Hoxie (University of Pennsylvania). Each cell line was grown in RPMI medium 1640 (GIBCO BRL) containing 10% FBS and supplemented with 2 mM L-glutamine, 2 mM Na pyruvate, 10 mM HEPES, and gentamicin (50 µg/ml) reagent solution. THP-1 cells were grown in RPMI containing the same supplements as above with the addition of 0.05 mM 2-mercaptoethanol.

Antiserum. Visna neutralizing antiserum (NN) was raised against whole visna virus in a goat (14). Newborn goat serum (NGS) was the control serum utilized in the blocking experiment. The antiserum to visna virus envelope glycoprotein was raised in guinea pigs and is designated gp2,3 (14). The MuLV capsid antibody, goat anti-MLV AKR capsid p30, was kindly provided by Paula Cannon, University of Southern California.

Plasmids. A truncated visna virus envelope gene was cloned by digestion from full-length visna virus clone LV1.1, an antigenic variant of visna virus 1514 (31), into the mammalian expression vector pCB6 (provided generously by Paul Bates, University of Pennsylvania). This clone, a 70-amino-acid cytoplasmic tail truncation, was designated LV1#61-2. Visna envelope expression was increased by utilizing PCR to allow the addition of a Kozak consensus sequence (CCACCATGG) at the 5' end of the envelope gene directly upstream and including the initiator ATG, and three stops were inserted at the 3' end into LV1#61-2. This PCR product was recloned into pCB6 at the BglII/MluI sites and designated VisEnv-70. Truncated envelope constructs (VisEnv-3, VisEnv-7, VisEnv-11, VisEnv-20, VisEnv-30, and VisEnv-46) were created with the QuikChange site-directed mutagenesis kit by Stratagene (La Jolla, Calif.), using the VisEnv-70 clone as a template. The MuLV gag-pol structural plasmid pCgp (20), the green fluorescent protein (GFP) packageable reporter plasmid pMX-GFP, and the VSV-G envelope plasmid pHIT/G were kindly provided by Paul Bates (University of Pennsylvania). Plasmid pE-GFP-N1, used to determine transfection efficiency, was obtained from Clontech (Palo Alto, Calif.).

Production of pseudotypes. A modification of a three-plasmid system for production of pseudotypes was utilized (36). Briefly, 293T cells were first plated at 30% confluence into a 10-cm-diameter tissue culture dish. The following morning a total of 10 µg of DNA was transfected, including 3.3 µg of each plasmid: a visna virus envelope, VSV-G or a GFP marker for transfection efficiency (no envelope control), pCgp (MuLV gag-pol structural construct), and the packageable pMX-GFP reporter plasmid. LipofectAMINE PLUS reagent package (GIBCO BRL) transfections were carried out as described in the product instructions using complexes of 65 µl of Plus Reagent, 10 µg of DNA, and 750 µl of serum-free DMEM dilution medium and 35 µl of LipofectAMINE and 750 µl of serum-free DMEM dilution medium for each 10-cm-diameter dish. Complexes were added to 293T cells in 7 ml of fresh serum-free DMEM and incubated at 37°C, for 3 h after which the concentration of FBS was brought to 10% by the addition of 7 ml of 20% 293T growth medium. At 7 h posttransfection, medium was removed and replaced with 8 ml of fresh 293T medium supplemented with 2.5% FBS. Transfected cells were transferred to 32°C at 21 to 24 h posttransfection to reduce visna virus envelope-induced fusion and increase pseudotype yield. Pseudotypes were harvested 48 h posttransfection by scraping cells, clarifying the viral supernatant by centrifugation for 10 min at 1,600 rpm in a Sorvall RT6000D tabletop centrifuge (cell pellets were retained for use in Western blot analysis described below), and filtering supernatant through a 0.45-µm-pore-size Millex-HV filter (Millipore, Malsheim, France). Pseudotype stocks were stored at 4°C or -80°C.

Envelope expression studies. 293T cells were transfected with each of the visna virus envelope clones with cytoplasmic tails of various lengths as described above for making pseudotypes. Photomicroscopy was performed on a Nikon inverted light microscope with Hoffman optics (magnification, ×200) at 48 h posttransfection to observe fusion. The 293T producer cell pellet, harvested 48 h posttransfection (see above), was lysed by rotating in 600 µl of cell lysis buffer {6.5 mM 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate [CHAPS], 50 mM Tris-HCl [pH 7.5], 0.15 M NaCl, containing complete protease inhibitor cocktail [Boehringer Mannheim, Indianapolis, Ind.]} for 1 h at 4°C and clarified by centrifugation for 10 min at 12,000 rpm in an Eppendorf model 5415C centrifuge, and supernatant was assayed for protein concentration using the microassay procedure of the Bio-Rad protein assay (Hercules, Calif.). For each sample, 50 µg of lysate was separated on a sodium dodecyl sulfate (SDS)-6% polyacrylamide gel. Late-stage visna virus-infected SCP cell lysates (50 µg) served as a positive control for visna virus envelope expression, and the negative control for visna virus envelope expression was MuLV/VSV-G 293T producer cell lysates. To detect visna virus envelope expression in 293T producer cells, the gel was transferred to Immobilon-P Transfer Membrane (Millipore) and Western blot analysis was performed with the anti-visna virus envelope antiserum gp2,3 (1:500) and a horseradish peroxidase-conjugated rabbit anti-guinea pig secondary antibody (1:1,000) (Dako, Carpinteria, Calif.).

Envelope incorporation into pseudotype particles. Incorporation of visna virus envelope onto MuLV cores was determined by Western blot analysis of MuLV/visna virus pseudotype particles. MuLV/visna virus or MuLV/VSV-G pseudotype supernatant harvested as described above (5 ml) was pelleted through a 20% sucrose-25 mM Tris (pH 8.0), 150 mM NaCl, and 2 mM EDTA (TNE) cushion in a Sorvall OTD65B Ultracentrifuge in a TH641 rotor for 2 h at 133,460 × g. In parallel, 5 ml of visna virus 1514 stock (5 × 107 50% tissue culture infective doses/ml) was pelleted in the same manner as the pseudotypes. All virus pellets were lysed with 100 µl of RIPA lysis buffer (140 mM NaCl, 10 mM Tris [pH 8.0], 5 mM EDTA, 1% sodium deoxycholate, 1% Triton X-100, 0.1% SDS). Pseudotype particle lysates (15 µl) were separated by SDS-6% polyacrylamide gel electrophoresis (PAGE) for detection of visna virus envelope and by SDS-12% PAGE for detection of MuLV capsid p30. Pelleted visna virus 1514 lysate (3.5 µl) was run on both gels. Gels were transferred to Immobilon-P transfer membrane (Millipore). Western blot analysis was performed on the 6% gel blot with the anti-visna virus envelope antiserum gp2,3 (1:500) and a secondary horseradish peroxidase-conjugated rabbit anti-guinea pig secondary antibody (1:1,000) (Dako). The 12% gel blot was probed with goat anti-MLV AKR capsid p30 (1:5,000) and a horseradish peroxidase-conjugated rabbit anti-goat secondary antibody (1:2,000) (Dako)

Pseudotype infections. MuLV/visna virus pseudotypes were harvested, and 10 µl of viral supernatant was used in a reverse transcriptase (RT) assay adapted from Clabough et al. (9) to quantitate relative virus amount. The RT assay was modified for MuLV, utilizing MnCl at a final concentration of 2.5 mM in the RT buffer (rather than MgCl utilized by HIV and SIV RT), and excluding EGTA from the reaction mixture. MuLV/visna virus pseudotype (0.5 ml) for each envelope tail length was used to infect SCP cells (5 × 104 cells per well) in triplicate wells of a 24-well plate. After 9 h, 0.5 ml of EMEM supplemented with 5% FBS was added and infections were carried out for 2 days at 37°C. GFP-transduced cells were counted using a Becton Dickinson FACSCalibur three-color flow cytometer. Three thousand events were counted, and a percentage of events in the FL1 channel for GFP fluorescent scatter was determined and used to calculate the number of infectious units (IU) of pseudotype per milliliter of supernatant.

Neutralization experiments. SCP cells were plated (5 × 104 cells/well) in a 24-well plate and infected the following morning, in duplicate, with 0.5 ml of MuLV/VisEnv-7 or MuLV/VSV-G pseudotype that had been preincubated for 2 h at 4°C with either NN (1:200), NGS (1:200), or no antiserum. After 6 h, 0.5 ml of EMEM-1% FBS was added, and incubation continued for 48 h. GFP-transduced cells were detected by fluorescence-activated cell sorter analysis as described above.

Visna tropism. Adherent cells were plated (5 × 104 cells/well) in a 24-well tissue culture plate. The following morning, cells were washed once with serum-free medium and infected with 0.5 ml of MuLV/VisEnv-7 or MuLV/VSV-G or with 0.5 ml of serum-free medium. After 7 h at 37°C, 0.5 ml of EMEM-5% FBS or 0.5 ml of DMEM-5% FBS was added to each well and incubation continued for a total of 48 h at 37°C. Nonadherent cells (105 cells) were pelleted (1,200 rpm in a tabletop microcentrifuge) and resuspended in 0.5 ml of viral pseudotype supernatant (MuLV/VisEnv-7 or MuLV/VSV-G). After 7 h of incubation, 0.5 ml of RPMI supplemented with 5% FBS was added and incubation continued for a total of 48 h at 37°C. GFP-transduced cells were detected by flow cytometry as described above, and titers (in IU per milliliter) were calculated.


    RESULTS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

Construction of visna virus envelope clones. The cytoplasmic tail of the transmembrane envelope gene from visna virus infectious molecular clone LV1.1 (an antigenic variant of visna virus 1514) was truncated to 70 amino acids, and the envelope gene was subcloned into the eukaryotic expression vector pCB6. The full-length envelope gene of LV1.1 has a 126-amino-acid cytoplasmic tail; however, despite numerous cloning attempts, a clone containing the full-length cytoplasmic tail was not obtained. However, a clone with a nonsense mutation resulting in a 70-amino-acid truncated cytoplasmic tail was obtained and named LV1.1#61.2. When LV1.1#61.2 was transfected into pseudotype producer 293T cells, visna virus envelope expression was not detected (data not shown).

Frequently, lentivirus genes require a Kozak consensus sequence upstream of the ATG to drive optimal expression when expressed outside of the context of the viral genome. With this in mind, PCR was utilized to insert a Kozak sequence at the 5' end of the LV1.1#61.2 clone; in addition, three consecutive stop codons were inserted at the 3' end of the gene. The resultant clone was named VisEnv-70, and the amino acid sequence of the TM protein is depicted in Fig. 1. When this clone was transfected into 293T cells, expression of the visna virus envelope was detected (Fig. 2 and 3A).


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  		FIG. 1.   Amino acid sequence for the visna virus envelope TM protein clones. The sites of the cytoplasmic tail truncations generated for this study, including VisEnv-3, VisEnv-7, VisEnv-11, VisEnv-20, VisEnv-30, VisEnv-46, and VisEnv-70 are noted in the sequence (as 3, 7, 11, 20, 30, 46, and 70, respectively). The arrows represent the truncation sites for the various envelope transmembrane lengths. The transmembrane domain of the protein is indicated by the dashed line above the sequence. Two YXXempty motifs are underlined; the Y of this motif serves as the last amino acid in the VisEnv-3 and in VisEnv-11 clones. The numbering to the left of the sequence represents amino acid numbers in respect to full-length (wild-type) visna virus envelope. Wild-type visna virus envelope has a 126-amino-acid cytoplasmic tail but has not been clonable to date and is not shown here.


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  		FIG. 2.   Visna virus envelope induced fusion in 293T producer cells. Human embryonic kidney 293T cells were transfected to produce MuLV/visna virus, each with different envelope constructs for incorporation into the viral pseudotype particles. Envelope-mediated fusion was observed 48 h posttransfection by photomicroscopy at a magnification of ×170 on a Hoffman inverted light microscope for each of the truncated envelope clones, including VisEnv-3, VisEnv-7, VisEnv-11, VisEnv-20, VisEnv-30, VisEnv-46, and VisEnv-70, along with the VSV G protein (VSV-G), or the no-envelope control. Envelope-induced fusion is seen in VisEnv-3-, VisEnv-7-, VisEnv-11-, VisEnv-46-, and VisEnv-70-transfected producer cells, while fusion is not observed in VisEnv-20 and VisEnv-30 transfections.


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  		FIG. 3.   VisEnv expression and incorporation into MuLV particles. (A) Cell lysates (50 µg) from 293T producer cells were subjected to Western blot analysis using the anti-visna virus envelope antiserum gp2,3 as described in Materials and Methods to detect the visna virus envelope glycoprotein. Infected SCP cells (lane 1) are the positive control for visna virus envelope expression. The truncations of the visna virus envelope tail are in lanes 2 through 8. Producer cells making MuLV/VSV-G pseudotypes were lysed and serve as a negative control for visna virus envelope expression (lane 9). Cell lysis conditions favor the solubilization of SU protein (arrow) but not the TM protein. A difference in processing of the glycoprotein in productively infected SCP cells compared to transfected 293T cells may account for the slower-migrating glycoprotein in SCP cell lysates (lane 1). (B) Harvested MuLV/visna virus and MuLV/VSV-G pseudotypes were pelleted in an ultracentrifuge and lysed and Western blot analysis for visna virus envelope with gp2,3 (top panel) and for MuLV capsid p30 (bottom panel) was performed. Visna virus 1514 stocks produced in SCP cells by productive infection were pelleted in parallel and lysed and serve as a positive control for visna virus envelope incorporation (top panel, lane 1) and a negative control for MuLV capsid p30 (bottom panel, lane 1). Pelleted pseudotype virions from each of the visna virus truncated envelopes are in lanes 2 through 8. Pelleted MuLV/VSV-G stocks serve as a negative control for visna virus envelope incorporation (top panel, lane 9) and as a positive control for MuLV capsid p30 (bottom panel, lane 9). Virus lysis conditions solubilize both SU and TM proteins (arrows).

Since the goal of these experiments was to obtain visna virus envelopes that would efficiently pseudotype with MuLV cores, a panel of envelope clones with truncated cytoplasmic tails was designed using the VisEnv-70 clone as a template in a PCR-based mutagenesis protocol (QuikChange; Stratagene) (Fig. 1). Truncated envelope clones were designed to eliminate each of the two YXXempty motifs (tyrosine, any amino acid, any amino acid, and an amino acid with a bulky hydrophobic side chain [empty ]) (underlined in Fig. 1) present in the membrane-proximal portion of the visna virus TM protein cytoplasmic tail, leaving the tyrosine of this sequence as the terminal amino acid in each instance. This motif has been implicated in the trafficking and endocytosis of membrane proteins (42). VisEnv-11 eliminates one of the two YXXempty motifs. VisEnv-3 truncates the cytoplasmic tail to eliminate both of the YXXempty motifs. Although VisEnv-7 does not eliminate the membrane-proximal YXXempty motif, it was examined because of its similarity in length to truncated envelopes of HIV type 1 (HIV-1) and HIV-2 that efficiently pseudotype MuLV. The cytoplasmic tails of the HIV-1 and HIV-2 TM proteins contain this motif where the tyrosine is positioned at amino acid 7. Elimination of the motif from HIV-1 and HIV-2 facilitated efficient pseudotyping of MuLV with these glycoproteins (21, 27). Arbitrary tail lengths between VisEnv-11 and VisEnv-70 were examined as well, including VisEnv-20, VisEnv-30, and VisEnv-46 (Fig. 1).

Expression of visna virus envelope protein by 293T producer cells. Production of MuLV/visna virus pseudotypes is dependent on the incorporation of the visna virus envelope glycoprotein onto the surface of the MuLV cores. As the MuLV cores bud from the cellular membrane, they incorporate the envelope onto the particle surface. Retroviruses, including MuLV, can bud in the absence of an envelope glycoprotein; however, this will result in noninfectious particles. To produce functional MuLV/visna virus pseudotypes, high surface expression of the heterologous envelope protein facilitates incorporation into MuLV particles.

A three-plasmid system for pseudotype production was utilized as described (36). An envelope plasmid was cotransfected into 293T producer cells in conjunction with the MuLV gag-pol structural plasmid, which contains the enzymatic and structural genes for particle formation (pCgp), and the replication-deficient packageable GFP reporter plasmid, pMX-GFP, which has the viral long terminal repeats for integration and the packaging signal for RNA incorporation into MuLV particles. It was observed that transfection of some of the visna virus envelope constructs in 293T cells can result in a visible induction of cell-cell fusion (Fig. 2). As early as 24 h posttransfection, and most notably at 48 h, multinucleated giant cells were observed in cells transfected with VisEnv-3, VisEnv-7, VisEnv-11, VisEnv-46, and VisEnv-70 (Fig. 2). The VisEnv-20 and the VisEnv-30 clones consistently do not induce cell-cell fusion (Fig. 2). Extensive cell-cell fusion was consistently observed for the shorter envelope constructs and only rarely observed for VisEnv-46 transfections.

The envelope protein (G) of VSV was used as a control for pseudotype production, and the phenotype of producer cells expressing this surface molecule is consistently a slightly rounded cell in comparison to the control transfection (Fig. 2). To control for transfection efficiency, the envelope plasmid was replaced by a reporter plasmid carrying GFP under the control of a cytomegalovirus promoter (pEGFP-N1). This plasmid was used to quantitate the percentage of cells transfected (routinely 60 to 90%) by observing GFP fluorescence, in addition to producing envelope-minus MuLV particles (Fig. 2).

Cell-cell fusion in producer cells is not an absolute indicator of envelope expression. Some retroviral envelopes have varied abilities to fuse cells, depending on the length of the transmembrane cytoplasmic tail (23, 29, 45); thus, the envelope may be expressed at appreciable levels in the absence of cell-cell fusion. To assess envelope expression in producer cells in addition to the functional observation of cell-cell fusion, cell lysates from 293T producer cells were used in Western blot analysis with an anti-visna virus envelope antibody. It was found that visna virus envelope constructs resulting in the most fusion as observed in Fig. 2, including VisEnv-3, VisEnv-7, VisEnv-11, and VisEnv-70, are expressed in 293T producer cells, at significant levels as detected by Western blot analysis of producer cell lysates (Fig. 3A). In repeated experiments VisEnv-3, VisEnv-7, and VisEnv-11 consistently expressed high levels of envelope. The Western blot analysis depicted in Fig. 3A shows slightly lower expression of VisEnv-7 than VisEnv-3 and VisEnv-11; however, this variability seen in one transfection does not represent results of multiple repeated experiments. Expression of VisEnv-20, VisEnv-30, and VisEnv-46 by Western blot analysis could not be detected when 50 µg of cell lysate was run (Fig. 3A, lanes 5 to 7) but could be detected when 75 µg of lysate was resolved on a gel (data not shown). Of these three low expressers, VisEnv-46 had the highest signal for visna virus envelope expression. Lower levels of expression of VisEnv-20, VisEnv-30, and VisEnv-46 by Western blot analysis are consistent with the lack or low level of fusion observed in producer cells with these constructs (Fig. 2).

Cytoplasmic tail length requirement for the production of functional MuLV/visna virus pseudotypes. Production of functional MuLV/visna virus pseudotypes requires efficient incorporation of the visna virus envelope constructs into MuLV cores. To examine this incorporation, supernatants from producer cells containing pseudotype virions were pelleted in an ultracentrifuge, lysed, separated by SDS-6% PAGE, and transferred to an Immobilon membrane, and Western blot analysis for visna virus envelope glycoprotein was performed. VisEnv-3, VisEnv-7, and VisEnv-11 each were detected in pseudotype pellets (Fig. 3B, top panel). VisEnv-20, VisEnv-30, and VisEnv-46 were not detected in the pseudotype virions (Fig. 3B, top panel). This result is probably due to a low level or lack of expression of these envelopes in producer cells as seen in Fig. 3A. In contrast, VisEnv-70 was not detected in pseudotype pellets (Fig. 3B, top panel), although it was expressed in producer cells (Fig. 3A). Lysis conditions for pseudotype particles favored the solubilization of both surface (SU) and TM, and both proteins are labeled in Fig. 3B (top panel). To confirm that virions were produced in the transfections, virus lysates were separated on a 12% gel and subjected to Western blot analysis for MuLV capsid p30. Each of the pseudotype preparations harvested had similar levels of MuLV capsid p30 (Fig. 3B, bottom panel), indicating that MuLV cores were efficiently expressed and budding from all transfected cells.

An RT assay was utilized to quantitate the relative number of particles produced since it was shown in Fig. 3B that all transfections produced MuLV cores. The RT enzyme is packaged into all MuLV particles, and the activity of this enzyme in pseudotype supernatants can be measured quantitatively. RT activity of MuLV/visna virus and MuLV/VSV-G pseudotype stocks is reported in Table 1. Each MuLV/visna virus transfection resulted in the production of similar amounts of RT activity, representing similar numbers of viral particles produced, further supporting data from Western blot analysis of virus pellets for MuLV capsid p30 (Fig. 3B, bottom panel). A lower RT activity was measured for VSV-G and no envelope transfections, suggesting that fewer particles were released from these producer cells. It should be noted that the pseudotype preparation may contain both enveloped particles and naked cores, and both particle types will be quantitated in the RT assay.

                              
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  		TABLE 1.   RT activity and titers of MuLV/visna virus and MuLV/VSV-G pseudotypes on SCP target cells

Each pseudotype, including the no-envelope and MuLV/VSV-G particles, has packaged a GFP reporter gene RNA flanked by MuLV long terminal repeats. Upon transduction by these viral particles, the RNA is released into the host cell cytoplasm and is reverse transcribed, and the DNA is integrated into the host cell genome. Transduced cells express GFP, fluoresce, and can be detected with a flow cytometer.

SCP cells are the classic susceptible cells used to study visna virus infection in vitro. To study the functionality of the various MuLV/visna virus pseudotypes produced, SCP cells in a 24-well plate were infected with viral pseudotype supernatant for 48 h, after which GFP-transduced cells were enumerated using a Becton Dickinson flow cytometer. The percentage of GFP-transduced cells determined by flow cytometry was used to calculate the number of IU per milliliter of pseudotypes, based on the total number of cells per well (Table 1). Transducing pseudotypes were produced when the envelope glycoprotein was VisEnv-3, VisEnv-7, VisEnv-11, or VSV-G. As expected, the low or nonexpressing visna virus envelope constructs as determined by Western blot analysis of producer cell lysates (Fig. 3A) including the VisEnv-20, VisEnv-30, and VisEnv-46 produced particles as measured by Western blotting for MuLV p30 (Fig. 3B, bottom panel) and by RT assay (Table 1) but did not result in the production of transducing pseudotypes (Table 1). VisEnv-70, expressed in 293T producer cells, but not incorporated into virus particles (Fig. 3B, top panel), did not result in the production of transducing particles (Table 1).

The specific infectivity of pseudotype stocks in infectious units per RT (IU/RT) value was calculated to determine if differences in titer are the consequence of the quantity of particles released from producer cells or from the inherent infectivity of those particles released. Using the RT activity and titer of each stock, IU/RT was calculated and is reported in the third column of Table 1. MuLV/VSV-G pseudotypes have the highest specific infectivity compared to the MuLV/visna virus pseudotypes. In repeated experiments, MuLV/VSV-G pseudotypes have a lower RT activity but always have the highest specific infectivity. This suggests that incorporation of VSV-G into MuLV cores is more efficient than the incorporation of the visna virus envelopes or that the VSV-G envelope is more fusogenic or functional than the visna virus envelope glycoprotein when incorporated into MuLV cores, resulting in a higher specific infectivity.

Visna virus NN neutralizes transduction by MuLV/visna virus pseudotypes. Pseudotype viruses should reflect the cell tropism and entry characteristics of the virus whose envelope is represented on the particle surface without the necessity for virus replication. MuLV/visna virus pseudotypes were able to transduce SCP cells, showing that the pseudotypes are able to transduce the classic in vitro host cell for visna virus infection and can serve as a model for visna virus entry. To further characterize this interaction as analogous to whole virus, we neutralized the pseudotype transduction with NN, a potent neutralizing antiserum specific for visna virus (14). MuLV/visna virus (VisEnv-7) and MuLV/VSV-G pseudotypes were preincubated with NN, NGS, or no treatment, and the virus-serum or virus alone mixture was used to transduce SCP cells as described in Materials and Methods. GFP-transduced cells were measured 48 h posttransduction as described above and used to calculate the number of IU per milliliter for each treatment. The NN significantly reduced the titer of MuLV/visna virus pseudotypes (a >100-fold reduction), while it did not have a significant inhibitory effect on the titer of MuLV/VSV-G pseudotypes, showing a specificity for visna virus envelope and no reactivity with an unrelated virus (Fig. 4). The serum control, NGS, did not affect the titer of either virus.


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  		FIG. 4.   Visna virus NN significantly reduces infectivity of MuLV/visna virus pseudotypes, while having a minimal effect on MuLV/VSV-G infectivity. SCP cells were infected with MuLV/VSV-G or MuLV/VisEnv-7 pseudotypes that were untreated or preincubated with either visna virus NN or NGS. Cells were infected for 48 h and the number of IU per milliliter was calculated as described in Table 1. Results shown are from one representative experiment. Error bars, standard deviations.

Visna virus cell tropism. MuLV/visna virus pseudotypes, as demonstrated above, provide a model to study visna virus host cell tropism. A variety of cell lines from different organisms were challenged with MuLV/visna virus (VisEnv-7) pseudotypes to study the distribution of the visna virus receptor. MuLV/VSV-G pseudotypes were used in parallel with the MuLV/visna virus pseudotypes as a positive control for transduction and GFP production in each of the various cell types. The receptor for VSV is known to be widespread, resulting in a fairly ubiquitous cell tropism for the virus (28). The usual target cells of visna virus infection, SCP and GSM cells, were both permissive to MuLV/visna virus particles at titers of 3.69 × 104 and 1.91 × 104 IU/ml, respectively (Fig. 5A). Results in Fig. 5A are from one representative experiment in which duplicate wells were infected. Multiple stocks of MuLV/VisEnv-7 have been produced for use in various pseudotype experiments, with titers in the range of 104 to 105 IU/ml obtained on SCP cells. To determine whether there was a species restriction to visna virus entry, human cells (HeLa, iBMEC, and 293T), hamster cells (BHK-21 and CHO-K1), mouse cells (RAW264.2 and NIH 3T3), monkey cells (COS-7), and quail cells (QT6) were each challenged with the same stock of MuLV/visna virus and titers were determined (Fig. 5A). Cells from all species tested were permissive (Table 2). All cell types, except mouse NIH 3T3 and hamster CHO cells, were permissive to visna virus entry, with significant titers comparable to titers on the known permissive SCP cells on the order of 104 IU/ml. Further, a panel of human nonadherent cells was examined (Fig. 5B). Lymphocyte lineage cells, CEM, CEMx174, and Jurkat cells, along with THP-1 human monocyte cells, were susceptible to MuLV/visna virus particle transduction, while U937 cells, monocytic human cells, were not transducible by MuLV/visna virus (Fig. 5B). All cells in this panel of nonadherent cells were challenged with the same stock of MuLV/VisEnv-7, whose titer on SCP cells was 1.86 × 104 IU/ml (Fig. 5B). A variety of cell types, adherent and nonadherent, were transducible by MuLV/visna virus, including epithelial, endothelial, fibroblast, monocyte, and lymphocyte cells (Table 2).


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  		FIG. 5.   Host cell tropism of MuLV/visna virus pseudotypes. (A) All cell types were plated in a 24-well plate at 5 × 104 cells/well the day before infection with 0.5 ml of MuLV/VisEnv-7 or MuLV/VSV-G pseudotype supernatant in duplicate wells. Infections were performed as described in Materials and Methods, and GFP-transduced cells were detected 48 h postinfection. SCP and GSM cells are sheep and goat cells; HeLa, iBMEC, and 293T are human cells; BHK-21 and CHO-K1 are hamster cells; RAW264.2 and NIH 3T3 are mouse cells; COS-7 are monkey cells; and QT6 are quail cells. Results reported are from one representative experiment in which duplicate wells were infected; the same trends have been observed in multiple replications of the same experiment. (B) Nonadherent human cells (105 cells/well) were challenged with MuLV/VisEnv-7 and MuLV/VSV-G, including CEM-E, CEMx174, U937, Jurkat, and THP-1 cells. SCP cells (5 × 104) were plated and infected the following day, in parallel with the nonadherent cells, to show the titer of this MuLV/visna virus stock on the natural in vitro sheep host cell.

                              
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  		TABLE 2.   Cells screened for MuLV/visna virus susceptibility


    DISCUSSION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

Viral pseudotypes serve as a powerful tool to measure virus entry independent of replication. In this study, we produced MuLV/visna virus pseudotypes that infected some, but not all, cells of sheep, goat, human, hamster, mouse, quail, and monkey origin. This provides a powerful system to examine virus entry and for identification of the visna virus receptor. Functional MuLV/visna virus particles were produced when the visna virus envelope TM protein had a truncated cytoplasmic tail. Susceptibility to neutralization by anti-visna virus serum provided further evidence of biologic relevance of this MuLV/visna virus pseudotype system to visna virus.

Visna virus, as a member of the lentivirus family of retroviruses, has a long transmembrane cytoplasmic tail on the envelope glycoprotein (126 amino acids) compared to the transmembrane tail length of wild-type MuLV glycoprotein (~20 to 30 amino acids). MuLV/HIV and MuLV/SIV pseudotype production has been described. However, for some strains of these lentiviruses, envelope incorporation into MuLV particles required truncations and/or alterations of the cytoplasmic tail of the TM protein (21, 27, 38). The underlying determinants for heterologous envelope incorporation into MuLV cores are unknown. It has been suggested that longer envelope transmembrane cytoplasmic tail lengths introduce a steric restriction on envelope incorporation into the MuLV core (21). When pseudotyping MuLV with visna virus envelope glycoprotein, the tails with shorter lengths, including VisEnv-3, VisEnv-7, and VisEnv-11 were incorporated into MuLV particles and subsequently resulted in the production of functional transducing MuLV/visna virus pseudotypes. No conclusions can be made concerning the intermediate-length envelope constructs, as the VisEnv-20, VisEnv-30, and VisEnv-46 envelopes are not expressed in producer cells to an appreciable level. However, the longer tail, VisEnv-70, was expressed at the surface, but was not incorporated into budding MuLV particles, suggesting a possible steric restriction for envelope incorporation.

Alternatively, one report suggests a sequence-specific requirement for incorporation or exclusion of heterologous envelope glycoproteins from MuLV particles (21). The visna virus envelope transmembrane cytoplasmic tail has two occurrences of the sequence YXXempty , a sequence thought to be important in membrane protein trafficking (42). The tyrosine of the implicated sequence occurs at both amino acid 11 and amino acid 3 of the cytoplasmic tail of the visna virus envelope TM protein. To address the possibility that disruption of this signal could increase surface expression and facilitate pseudotype production, VisEnv-3 and VisEnv-11 truncations were made. These constructs leave the tyrosine of the motif as the C-terminal amino acid of each respective protein construct, as had been done to HIV and SIV envelopes to produce functional MuLV pseudotypes. Of note, the expression of VisEnv-3 (with no YXXempty motif), VisEnv-7 (with one YXXempty ), and VisEnv-11 (with one YXXempty ) was much greater (Fig. 2 and 3A) than expression of VisEnv-20, VisEnv-30, and VisEnv-46 (each with two YXXempty motifs). Thus, our results could support the suggestion by Hohne et al. that the removal of one or both of these motifs could reduce the trafficking of the envelope from the cellular membrane and increase the surface expression facilitating incorporation into MuLV pseudotypes (21). The VisEnv-20, VisEnv-30, and VisEnv-46 envelopes have two such sequences close to the C-terminal end of the glycoprotein and are not expressed at detectable levels. This theory does not, however, account for the surface expression of the VisEnv-70 protein with two YXXempty motifs, unless the location of the signal sequence in relation to the C-terminal end of the protein is important or the structure of the full-length protein masks one or both of these motifs. Our data support the possibility that the shorter TM protein cytoplasmic tail length could optimize steric conditions for incorporation of heterologous envelopes into MuLV cores, while sequence specificity of the cytoplasmic tail may be important for adequate expression of the envelope for incorporation into budding virions.

Contrary to what we have reported here, Zeilfelder and Bosch recently reported the inability to produce functional MuLV/visna virus particles (44). Similar to our experiments, Zeilfelder and Bosch truncated the cytoplasmic tail of the TM protein. It is possible that their inability to detect transducing pseudotype particle production was the result of a less sensitive detection system (beta -galactosidase) and suboptimal conditions for production and titration of their viral stocks. We found optimal conditions both for the production of high-titer MuLV/visna virus and for the titration of the pseudotypes stocks to be dependent on low concentrations in serum in cell medium. To maximize MuLV/visna virus titers, medium on transfected producer cells is changed to DMEM supplemented with 2.5% FBS approximately 7 h posttransfection. When producer cell medium is supplemented with 10% FBS rather than 2.5% FBS, at least a 1-log reduction in pseudotype titer is observed (data not shown). In addition, titration of stocks on a cell of sheep origin, such as SCP cells, is ideal for determining MuLV/visna virus pseudotype titer. Together, these technical optimizations may explain the discrepancy between our results and those reported by Zeilfelder and Bosch (44).

Reports on visna virus cell tropism and receptor expression focus on cells of sheep or goat lineage, with only a few reports studying cells from other species. Our MuLV/visna virus pseudotypes provide an optimal, sensitive system to quantitate visna virus envelope-mediated entry into host cells. Our results on the widespread expression of the visna virus receptor in many species confirm some cell culture-based reports on receptor distribution (3, 18, 24, 26, 44); however, there are a few discrepancies. For example, Lyall et al. report that NIH 3T3 cells express the visna virus receptor, as they fuse when subjected to a coculture assay with vaccinia virus-driven visna virus envelope-expressing BHK-21 cells (24). Zeilfelder and Bosch also report that NIH 3T3 cells form multinucleated cells when cocultured with visna virus envelope-expressing HeLa cells (44). Contrary to these observations, we report here that NIH 3T3 cells do not express the visna virus receptor, as MuLV/visna virus pseudotypes do not transduce NIH 3T3 cells at any detectable level in our assay. Further, pseudotype experiments support previous reports from our laboratory using a PCR assay for detection of gag transcripts where NIH 3T3 cells were shown to be nonpermissive to visna virus infection (4).

Our work supports that of Lyall et al., demonstrating both the presence of the visna virus receptor on COS-7, HeLa, BHK-21, and 293T cells and the lack of receptor on CHO cells (24). Zeilfelder and Bosch, in an independent work, conclude that HeLa cells lack visna virus receptor because no multinucleated cells were observed in an experiment in which visna virus envelope was transfected into HeLa cells and cells were inspected for cell-cell fusion. Our results do not support this observation, as MuLV/visna virus pseudotypes efficiently transduced HeLa cells (titer, 1.26 × 104), demonstrating the presence of the visna virus receptor.

Interestingly, visna virus did not demonstrate a species restriction for entry in these studies. RAW264.2, a mouse macrophage-like cell line, was permissive to MuLV/visna virus entry. However NIH 3T3, another mouse cell line, was not permissive to entry. Along the same line, BHK-21, a hamster cell line, was susceptible to MuLV/visna virus pseudotypes, but CHO (hamster ovary) cells were not. All human cell lines tested, excluding the human promonocyte-like U937 cells, were permissive to MuLV/visna virus entry. Future comparative studies will use these nonpermissive cell lines to identify the feature that delineates them from virus-permissive cells.

The production of high-titer MuLV/visna virus pseudotypes has opened the door for studying visna virus entry and receptor distribution in a relevant, sensitive, and quantitative manner. Further, this powerful tool, in combination with receptor distribution information reported here, will be utilized to screen a susceptible cell cDNA library for the visna virus receptor.


    ACKNOWLEDGMENTS

This research was supported by grants from the National Institutes of Health (NIH T32 NS07392 and NS23039).

We thank Paul Bates of the University of Pennsylvania for his insightful suggestions and advice throughout the work, Debbie Hauer for her technical assistance in the cloning of the visna virus envelope constructs, and Lee Blosser for his assistance with the fluorescence-activated cell sorter analysis. We also thank Sheila Barber and Jason Roos for insightful discussions and scientific suggestions incorporated into this work.


    FOOTNOTES

* Corresponding author. Mailing address: 600 N. Wolfe St., Jefferson St. Bldg. 3-127, Baltimore, MD 21287. Phone: (410) 955-9770. Fax: (410) 955-9823. E-mail: jclement{at}jhmi.edu.


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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

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Journal of Virology, December 2001, p. 11464-11473, Vol. 75, No. 23
0022-538X/01/$04.00+0   DOI: 10.1128/JVI.75.23.11464-11473.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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