scholarly journals Replacement of the Ectodomains of the Hemagglutinin-Neuraminidase and Fusion Glycoproteins of Recombinant Parainfluenza Virus Type 3 (PIV3) with Their Counterparts from PIV2 Yields Attenuated PIV2 Vaccine Candidates

2000 ◽  
Vol 74 (14) ◽  
pp. 6448-6458 ◽  
Author(s):  
Tao Tao ◽  
Mario H. Skiadopoulos ◽  
Fatemeh Davoodi ◽  
Jeffrey M. Riggs ◽  
Peter L. Collins ◽  
...  
Keyword(s):  
Virus Type ◽  
Vaccine Candidate ◽  
Cytoplasmic Tail ◽  
Full Length ◽  
Parainfluenza Virus ◽  
Tail Domain ◽  
Wild Type ◽  
Vaccine Candidates

ABSTRACT We sought to develop a live attenuated parainfluenza virus type 2 (PIV2) vaccine strain for use in infants and young children, using reverse genetic techniques that previously were used to rapidly produce a live attenuated PIV1 vaccine candidate. The PIV1 vaccine candidate, designated rPIV3-1cp45, was generated by substituting the full-length HN and F proteins of PIV1 for those of PIV3 in the attenuatedcp45 PIV3 vaccine candidate (T. Tao et al., J. Virol. 72:2955–2961, 1998; M. H. Skiadopoulos et al., Vaccine 18:503–510, 1999). However, using the same strategy, we failed to recover recombinant chimeric PIV3-PIV2 isolate carrying the full-length PIV2 glycoproteins in a wild-type PIV3 backbone. Viable PIV3-PIV2 chimeras were recovered when chimeric HN and F open reading frames (ORFs) rather than complete PIV2 F and HN ORFs were used to construct the full-length cDNA. The recovered viruses, designated rPIV3-2CT, in which the PIV2 ectodomain and transmembrane domain were fused to the PIV3 cytoplasmic domain, and rPIV3-2TM, in which the PIV2 ectodomain was fused to the PIV3 transmembrane and cytoplasmic tail domain, possessed similar in vitro and in vivo phenotypes. Thus, it appeared that only the cytoplasmic tail of the HN or F glycoprotein of PIV3 was required for successful recovery of PIV3-PIV2 chimeras. Although rPIV3-2CT and rPIV3-2TM replicated efficiently in vitro, they were moderately to highly attenuated for replication in the respiratory tracts of hamsters, African green monkeys (AGMs), and chimpanzees. This unexpected finding indicated that chimerization of the HN and F proteins of PIV2 and PIV3 itself specified an attenuation phenotype in vivo. Despite this attenuation, these viruses were highly immunogenic and protective against challenge with wild-type PIV2 in hamsters and AGMs, and they represent promising candidates for clinical evaluation as a vaccine against PIV2. These chimeric viruses were further attenuated by the addition of 12 mutations of PIV3cp45 which lie outside of the HN and F genes. The attenuating effects of these mutations were additive with that of the chimerization, and thus inclusion of all or some of the cp45 mutations provides a means to further attenuate the PIV3-PIV2 chimeric vaccine candidates if necessary.

scholarly journals Role of Interferon in the Replication of Human Parainfluenza Virus Type 1 Wild Type and Mutant Viruses in Human Ciliated Airway Epithelium

2008 ◽  
Vol 82 (16) ◽  
pp. 8059-8070 ◽  
Author(s):  
Emmalene J. Bartlett ◽  
Margaret Hennessey ◽  
Mario H. Skiadopoulos ◽  
Alexander C. Schmidt ◽  
Peter L. Collins ◽  
...  
Keyword(s):  
Virus Type ◽  
Airway Epithelium ◽  
Parainfluenza Virus ◽  
Progeny Virus ◽  
Wild Type ◽  
Vaccine Candidates ◽  
C Proteins ◽  
Human Parainfluenza Virus

ABSTRACT Human parainfluenza virus type 1 (HPIV1) is a significant cause of pediatric respiratory disease in the upper and lower airways. An in vitro model of human ciliated airway epithelium (HAE), a useful tool for studying respiratory virus-host interactions, was used in this study to show that HPIV1 selectively infects ciliated cells within the HAE and that progeny virus is released from the apical surface with little apparent gross cytopathology. In HAE, type I interferon (IFN) is induced following infection with an HPIV1 mutant expressing defective C proteins with an F170S amino acid substitution, rHPIV1-CF170S, but not following infection with wild-type HPIV1. IFN induction coincided with a 100- to 1,000-fold reduction in virus titer, supporting the hypothesis that the HPIV1 C proteins are critical for the inhibition of the innate immune response. Two recently characterized live attenuated HPIV1 vaccine candidates expressing mutant C proteins were also evaluated in HAE. The vaccine candidates, rHPIV1-CR84G/Δ170HNT553ALY942A and rHPIV1-CR84G/Δ170HNT553ALΔ1710-11, which contain temperature-sensitive (ts) attenuating (att) and non-ts att mutations, were highly restricted in growth in HAE at permissive (32°C) and restrictive (37°C) temperatures. The viruses grew slightly better at 37°C than at 32°C, and rHPIV1-CR84G/Δ170HNT553ALY942A was less attenuated than rHPIV1-CR84G/Δ170HNT553ALΔ1710-11. The level of replication in HAE correlated with that previously observed for African green monkeys, suggesting that the HAE model has potential as a tool for the preclinical evaluation of HPIV1 vaccines, although how these in vitro data will correlate with vaccine virus replication in seronegative human subjects remains to be seen.

scholarly journals Rab27a Is an Essential Component of Melanosome Receptor for Myosin Va

2002 ◽  
Vol 13 (5) ◽  
pp. 1735-1749 ◽  
Author(s):  
Xufeng Wu ◽  
Fei Wang ◽  
Kang Rao ◽  
James R. Sellers ◽  
John A. Hammer
Keyword(s):  
Full Length ◽  
Tail Domain ◽  
Wild Type ◽  
Essential Component ◽  
Myosin Va ◽  
Alternatively Spliced ◽  
Additional Protein ◽  
The Brain ◽  
Alternatively Spliced Exons

Melanocytes that lack the GTPase Rab27a (ashen) are disabled in myosin Va-dependent melanosome capture because the association of the myosin with the melanosome surface depends on the presence of this resident melanosomal membrane protein. One interpretation of these observations is that Rab27a functions wholly or in part as the melanosome receptor for myosin Va (Myo5a). Herein, we show that the ability of the myosin Va tail domain to localize to the melanosome and generate a myosin Va null (dilute) phenotype in wild-type melanocytes is absolutely dependent on the presence of exon F, one of two alternatively spliced exons present in the tail of the melanocyte-spliced isoform of myosin Va but not the brain-spliced isoform. Exon D, the other melanocyte-specific tail exon, is not required. Similarly, the ability of full-length myosin Va to colocalize with melanosomes and to rescue their distribution indilute melanocytes requires exon F but not exon D. These results predict that an interaction between myosin Va and Rab27a should be exon F dependent. Consistent with this, Rab27a present in detergent lysates of melanocytes binds to beads coated with purified, full-length melanocyte myosin Va and melanocyte myosin Va lacking exon D, but not to beads coated with melanocyte myosin Va lacking exon F or brain myosin Va. Moreover, the preparation of melanocyte lysates in the presence of GDP rather than guanosine-5′-O-(3-thio)triphosphate reduces the amount of Rab27a bound to melanocyte myosin Va-coated beads by approximately fourfold. Finally, pure Rab27a does not bind to myosin Va-coated beads, suggesting that these two proteins interact indirectly. Together, these results argue that Rab27a is an essential component of a protein complex that serves as the melanosome receptor for myosin Va, suggest that this complex contains at least one additional protein capable of bridging the indirect interaction between Rab27a and myosin Va, and imply that the recruitment of myosin Va to the melanosome surface in vivo should be regulated by factors controlling the nucleotide state of Rab27a.

Functions of the cytoplasmic domain of the beta PS integrin subunit during Drosophila development

Development ◽  
1994 ◽  
Vol 120 (1) ◽  
pp. 91-102 ◽  
Author(s):  
Y. Grinblat ◽  
S. Zusman ◽  
G. Yee ◽  
R.O. Hynes ◽  
F.C. Kafatos
Keyword(s):  
Germ Line ◽  
Cytoplasmic Tail ◽  
Cytoplasmic Domain ◽  
P Element ◽  
Integrin Subunit ◽  
Wild Type ◽  
Mutant Proteins ◽  
Adhesive Interactions

Integrins constitute a family of membrane-spanning, heterodimeric proteins that mediate adhesive interactions between cells and surrounding extracellular matrices (or other cells) and participate in signal transduction. We are interested in assessing integrin functions in the context of developing Drosophila melanogaster. This report, using mutants of the beta PS subunit encoded by the myospheroid (mys) locus, analyzes the relationships between integrin protein structure and developmental functions in an intact organism. As a first step in this analysis, we demonstrated the ability of a fragment of wild-type mys genomic DNA, introduced into the germ line in a P-element vector P[mys+], to rescue phenotypes attributed to lack of (or defects in) the endogenous beta PS during several discrete morphogenetic events. We then produced in vitro a series of modifications of the wild-type P[mys+] transposon, which encode beta PS derivatives with mutations within the small and highly conserved cytoplasmic domain. In vivo analysis of these mutant transposons led to the following conclusions. (1) The cytoplasmic tail of beta PS is essential for all developmental functions of the protein that were assayed. (2) An intron at a conserved position in the DNA sequence encoding the cytoplasmic tail is thought to participate in important alternative splicing events in vertebrate beta integrin subunit genes, but is not required for the developmental functions of the mys gene assayed here. (3) Phosphorylation on two conserved tyrosines found in the C terminus of the beta PS cytoplasmic tail is not necessary for the tested developmental functions. (4) Four highly conserved amino acid residues found in the N-terminal portion of the cytoplasmic tail are important but not critical for the developmental functions of beta PS; furthermore, the efficiencies with which these mutant proteins function during different morphogenetic processes vary greatly, strongly suggesting that the cytoplasmic interactions involving PS integrins are developmentally modulated.

scholarly journals Human Parainfluenza Virus Type 1 C Proteins Are Nonessential Proteins That Inhibit the Host Interferon and Apoptotic Responses and Are Required for Efficient Replication in Nonhuman Primates

2008 ◽  
Vol 82 (18) ◽  
pp. 8965-8977 ◽  
Author(s):  
Emmalene J. Bartlett ◽  
Ann-Marie Cruz ◽  
Janice Esker ◽  
Adam Castaño ◽  
Henrick Schomacker ◽  
...  
Keyword(s):  
Virus Type ◽  
Parainfluenza Virus ◽  
Airway Epithelial ◽  
Efficient Replication ◽  
C Proteins ◽  
Induced Apoptosis ◽  
Human Parainfluenza Virus

ABSTRACT Recombinant human parainfluenza virus type 1 (rHPIV1) was modified to create rHPIV1-P(C−), a virus in which expression of the C proteins (C′, C, Y1, and Y2) was silenced without affecting the amino acid sequence of the P protein. Infectious rHPIV1-P(C−) was readily recovered from cDNA, indicating that the four C proteins were not essential for virus replication. Early during infection in vitro, rHPIV1-P(C−) replicated as efficiently as wild-type (wt) HPIV1, but its titer subsequently decreased coincident with the onset of an extensive cytopathic effect not observed with wt rHPIV1. rHPIV1-P(C−) infection, but not wt rHPIV1 infection, induced caspase 3 activation and nuclear fragmentation in LLC-MK2 cells, identifying the HPIV1 C proteins as inhibitors of apoptosis. In contrast to wt rHPIV1, rHPIV1-P(C−) and rHPIV1-CF170S, a mutant encoding an F170S substitution in C, induced interferon (IFN) and did not inhibit IFN signaling in vitro. However, only rHPIV1-P(C−) induced apoptosis. Thus, the anti-IFN and antiapoptosis activities of HPIV1 were separable: both activities are disabled in rHPIV1-P(C−), whereas only the anti-IFN activity is disabled in rHPIV1-CF170S. In African green monkeys (AGMs), rHPIV1-P(C−) was considerably more attenuated than rHPIV1-CF170S, suggesting that disabling the anti-IFN and antiapoptotic activities of HPIV1 had additive effects on attenuation in vivo. Although rHPIV1-P(C−) protected against challenge with wt HPIV1, its highly restricted replication in AGMs and in primary human airway epithelial cell cultures suggests that it might be overattenuated for use as a vaccine. Thus, the C proteins of HPIV1 are nonessential but have anti-IFN and antiapoptosis activities required for virulence in primates.

scholarly journals Requirement of the NPXY motif in the integrin beta 3 subunit cytoplasmic tail for melanoma cell migration in vitro and in vivo.

1995 ◽  
Vol 130 (2) ◽  
pp. 441-450 ◽  
Author(s):  
E J Filardo ◽  
P C Brooks ◽  
S L Deming ◽  
C Damsky ◽  
D A Cheresh
Keyword(s):  
Cell Migration ◽  
Cytoplasmic Tail ◽  
Melanoma Cells ◽  
Structural Motif ◽  
Wild Type ◽  
Npxy Motif ◽  
Alpha V Beta 3 ◽  
Integrin Beta 3

The NPXY sequence is highly conserved among integrin beta subunit cytoplasmic tails, suggesting that it plays a fundamental role in regulating integrin-mediated function. Evidence is provided that the NPXY structural motif within the beta 3 subunit, comprising residues 744-747, is essential for cell morphological and migratory responses mediated by integrin alpha v beta 3 in vitro and in vivo. Transfection of CS-1 melanoma cells with a cDNA encoding the wild-type integrin beta 3 subunit, results in de novo alpha v beta 3 expression and cell attachment, spreading, and migration on vitronectin. CS-1 cells expressing alpha v beta 3 with mutations that disrupt the NPXY sequence interact with soluble vitronectin or an RGD peptide, yet fail to attach, spread, or migrate on immobilized ligand. The biological consequences of these observations are underscored by the finding that CS-1 cells expressing wild-type alpha v beta 3 acquire the capacity to form spontaneous pulmonary metastases in the chick embryo when grown on the chorioallantoic membrane. However, migration-deficient CS-1 cells expressing alpha v beta 3 with mutations in the NPXY sequence lose this ability to metastasize. These findings demonstrate that the NPXY motif within the integrin beta 3 cytoplasmic tail is essential for alpha v beta 3-dependent post-ligand binding events involved in cell migration and the metastatic phenotype of melanoma cells.

N-Linked Glycans within the A1A2A3 Domains of VWF Play a Critical Role in Modulating Macrophage-Mediated Clearance

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 469-469
Author(s):  
Alain Chion ◽  
Jamie O'Sullivan ◽  
Gudmundur Bergsson ◽  
Sean Keyes ◽  
Orla Rawley ◽  
...  
Keyword(s):  
Plasma Clearance ◽  
Full Length ◽  
Type I ◽  
Dependent Manner ◽  
Wild Type ◽  
Macrophage Receptors ◽  
A2 Domain

Abstract Enhanced plasma clearance of von Willebrand factor (VWF) plays an important role in the etiology of both type 1 and type 2 VWD. Nevertheless, although significant progress has been achieved in understanding the structure and functional properties of VWF, the mechanism(s) responsible for modulating VWF clearance from the plasma remain poorly understood. Accumulating recent data suggests that hepatic and splenic macrophages play key roles in modulating VWF clearance. A number of putative macrophage receptors for VWF have been also been described, including LRP1, β2-integrins and Siglec-5. In addition, it is well recognised that variation in VWF glycan expression significantly influences its clearance rate. In particular, terminal ABO(H) blood group determinants which are predominantly expressed on the N-linked glycans of human VWF significantly modulate its rate of clearance. Critically however, the molecular mechanisms through which specific macrophage receptors interact with particular regions of the complex VWF glycoprotein have not been defined. To investigate the role of VWF glycans and specific VWF domains in regulating VWF clearance, we expressed and purified a series of recombinant VWF variants and truncations with/without specific glycan sites. In addition, VWF glycosylation was modified using specific exoglycosidase digestions. Subsequently, recombinant VWF variants and glycoforms thereof were injected into VWF-/-mice, and plasma VWF clearance rates determined by ELISA. VWF-macrophage interactions were also quantified in vitro using phorbol ester-differentiated monocytic THP-1 cells, and primary human monocytes, in a High Content Analysis Imaging system. In keeping with previous reports, we observed that clearance of a truncated VWFA1A2A3 fragment in VWF-/-mice was very similar to that of full-length wild type (WT-) VWF (VWFA1A2A3; t1/2 = 6.3 min versus rWT-VWF; t1/2 = 7.9 min). Furthermore, chemical depletion of macrophages using clodronate liposomes administration significantly inhibited A1A2A3 clearance in vivo (1.7-fold at 10 min time point) to a similar extent to that observed with full length VWF. In vitro binding experiments confirmed that A1A2A3 bound to differentiated THP-1 cells in a dose- and time- dependent manner. Interestingly, this binding was significantly enhanced in the presence of ristocetin. Cumulatively, these data demonstrate that the A1A2A3 domains of VWF contain a critical receptor-binding site for macrophage-mediated clearance. Interestingly, we observed that the half-life of infused human plasma-derived VWF and recombinant VWF expressed in HEK293T cells in VWF-/- mice were significantly different. Furthermore, treatment with PNGase F to completely remove N-linked glycan structures markedly enhanced the clearance of full length VWF (t1/2 2.1 min; p<0.05). Collectively, these findings highlight the essential roles played by N-glycans in regulating VWF survival. Two N-linked glycan sites are located within A1A2A3 at N1515 and N1574 respectively. Importantly, we found that PNGase digestion of A1A2A3 resulted in markedly enhanced macrophage binding in vitro. Consequently we hypothesized that the two N-glycans located within the A2 domain might be important in regulating VWF clearance by macrophages. Targeted disruption of these individual N-glycan sites by site-directed mutagenesis (A1A2A3-N1515Q and A1A2A3-N1574Q respectively) resulted in significantly enhanced macrophage binding in vitro compared to wild type A1A2A3. Furthermore, following tail vein infusion in VWF-/-mice, full length VWFN1515Q and VWFN1574Q both demonstrated markedly reduced half-lives compared to wild type VWF (VWFN1515Q; t1/2 = 3.7 min, VWFN1574Q; t1/2 = 5.5 min). Finally, introduction of the N1515Q point mutation into truncated A1A2A3 also served to significantly enhance plasma clearance, (A1A2A3N1515Q-VWF; t1/2 = 3.1 min versus A1A2A3-VWF; t1/2 = 6.3 min). In conclusion, our novel data identify a crucial role of the VWF A domains in regulating macrophage-mediated VWF clearance. In addition, we further demonstrate that the N-linked glycans structures located at N1515 and N1574 within the A2 domain play specific roles in protecting VWF against in vivo clearance by macrophages. Given the important role played by enhanced VWF clearance in the etiology of type I VWD, these findings are of direct clinical importance. Disclosures No relevant conflicts of interest to declare.

scholarly journals A Triad of Subunits from the Gal11/Tail Domain of Srb Mediator Is an In Vivo Target of Transcriptional Activator Gcn4p

2004 ◽  
Vol 24 (15) ◽  
pp. 6871-6886 ◽  
Author(s):  
Fan Zhang ◽  
Laarni Sumibcay ◽  
Alan G. Hinnebusch ◽  
Mark J. Swanson
Keyword(s):  
Rna Polymerase Ii ◽  
Tata Binding Protein ◽  
Tail Domain ◽  
Transcriptional Coactivator ◽  
Wild Type ◽  
High Level ◽  
Target Promoters ◽  
Transcriptional Induction

ABSTRACT The Srb mediator is an important transcriptional coactivator for Gcn4p in the yeast Saccharomyces cerevisiae. We show that three subunits of the Gal11/tail domain of mediator, Gal11p, Pgd1p, and Med2p, and the head domain subunit Srb2p make overlapping contributions to the interaction of mediator with recombinant Gcn4p in vitro. Each of these proteins, along with the tail subunit Sin4p, also contributes to the recruitment of mediator by Gcn4p to target promoters in vivo. We found that Gal11p, Med2p, and Pgd1p reside in a stable subcomplex in sin4Δ cells that interacts with Gcn4p in vitro and that is recruited independently of the rest of mediator by Gcn4p in vivo. Thus, the Gal11p/Med2p/Pgd1p triad is both necessary for recruitment of intact mediator and appears to be sufficient for recruitment by Gcn4p as a free subcomplex. The med2Δ mutation impairs the recruitment of TATA binding protein (TBP) and RNA polymerase II to the promoter and the induction of transcription at ARG1, demonstrating the importance of the tail domain for activation by Gcn4p in vivo. Even though the Gal11p/Med2p/Pgd1p triad is the only portion of Srb mediator recruited efficiently to the promoter in the sin4Δ strain, this mutant shows high-level TBP recruitment and wild-type transcriptional induction at ARG1. Hence, the Gal11p/Med2p/Pgd1p triad may contribute to TBP recruitment independently of the rest of mediator.

scholarly journals New Insight into Filamentous Hemagglutinin Secretion Reveals a Role for Full-Length FhaB in Bordetella Virulence

mBio ◽  
2015 ◽  
Vol 6 (4) ◽  
Author(s):  
Jeffrey A. Melvin ◽  
Erich V. Scheller ◽  
Christopher R. Noël ◽  
Peggy A. Cotter
Keyword(s):  
Respiratory Tract ◽  
Lower Respiratory Tract ◽  
Primary Component ◽  
Full Length ◽  
Wild Type ◽  
Content Type ◽  
Filamentous Hemagglutinin ◽  
Mutant Strains

ABSTRACTBordetellafilamentous hemagglutinin (FHA), a primary component of acellular pertussis vaccines, contributes to virulence, but how it functions mechanistically is unclear. FHA is first synthesized as an ~370-kDa preproprotein called FhaB. Removal of an N-terminal signal peptide and a large C-terminal prodomain (PD) during secretion results in “mature” ~250-kDa FHA, which has been assumed to be the biologically active form of the protein. Deletion of two C-terminal subdomains of FhaB did not affect production of functional FHA, and the mutant strains were indistinguishable from wild-type bacteria for their ability to adhere to the lower respiratory tract and to suppress inflammation in the lungs of mice. However, the mutant strains, which produced altered FhaB molecules, were eliminated from the lower respiratory tract much faster than wild-typeB. bronchiseptica, suggesting a defect in resistance to early immune-mediated clearance. Our results revealed, unexpectedly, that full-length FhaB plays a critical role inB. bronchisepticapersistence in the lower respiratory tract.IMPORTANCETheBordetellafilamentous hemagglutinin (FHA) is a primary component of the acellular pertussis vaccine and an important virulence factor. FHA is initially produced as a large protein that is processed during secretion to the bacterial surface. As with most processed proteins, the mature form of FHA has been assumed to be the functional form of the protein. However, our results indicate that the full-length form plays an essential role in virulencein vivo. Furthermore, we have found that FHA contains intramolecular regulators of processing and that this control of processing is integral to its virulence activities. This report highlights the advantage of studying protein maturation and function simultaneously, as a role for the full-length form of FHA was evident only fromin vivoinfection studies and not fromin vitrostudies on the production or maturation of FHA or even fromin vitrovirulence-associated activity assays.

scholarly journals Construction and Characterization of a Full-Length Infectious Simian T-Cell Lymphotropic Virus Type 3 Molecular Clone

2007 ◽  
Vol 81 (12) ◽  
pp. 6276-6285 ◽  
Author(s):  
Sébastien Alain Chevalier ◽  
Marine Walic ◽  
Sara Calattini ◽  
Adeline Mallet ◽  
Marie-Christine Prévost ◽  
...  
Keyword(s):  
T Cell ◽  
Virus Type ◽  
Fluorescent Protein ◽  
Experimental Studies ◽  
Full Length ◽  
Cell Culture Supernatant ◽  
Molecular Clone ◽  
Human T Cell

ABSTRACT Together with their simian T-cell lymphotropic virus (STLV) equivalent, human T-cell lymphotropic virus type 1 (HTLV-1), HTLV-2, and HTLV-3 form the primate T-cell lymphotropic virus (PTLV) group. Over the years, understanding the biology and pathogenesis of HTLV-1 and HTLV-2 has been widely improved by the creation of molecular clones. In contrast, so far, PTLV-3 experimental studies have been restricted to the overexpression of the tax gene using reporter assays. We have therefore decided to construct an STLV-3 molecular clone. We generated a full-length STLV-3 proviral clone (8,891 bp) by PCR amplification of overlapping fragments. This STLV-3 molecular clone was then transfected into 293T cells. Reverse transcriptase PCR experiments followed by sequence analysis of the amplified products allowed us to establish that both gag and tax/rex mRNAs were transcribed. Western blotting further demonstrated the presence of the STLV-3 p24 gag protein in the cell culture supernatant from transfected cells. Transient transfection of 293T cells and of 293T-long terminal repeat-green fluorescent protein cells with the STLV-3 clone promoted syncytium formation, a hallmark of PTLV Env expression, as well as the appearance of fluorescent cells, also demonstrating that the Tax3 protein was expressed. Virus particles were visible by electron microscopy. These particles are infectious, as demonstrated by our cell-free-infection experiments with purified virions. All together, our data demonstrate that the STLV-3 molecular clone is functional and infectious. This clone will give us a unique opportunity to study in vitro the different pX transcripts and the putative presence of antisense transcripts and to evaluate the PTLV-3 pathogenicity in vivo.

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