SLX4 interacts with RTEL1 to prevent transcription-mediated DNA replication perturbations.

The SLX4 tumor suppressor is a scaffold that plays a pivotal role in several aspects of genome protection, including homologous recombination, interstrand DNA crosslink repair and the maintenance of common fragile sites and telomeres. Here, we unravel an unexpected direct interaction between SLX4 and the DNA helicase RTEL1, which, until now, were viewed as having independent and antagonistic functions. We identify cancer and Hoyeraal-Hreidarsson syndrome-associated mutations in SLX4 and RTEL1, respectively, that abolish SLX4-RTEL1 complex formation. We show that both proteins are recruited to nascent DNA, tightly co-localize with active RNA pol II, and that SLX4, in complex with RTEL1, promotes FANCD2/RNA pol II co-localization. Importantly, disrupting the SLX4-RTEL1 interaction leads to DNA replication defects in unstressed cells, which are rescued by inhibiting transcription. Our data demonstrate that SLX4 and RTEL1 interact to prevent replication-transcription conflicts and provide evidence that this is independent of the nuclease scaffold function of SLX4.

Vascular endothelial growth factor receptor 1 expression in nasal polyp tissue.

BACKGROUND: Edema represents a key feature of nasal polyp (NP) disease. Members of the vascular endothelial growth factor (VEGF) family may be involved, but the precise role of VEGF-A, VEGF-B, placental growth factor (PlGF), and their receptors VEGFR1 and VEGFR2 in NP edema formation remains elusive. OBJECTIVE: Exploring the expression of VEGF family members and their receptors and their correlation with clinical, radiological, and edema markers in NP. METHODS: The expression of VEGF-A, VEGF-B, PlGF, VEGFR1, and VEGFR2 was measured in NP (n = 23) and control tissue (n = 22) at mRNA and protein level. Edema was evaluated by measuring albumin levels and wet/dry ratios. Computed tomography (CT) scans were scored using the Lund-Mackay scoring system. IL-5 mRNA expression was determined by real-time RT-PCR. Cell suspensions from NP (n = 10) and control tissue (n = 12) were stimulated in vitro with IL-1ß or TNFα. RESULTS: mRNA expression of VEGFR1 and VEGF-B was significantly higher in NP compared with control tissue. Expression levels of VEGF-B and VEGFR1 significantly correlated with NP albumin content (VEGF-B: P = 0.0208; VEGFR1: P = 0.0293), CT scan scores (VEGF-B: P = 0.0075; VEGFR1: P = 0.0068), and IL-5 mRNA (VEGF-B: P = 0.0027; VEGFR1: P = 0.0001). In vitro stimulation of control and NP tissue cell suspensions with IL-1ß or TNFα significantly reduced the expression of VEGFR2 in control tissue, without altering VEGFR1 and VEGF-B expression. hVEGF-B induced nitric oxide production in NP macrophages (P < 0.05). CONCLUSION: Expression levels of VEGFR1 and VEGF-B correlate with edema and clinical markers of NP disease and therefore represent potential therapeutic targets.

Staphylococcus aureus enterotoxin B augments granulocyte migration and survival via airway epithelial cell activation.

BACKGROUND: Staphylococcus aureus enterotoxin B (SEB) has recently been postulated to be involved in the pathology of granulocyte-dominated disease. Studying the immunologic interaction between SEB and airway epithelial cells in immortalized cell lines or long-term epithelial cell cultures has obvious disadvantages. METHODS: We used a novel technique of freshly isolated and purified human nasal epithelial cells (HNEC) from healthy, nonallergic individuals, which were incubated for 24 h without/with SEB at different concentrations. Chemokine production was evaluated in the supernatant using Cytometric Bead Array. The chemotactic activity of the supernatant was studied in vitro using a Boyden chamber. Survival was evaluated with flow cytometry, using propidium iodide to identify dead cells. RESULTS: Staphylococcus aureus enterotoxin B showed a dose-dependent induction of interferon-inducible protein-10, monokine induced by interferon-gamma, regulated upon activation normal T cell expressed and secreted, monocyte chemoattractant protein 1 (MCP-1) and granulocyte colony stimulating factor production by epithelial cells in vitro. The supernatant of epithelial cells had chemotactic activity for granulocytes in vitro, which was enhanced in the supernatant of SEB-stimulated epithelial cells. Reduced number of propidium iodide positive granulocytes was found in the conditions where supernatant of SEB-stimulated epithelial cells was applied. CONCLUSION: Staphylococcus aureus enterotoxin B exerts a direct pro-inflammatory effect on HNEC, with induction of chemokine and growth factor release, resulting in the migration and prolonged survival of granulocytes in vitro.

Dexamethasone-induced apoptosis of freshly isolated human nasal epithelial cells concomitant with abrogation of IL-8 production.

BACKGROUND: Human nasal epithelial cells (hNECs) are the first line of immune defense and are able to produce mediators that recruit, activate and prolong survival of immune cells, among which IL-8 takes an important place. Studies on IL-8 and effects of dexamethasone on hNECs have been hampered by methodological shortcomings. The purpose of the study is to investigate the contribution of freshly isolated hNECs to IL-8 production in chronic rhinosinusitis with nasal polyps (CRSwithNP). Secondly, the effects of dexamethasone treatment on hNEC apoptosis and IL-8 production are investigated. METHODOLOGY: hNECs were freshly isolated from nasal polyp tissue and healthy inferior turbinate of NP patients (n=12) and from inferior turbinates of healthy donors (n=19) by protease treatment and two negative selection procedures. hNECs were incubated with IL-1ß (10ng/ml), TNFα (10ng/ml) or dexamethasone (10, 100 and 1000 Amicrog/ml). After 24h, IL-8 levels were determined in the supernatants by ELISA. Finally, hNECs were incubated with increasing doses of dexamethasone and stained with trypan-blue and annexin-FITC/PI to study apoptosis. RESULTS: hNECs isolated from nasal turbinates of healthy and NP patients and polyp tissue from NP patients produced similar levels of IL-8. IL-1ß induced higher levels of IL-8 production in all types of hNECs without differences between control and NP tissue. Dexamethasone induced apoptosis of hNECs concomitant with abrogation of IL-8 production by hNECs. CONCLUSIONS: IL-8 production by human nasal epithelial cells does not differ between NP and healthy tissue under baseline nor stimulatory conditions. Dexamethasone induces apoptosis of hNECs and abrogates IL-8 production.

Atomic model of human cystic fibrosis transmembrane conductance regulator: membrane-spanning domains and coupling interfaces.

We describe herein an atomic model of the outward-facing three-dimensional structure of the membrane-spanning domains (MSDs) and nucleotide-binding domains (NBDs) of human cystic fibrosis transmembrane conductance regulator (CFTR), based on the experimental structure of the bacterial transporter Sav1866. This model, which is in agreement with previous experimental data, highlights the role of some residues located in the transmembrane passages and directly involved in substrate translocation and of some residues within the intracellular loops (ICL1-ICL4) making MSD/NBD contacts. In particular, our model reveals that D173 ICL1 and N965 ICL3 likely interact with the bound nucleotide and that an intricate H-bond network (involving especially the ICL4 R1070 and the main chain of NBD1 F508) may stabilize the interface between MSD2 and the NBD1F508 region. These observations allow new insights into the ATP-binding sites asymmetry and into the molecular consequences of the F508 deletion, which is the most common cystic fibrosis mutation.

Hydrophobic cluster analysis and modeling of the human Rh protein three-dimensional structures.

Rh (Rhesus) is a major blood group system in man, which is clinically significant in transfusion medicine. Rh antigens are carried by an oligomer of two major erythroid specific polypeptides, the Rh (D and CcEe) proteins and the RhAG glycoprotein, that shared a common predicted structure with 12 transmembrane a-helices (M0 to M11). Non erythroid homologues of these proteins have been identified (RhBG and RhCG), notably in diverse organs specialized in ammonia production and excretion, such as kidney, liver and intestine. Phylogenetic studies and experimental evidence have shown that these proteins belong to the Amt/Mep/Rh protein superfamily of ammonium/methylammonium permease, but another view suggests that Rh proteins might function as CO2 gas channels. Until recently no information on the structure of these proteins were available. However, in the last two years, new insight has been gained into the structural features of Rh proteins (through the determination of the crystal structures of bacterial AmtB and archeaebacterial Amt-1. Here, models of the subunit and oligomeric architecture of human Rh proteins are proposed, based on a refined alignment with and crystal structure of the bacterial ammonia transporter AmtB, a member of the Amt/Mep/Rh superfamily. This alignment was performed considering invariant structural features, which were revealed through Hydrophobic Cluster Analysis, and led to propose alternative predictions for the less conserved regions, particularly in the N-terminal sequences. The Rh models, on which an additional Rh-specific, N-terminal helix M0 was tentatively positioned, were further assessed through the consideration of biochemical and immunochemical data, as well as of stereochemical and topological constraints. These models highlighted some Rh specific features that have not yet been reported. Among these, are the prediction of some critical residues, which may play a role in the channel function, but also in the stability of the subunit structure and oligomeric assembly. These results provide a basis to further understand the structure/function relationships of Rh proteins, and the alterations occurring in variant phenotypes.

Nucleotide binding domains of human CFTR: a structural classification of critical residues and disease-causing mutations.

Defective function of the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) causes CF, the most frequent lethal inherited disease among the Caucasian population. The structure of this chloride ion channel includes two nucleotide-binding domains (NBDs), whose ATPase activity controls channel gating. Recently, the experimental structures of mouse and human CFTR NBD1 and our model of the human CFTR NBD1/NBD2 heterodimer have provided new insights into specific structural features of the CFTR NBD dimer. In the present work, we provide a structural classification of CF-causing mutations which may complement the existing functional classification. Our analysis also identified amino acid residues which may play a critical role in interdomain interaction and are located at the NBD1-NBD2 interface or on the surface of the dimer. In particular, a cluster of aromatic amino acids, which includes F508 and straddles the two NBDs, might be directly involved in the interaction of the NBD1/NBD2 heterodimer with the channel-forming membrane-spanning domains.

Nucleotide-binding domains of human cystic fibrosis transmembrane conductance regulator: detailed sequence analysis and three-dimensional modeling of the heterodimer.

The cystic fibrosis transmembrane conductance regulator (CFTR) protein is encoded by the gene that is defective in cystic fibrosis, the most common lethal inherited disease among the Caucasian population. CFTR belongs to the ABC transporter superfamily, whose members form macromolecular architectures composed of two membrane-spanning domains and two nucleotide-binding domains (NBDs). The experimental structures of NBDs from several ABC transporters have recently been solved, opening new avenues for understanding the structure/function relationships and the consequences of some disease-causing mutations of CFTR. Based on a detailed sequence/structure analysis, we propose here a three-dimensional model of the human CFTR NBD heterodimer. This model, which is in agreement with recent experimental data, highlights the specific features of the CFTR asymmetric active sites located at the interface between the two NBDs. Moreover, additional CFTR-specific features can be identified at the subunit interface, which may play critical roles in active site interdependence and are uncommon in other NBD dimers.

uDENN, DENN, and dDENN: indissociable domains in Rab and MAP kinases signaling pathways.

DENN domains are found in a variety of signaling proteins but their exact function remains undefined. Some of the DENN-containing proteins, such as rat Rab3GEP (Rab3 GDP/GTP exchange protein) or mouse Rab6IP1 (Rab6 interacting protein 1) interact with GTPases of the Rab family. Others, such as human MADD (MAP (Mitogen-activated protein) kinase activating protein containing death domain) and human ST5 (Suppressor of tumoreginicity 5) gene products are involved in regulation of MAPKs (Mitogen-activated protein kinases) signaling pathways. Using a combination of profile-based and bidimensional analyses, we show here that DENN domains are much larger than described to date in domain databases, always encircled on both sides by more divergent domains, that we called uDENN and dDENN. These however share conserved amino acids which could play a key role in the DENN functions.

Structural characterization of the LEM motif common to three human inner nuclear membrane proteins.

BACKGROUND: Integral membrane proteins of the inner nuclear membrane are involved in chromatin organization and postmitotic reassembly of the nucleus. The discovery that mutations in the gene encoding emerin causes X-linked Emery-Dreifuss muscular dystrophy has enhanced interest in such proteins. A common structural domain of 50 residues, called the LEM domain, has been identified in emerin MAN1, and lamina-associated polypeptide (LAP) 2. In particular, all LAP2 isoforms share an N-terminal segment composed of such a LEM domain that is connected to a highly divergent LEM-like domain by a linker that is probably unstructured. RESULTS: We have determined the three-dimensional structures of the LEM and LEM-like domains of LAP2 using nuclear magnetic resonance and molecular modeling. Both domains adopt the same fold, mainly composed of two large parallel alpha helices. CONCLUSIONS: The structural LEM motif is found in human inner nuclear membrane proteins and in protein-protein interaction domains from bacterial multienzyme complexes. This suggests that LEM and LEM-like domains are protein-protein interaction domains. A region conserved in all LEM domains, at the surface of helix 2, could mediate interaction between LEM domains and a common protein partner.

Normal membrane localization and actin association of the NF2 tumor suppressor protein are dependent on folding of its N-terminal domain.

The neurofibromatosis type 2 (NF2) tumor suppressor protein, known as schwannomin or merlin, is involved in linking membrane proteins to the cytoskeleton. Like the related ERM proteins, schwannomin has long been suspected of exhibiting a complex 3D organization caused by the association of different regions within the protein. Intramolecular interactions characterized to date are linking N-terminal sequences of the protein to C-terminal sequences. Here, we demonstrate, by a biochemical approach, the existence of a structured domain entirely contained within the N-terminal half of schwannomin. This structure, which is resistant to chymotryptic digestion, encompasses the FERM domain (residues 19-314), but excludes the 18 extreme N-terminal residues specific to schwannomin. The structure is disrupted by some, but not all, naturally occurring NF2 mutations. We investigated the significance of this structured domain in schwannomin cellular functions and found that normal schwannomin localization beneath the plasma membrane is directly dependent on proper folding of the N-terminal domain. In addition, folding of the N-terminal domain influences schwannomin interaction with actin through two novel actin-binding sites located in this region. These results suggest that loss of activity of several naturally occurring schwannomin mutants is due to disruption of the fold of the N-terminal domain, leading to loss of both membrane localization and actin association.

Study of the structure-activity relationships for the pyrazinamidase (PncA) from Mycobacterium tuberculosis.

In an attempt to investigate the molecular basis of pyrazinamide hydrolysis by the PncA protein from Mycobacterium tuberculosis, we determined the pyrazinamidase activity of nine PncA mutants bearing a single amino acid substitution. Among them, three mutants (D8G, K96T and S104R) had virtually no activity (< or =0.004 unit/mg), five (F13S, T61P, P69L, Y103S and A146V) retained a low level of activity (0.06-0.25 unit/mg) and one (T167L) exhibited a wild-type activity (1.51 units/mg). The possible structural effects of these substitutions were assessed by analysing a three-dimensional model of the PncA protein constructed on the basis of the crystal structure of the N-carbamoylsarcosine amidohydrolase (CSHase) from Arthrobacter sp., an amidohydrolase which was found by hydrophobic cluster analysis to be closely related to PncA. In the PncA model, five of the mutated residues, Asp-8, Phe-13, Lys-96, Tyr-103 and Ser-104, were located within a 6 A sphere around the cysteine residue Cys-138, which could be the counterpart of the active cysteine residue Cys-177 found in the CSHase. Among the remaining mutated residues, Thr-61, Pro-69 and Ala-146 were found to be more distant from Cys-138 but were associated with structural elements contributing to the catalytic centre, whereas Thr-167 was situated in an alpha-helix located far from the putative active site. These data suggest that the decrease in pyrazinamidase activity observed in the PncA mutant proteins is well correlated with the structural modifications the mutations can cause in the environment of the putative active cysteine Cys-138.

Polydom: a secreted protein with pentraxin, complement control protein, epidermal growth factor and von Willebrand factor A domains.

To identify extracellular proteins with epidermal growth factor (EGF) domains that are potentially involved in the control of haemopoiesis, we performed degenerate reverse-transcriptase-mediated PCR on the murine bone-marrow stromal cell line MS-5 and isolated a new partial cDNA encoding EGF-like domains related to those in the Notch proteins. Cloning and sequencing of the full-length cDNA showed that it encoded a new extracellular multi-domain protein that we named polydom. This 387 kDa mosaic protein contained a signal peptide followed by a new association of eight different protein domains, including a pentraxin domain and a von Willebrand factor type A domain, ten EGF domains, and 34 complement control protein modules. The human polydom mRNA is strongly expressed in placenta, its expression in the other tissues being weak or undetectable. The particular multidomain structure of the encoded protein suggests an important biological role in cellular adhesion and/or in the immune system.

MAN1, an inner nuclear membrane protein that shares the LEM domain with lamina-associated polypeptide 2 and emerin.

The "MAN antigens" are polypeptides recognized by autoantibodies from a patient with a collagen vascular disease and localized to the nuclear envelope. We now show that one of the human MAN antigens termed MAN1 is a 82.3-kDa protein with an amino-terminal domain followed by two hydrophobic segments and a carboxyl-terminal tail. The MAN1 gene contains seven protein-coding exons and is assigned to human chromosome 12q14. Its mRNA is approximately 5.5 kilobases and is detected in several different cell types that were examined. Cell extraction experiments show that MAN1 is an integral membrane protein. When expressed in transfected cells, MAN1 is exclusively targeted to the nuclear envelope, consistent with an inner nuclear membrane localization. Protein sequence analysis reveals that MAN1 shares a conserved globular domain of approximately 40 amino acids, which we term the LEM module, with inner nuclear membrane proteins lamina-associated polypeptide 2 and emerin. The LEM module is also present in two proteins of Caenorhabditis elegans. These results show that MAN1 is an integral protein of the inner nuclear membrane that shares the LEM module with other proteins of this subcellular localization.

Molecular organization of a zinc binding n-terminal modulatory domain in a NMDA receptor subunit.

Ionotropic glutamate receptors (iGluRs) bind agonists in a domain that has been crystallized and shown to have a bilobed structure. Eukaryotic iGluRs also possess a second extracellular N-terminal domain related to the bacterial periplasmic binding protein LIVBP. In NMDA receptors, the high-affinity Zn inhibition is eliminated by mutations in the LIVBP-like domain of the NR2A subunit. Using LIVBP structure, we have modeled this domain as two lobes connected by a hinge and show that six residues controlling Zn inhibition form two clusters facing each other across a central cleft. Upon Zn binding the two lobes close tightly around the divalent cation. Thus, the extracellular region of NR2A consists of a tandem of Venus flytrap domains, one binding the agonist and the other a modulatory ligand. Such a functional organization may apply to other eukaryotic iGluRs.

Molecular cloning, expression analysis, and chromosomal localization of human syntaxin 8 (STX8).

We report the cloning of a cDNA encoding human syntaxin 8 (STX8), using the regulator (R) domain of the cystic fibrosis transmembrane conductance regulator (CFTR) as a bait to screen a human fetal lung cDNA library by the yeast two-hybrid system. This gene was found broadly transcribed and its mRNA size is about 1.3 kb. The STX8 gene maps to chromosomal band 17p12 and it encodes a 236-amino-acid protein. Syntaxin 8 contains in its C-terminal half a coiled-coil domain found highly conserved in the t-SNARE (SNAP receptor on target membrane) superfamily of proteins, which are involved in vesicular trafficking and docking. In syntaxin 8, a C-terminal hydrophobic domain may constitute a transmembrane anchor. It was recently shown that CFTR-mediated chloride currents can be regulated by syntaxin 1A, a t-SNARE family member, through direct protein-protein interaction. This raises the possibility that syntaxin 8 may also be involved in such regulations.

An homologue of the human 100-kDa protein (p100) is differentially expressed by Histoplasma capsulatum during infection of murine macrophages.

Using differential display reverse transcription-PCR (DDRT-PCR) we have identified several sequences that are specifically expressed by Histoplasma capsulatum during infection of murine macrophages (MPhi). Here, we report the characterization of a clone, pHc12, identified as a differentially expressed gene 1 hour after infection of MPhi. Screening of a cDNA library of H. capsulatum allowed us to isolate a clone, pHc12-E, that contains the complete coding sequence. We show that after infection the level of transcription of this gene increases about 5 fold. Analysis of its sequence revealed the presence of an open reading frame of 890 aa (ORF890) that shares respectively 30 and 33% identity with human and Caenorhabditis elegans p100 kD and rat p105 kD co-activator proteins. Using the two-dimensional Hydrophobic Cluster Analysis (HCA) method, we showed that H. capsulatum ORF890 and p100 kD co-activator proteins are clearly related. The H. capsulatum protein consists of a four-fold repeated module (domains I to IV) like the p100 kD co-activator proteins, whose three-dimensional (3D) structure is related to staphylococcal thermonuclease, followed by a modified fifth "hybrid" domain which partially resembles the structure of the tudor domain found in multiple copies in the Drosophila melanogaster tudor protein. These data strongly suggest that ORF890 is homologous to human p100 kD and that this protein, named Hcp100, may play an essential role during infection by co-activating the expression of specific genes.

The characterization of murine BCMA gene defines it as a new member of the tumor necrosis factor receptor superfamily.

The BCMA gene is a new gene discovered by the molecular analysis of a t(4;16) translocation, characteristic of a human T cell lymphoma. It has no significant similarity with any known protein or motif, so that its function was unknown. This report describes the cloning of murine BCMA cDNA and its genomic counterpart. The mouse gene is organized into three exons, like the human gene, and lies in murine chromosome 16, in the 16B3 band, the counterpart of the human chromosome 16p13 band, where the human gene lies. Murine BCMA cDNA encodes a 185 amino acids protein (184 residues for the human), has a potential central transmembrane segment like the human protein and is 62% identical to it. The murine BCMA mRNA is found mainly in lymphoid tissues, as is human BCMA mRNA. Alignment of the murine and human BCMA protein sequences revealed a conserved motif of six cysteines in the N-terminal part, which strongly suggests that the BCMA protein belongs to the tumor necrosis factor receptor (TNFR) superfamily. Human BCMA is the first member of the TNFR family to be implicated in a chromosomal translocation.

Kx, a quantitatively minor protein from human erythrocytes, is palmitoylated in vivo.

Kx is a quantitatively minor blood group protein of human erythrocytes which is thought to be a membrane transporter. In the red cell membrane, Kx forms a complex stabilized by a disulfide bond with the Kell blood group membrane protein which might function as a metalloprotease. The palmitoylation status of these proteins was studied by incubating red cells with [3H] palmitic acid. Purification of the Kell-Kx complex, by immunochromatography on an immobilized human monoclonal antibody of Kell blood group specificity demonstrated that the Kx but not the Kell protein is palmitoylated. Six cysteines in Kx are predicted to be intracytoplasmic and might be targets for palmitoylation. Three of these cysteines are present in a portion of sequence which is predicted to form an amphipathic alpha helix. Palmitoylation of one or several of these cysteines might contribute to anchor the cytoplasmic portion of the Kx protein to the inner surface of red cell membrane.

Conservative mutations in the immunosuppressive region of the bovine leukemia virus transmembrane protein affect fusion but not infectivity in vivo.

Many retroviruses, including bovine leukemia virus (BLV), contain a highly conserved region located about 40 amino acids downstream from the fusion peptide within the sequence of the external domain of the transmembrane (TM) protein. This region is notably thought to be involved in the presentation of the NH2-terminal peptide to allow cell fusion. By using hydrophobic cluster analysis and by analogy with the influenza A hemagglutinin structures, the core of the TM structure including this particular region was predicted to consist, in the BLV and other retroviral envelope proteins, of an alpha-helix followed by a loop region, both docked against a subsequent alpha-helix that forms a triple-stranded coiled coil. The loop region could undergo, as in hemagglutinin, a major refolding into an alpha-helix integrating the coiled coil structure and putting the fusion peptide to one tip of the molecule. Based on this model, we have identified amino acids that may be essential to the BLV TM structure, and a series of mutations were introduced in the BLV env gene of an infectious molecular clone. A first series of mutations was designed to disturb the coiled coil structure (substitutions with proline residues), whereas others would maintain the general TM structure. When expressed by Semliki Forest virus recombinants, all the mutated envelope proteins were stable and efficiently synthesized in baby hamster kidney cells. Both proline-substituted and conservative mutants were strongly affected in their capacity to fuse to CC81 indicator cells. In addition, it appeared that the integrity of the TM coiled coil structure is essential for envelope protein multimerization, as analyzed by metrizamide gradient centrifugation. Finally, to gain insight into the role of this coiled coil in the infectious potential of BLV in vivo, the mutated TM genes were introduced in an infectious and pathogenic molecular clone and injected into sheep. It appeared that only the conservative mutations (A60V and A64S) allowed maintenance of viral infectivity in vivo. Since these mutations destroyed the ability to induce syncytia, we conclude that efficient fusion capacity of the recombinant envelopes is not a prerequisite for the infectious potential of BLV in vivo. Viral propagation of these mutants was strongly affected in some of the infected sheep. However, the proviral loads within half of the infected animals (2 out of 2 for A60V and 1 out of 4 for A64S) were close to the wild-type levels. In these sheep, it thus appears that the A60V and A64S mutants propagate efficiently despite being unable to induce syncytia in cell culture.