The YXXL Sequences of a Transmembrane Protein of Bovine Leukemia Virus Are Required for Viral Entry and Incorporation of Viral Envelope Protein into Virions

The cytoplasmic domain of an envelope transmembrane glycoprotein (gp30) of bovine leukemia virus (BLV) has two overlapping copies of the (YXXL)₂ motif. The N-terminal motif has been implicated in in vitro signal transduction pathways from the external to the intracellular compartment and is also involved in infection and maintenance of high viral loads in sheep that have been experimentally infected with BLV. To determine the role of YXXL sequences in the replication of BLV in vitro, we changed the tyrosine or leucine residues of the N-terminal motif in an infectious molecular clone of BLV, pBLV-IF, to alanine to produce mutated proviruses designated Y487A, L490A, Y498A, L501A, and Y487/498A. Transient transfection of African green monkey kidney COS-1 cells with proviral DNAs that encoded wild-type and mutant sequences revealed that all of the mutated proviral DNAs synthesized mature envelope proteins and released virus particles into the growth medium. However, serial passages of fetal lamb kidney (FLK) cells, which are sensitive to infection with BLV, after transient transfection revealed that mutation of a second tyrosine residue in the N-terminal motif completely prevented the propagation of the virus. Similarly, Y498A and Y487/498A mutant BLV that was produced by the stably transfected COS-1 cells exhibited significantly reduced levels of cell-free virion-mediated transmission. Analysis of the protein compositions of mutant viruses demonstrated that lower levels of envelope protein were incorporated by two of the mutant virions than by wild-type and other mutant virions. Furthermore, a mutation of a second tyrosine residue decreased the specific binding of BLV particles to FLK cells and the capacity for viral penetration. Our data indicate that the YXXL sequences play critical roles in both viral entry and the incorporation of viral envelope protein into the virion during the life cycle of BLV.     

Dileucine and YXXL motifs in the cytoplasmic tail of the bovine leukemia virus transmembrane envelope protein affect protein expression on the cell surface

Several retroviruses downmodulate the cell surface expression of envelope (Env) proteins through peptide sequences located in the cytoplasmic tail of the transmembrane (TM) subunit. We investigated whether cell surface expression of a chimeric protein containing the cytoplasmic domain of the TM protein (CTM) of bovine leukemia virus (BLV) was regulated by two membrane-proximal dileucine motifs or by tyrosine Y487 or Y498 in YXXL motifs. A chimeric protein composed of the extracellular and membrane-spanning portions of human CD8-alpha plus a wild-type (wt) BLV CTM was detectable on the surface of only 40% of the cells in which it was transiently expressed. Replacement of either dileucine pair with alanines increased the level of surface display of chimeric proteins. Nearly all cells became surface positive when both dileucine motifs were altered simultaneously and when either an N-terminal segment containing both dileucine motifs or a C-terminal segment containing all YXXL motifs was deleted. In contrast, replacement of Y487 or Y498 with alanine or phenylalanine enabled only small increases in surface display compared with wt levels. Chimeric proteins had similar stabilities but were downmodulated from the cell surface at three different rates. Point mutants segregated into each of the three groups of proteins categorized according to these different rates. Interestingly, Y487 mutants were downmodulated less efficiently than Y498 mutants, which behaved like wt. CD8-CTM chimeric proteins were phosphorylated on serine residues, but the native BLV Env protein was not phosphorylated either in transfected cells or in a lymphoid cell line constitutively producing BLV. Thus, both dileucine and YXXL motifs within the BLV CTM contribute to downmodulation of a protein containing this domain. Interactions with other proteins may influence surface exposure of Env protein complexes in virus-infected cells, assisting in viral evasion of adaptive immunity.     

Even attenuated bovine leukemia virus proviruses can be pathogenic in sheep

Based on a reverse genetics approach, we previously reported that bovine leukemia virus (BLV) mutants harboring deletions in the accessory R3 and G4 genes persist at very low proviral loads and are unable to induce leukemia or lymphoma in sheep, indicating that these R3 and G4 gene sequences are required for pathogenesis. We now show that lymphoma can occur, albeit infrequently (1 case of 20) and after extended periods of latency (7 years). Direct sequencing and reinfection experiments demonstrated that lymphomagenesis was not due to the reversion of the mutant to the wild type. Similar observations with another type of attenuated mutant impaired in the transmembrane protein (TM) YXXL signaling motifs were made. We conclude that the R3 and G4 genes and the TM YXXL motifs are not strictly required for pathogenesis but that their integrity contributes to disease frequency and latency.     

Both the PPPY and PTAP motifs are involved in human T-cell leukemia virus type 1 particle release

In retroviruses, the late (L) domain has been defined as a conserved motif in the Gag polyprotein precursor that, when mutated, leads to the emergence of virus particles that fail to pinch off from the plasma membrane. These domains have been observed to contain the PPXY, PTAP, or YXXL motifs. The deltaretroviruses, which include bovine leukemia virus (BLV) and human T-cell leukemia virus type 1 (HTLV-1) and HTLV-2, have a conserved PPPY motif in the C-terminal region of the matrix (MA) domain of Gag, while HTLV-1 also encodes a PTAP motif in MA. In this study, we analyzed the roles of the PPPY and PTAP motifs in the C terminus of MA in HTLV-1 particle release. Mutation of either motif (i.e., PPPY changed to APPY or PTAP changed to PTRP) reduced budding efficiencies. Particle buds and electron-dense regions of plasma membrane were observed by electron microscopy. When the locations of PPPY and PTAP were switched, particle release was eliminated. Intriguingly, the replacement of the PTAP motif with either the PPPY or YPDL motifs did not influence the release of virus particles, but the replacement of the PPPY motif with either PTAP or YPDL eliminated particle production. This indicates that the role that PPPY plays in HTLV-1 budding cannot be replaced with either PTAP or YPDL. A similar observation was made with the BLV PPPY motif. Finally, HTLV-1 particle release was found to be sensitive to proteasome inhibitors, implicating a role for ubiquitin in HTLV-1 budding. In summary, our observations indicate that (i) the PPPY motif plays a crucial role in virus budding and (ii) the PTAP motif plays a more subtle role in HTLV-1 particle release. Each of these motifs may play an important role in virus release from specific cell types and therefore be important in efficient virus spread and transmission.     

The (YXXL/I)2 signalling motif found in the cytoplasmic segments of the bovine leukaemia virus envelope protein and Epstein-Barr virus latent membrane protein 2A can elicit early and late lymphocyte activation events.

The cytoplasmic domains of the transducing subunits associated with B and T cell antigen receptors contain a common amino acid motif consisting of two precisely spaced Tyr-X-X-Leu/Ile sequences (where X corresponds to a variable residue). Expression of a single copy of this motif suffices to initiate B or T cell activation. The bovine leukaemia virus (BLV) is a B cell lymphotropic retrovirus which causes a non-neoplasic proliferation of B cells. The cytoplasmic domain of the BLV transmembrane envelope glycoprotein, gp30, possesses two overlapping copies of the Tyr-X-X-Leu/Ile-containing motif which could participate in the induction of B cell activation. Similarly, the N-terminal cytoplasmic domain of the latent membrane protein 2A (LMP2A) of the Epstein-Barr virus (EBV) contains a single copy of the Tyr-X-X-Leu/Ile-containing motif which could play a critical role in B cell transformation. To determine whether these two virus-encoded cytoplasmic domains are endowed with signalling functions, we constructed chimeric proteins by replacing the cytoplasmic tail of CD8-alpha with that of either BLV gp30 or EBV LMP2A. We show here that, once separately expressed in B or T cell lines, these chimeras are capable of triggering both calcium responses and cytokine production when cross-linked with an antibody to CD8-alpha. Furthermore, using site-directed mutagenesis, we demonstrated unequivocally that this signalling function may be accounted for by the Tyr-X-X-Leu/Ile motifs they contain.(ABSTRACT TRUNCATED AT 250 WORDS)     

Role of the proline-rich motif of bovine leukemia virus transmembrane protein gp30 in viral load and pathogenicity in sheep

The cytoplasmic tail of bovine leukemia virus (BLV) transmembrane protein gp30 has multiple amino acid motifs that mimic those present in signaling proteins associated with B-cell and T-cell receptors. The proline-rich motif of gp30, PX(2)PX(4-5)P, is analogous to the recognition site of Src homology 3 (SH3) domains of signaling molecules. Using site-directed mutagenesis of an infectious molecular clone of BLV, point mutations were introduced which changed three of the prolines of the motif to alanines. The influence of these mutations on the pathogenicity of BLV was studied in sheep which received either (i) plasmid DNA with provirus containing proline-to-alanine mutations (pppBLV), (ii) plasmid DNA with wild-type provirus (wtBLV), or (iii) transfection reagent alone. Although all of the BLV-injected animals seroconverted at approximately the same time, viral loads at later time points were high in five of five of the wtBLV group and two of five of the pppBLV group but low in three of five of the pppBLV group, as determined by semiquantitative PCR. Viral expression was lower in the pppBLV-transfected sheep, as measured by p24 antigen enzyme-linked immunosorbent assay in cultured cells, and serologic titers were lower. Thirty-one months after transfection, four of four wtBLV-transfected sheep had died of leukemia and lymphoma, and all five of the pppBLV-transfected sheep were clinically healthy and had normal peripheral blood lymphocyte counts. These data indicate that the proline-rich motif of gp30 is not required for viral infectivity but is important for high viral load in vivo, suggesting that SH3-mediated gp30 interactions are critical for viral pathogenesis following infection. Absence of interactions with the proline-rich motif may prevent or delay tumorigenesis in sheep.     

Polarized human immunodeficiency virus budding in lymphocytes involves a tyrosine-based signal and favors cell-to-cell viral transmission

Maturation and release of human immunodeficiency virus type 1 (HIV-1) is targeted at the pseudopod of infected mononuclear cells. However, the intracellular mechanism or targeting signals leading to this polarized viral maturation are yet to be identified. We have recently demonstrated the presence of a functional YXXL motif for specific targeting of HIV-1 virions to the basolateral membrane surface in polarized epithelial Madin-Darby canine kidney cells (MDCK). Site-directed mutagenesis was used to demonstrate that the membrane-proximal tyrosine in the intracytoplasmic tail of the HIV-1 transmembrane glycoprotein (gp41) is an essential component of this signal. In the present study, immunolocalization of viral budding allowed us to establish that this tyrosine-based signal is involved in determining the exact site of viral release at the surface of infected mononuclear cells. Substitution of the critical tyrosine residue was also shown to increase the amount of envelope glycoprotein at the cell surface, supporting previous suggestions that the tyrosine-based motif can promote endocytosis. Although alteration of the dual polarization-endocytosis motif did not affect the infectivity of cell-free virus, it could play a key role in cell-to-cell viral transmission. Accordingly, chronically infected lymphocytes showed a reduced ability to transmit the mutant virus to a cocultivated cell line. Overall, our data indicate that the YXXL targeting motif of HIV is active in various cell types and could play an important role in viral propagation; this may constitute an alternative target for HIV therapeutics and vaccine development.     

In vivo infection of sheep by bovine leukemia virus mutants.

Direct inoculation of a cloned bovine leukemia virus (BLV) provirus into sheep has allowed study of the viral infectivity of genetic mutants in vivo. Three BLV variants cloned from BLV-induced tumors and 12 in vitro-modified proviruses were isolated and analyzed for viral expression in cell culture. The proviruses were then inoculated into sheep in order to assess viral infectivity in vivo. Of three variants cloned from BLV-induced tumors (344, 395, and 1345), one (344) was found infectious in vivo. This particular provirus was used to engineer 12 BLV mutants. A hybrid between the 5' region of the complete but noninfectious provirus 395 and the 3' end of mutant 344 was infectious in vivo, suggesting that the tax/rex sequences were altered in virus 395. As expected, several regions of the BLV genome appeared to be essential for viral infection: the protease, pol, and env genes. Even discrete modifications in the fusion peptide located at the NH2 end of the transmembrane gp30 glycoprotein destroyed the infectious potential. In contrast, mutations and deletions in the X3 region present between the env gene and the 3' tax/rex region did not interfere with viral infection in vivo. This region of unknown function could thus be used to introduce foreign sequences. A BLV recombinant carrying a ribozyme directed against the tax/rex sequences was still infectious in vivo. Cotransfection of two noninfectious mutants carrying deletions led to infection in two of four independent injections, the infectious virus being then a recombinant between the two deletants. The experimental approach described here should help to gain insight into essential mechanisms such as in vivo viral replication, cooperation between deletants for viral infectivity, and viral superinfections. The gene products in the X3 and X4 region which are dispensable for in vivo infection could be involved in leukemogenesis, and thus proviruses deleted in these sequences could constitute the basis for a live attenuated vaccine.     

The YXXL motif, but not the two NPXY motifs, serves as the dominant endocytosis signal for low density lipoprotein receptor-related protein

All members of the low density lipoprotein (LDL) receptor family contain at least one copy of the NPXY sequence within their cytoplasmic tails. For the LDL receptor, it has been demonstrated that the NPXY motif serves as a signal for rapid endocytosis through coated pits. Thus, it is generally believed that the NPXY sequences function as endocytosis signals for all the LDL receptor family members. The primary aim of this study is to define the endocytosis signal(s) within the cytoplasmic tail of LDL receptor-related protein (LRP). By using LRP minireceptors, which mimic the function and trafficking of full-length endogenous LRP, we demonstrate that the YXXL motif, but not the two NPXY motifs, serves as the dominant signal for LRP endocytosis. We also found that the distal di-leucine motif within the LRP tail contributes to its endocytosis, and its function is independent of the YXXL motif. Although the proximal NPXY motif and the proximal di-leucine motif each play a limited role in LRP endocytosis in the context of the full-length tail, these motifs were functional within the truncated receptor tail. In addition, we show that LRP minireceptor mutants defective in endocytosis signal(s) accumulate at the cell surface and are less efficient in delivery of ligand for degradation.     

A mutant form of the tax protein of bovine leukemia virus (BLV), with enhanced transactivation activity,increases expression and propagation of BLV in vitro but not in vivo

In a previous study, we identified an interesting mutant form of the Tax protein of bovine leukemia virus (BLV), designated D247G. This mutant protein strongly transactivated the long terminal repeat of BLV and was also able to transactivate the cellular proto-oncogene c-fos. This finding suggested that BLV that encode the mutant protein might propagate and induce lymphoma more efficiently than wild-type BLV. To characterize the effects of the strong transactivation activity of the mutant Tax protein, we constructed an infectious molecular clone of BLV that encoded D247G and examined the replication and propagation of the virus in vitro and in vivo. Cultured cells were transfected with the wild-type and mutant BLV, and then levels of viral proteins and particles and the propagation of viruses were compared. As expected, in vitro, mutant BLV produced more viral proteins and particles and was transmitted very effectively. We injected the wild-type and mutant BLV into sheep, which are easily infected with BLV, and monitored the proportion of BLV-positive cells in the blood and the expression of BLV RNA for 28 weeks. By contrast to the results of our analyses in vitro, we found no significant difference in the viral load or the expression of viral RNA between sheep inoculated with wild-type or mutant BLV. Our observations indicate that the mutant D247G Tax protein does not enhance the expansion of BLV and that there might be a dominant mechanism for regulation of the expression of BLV in vivo.     

Transcription factor AP-4 participates in activation of bovine leukemia virus long terminal repeat by p34 Tax.

Three 21bp repeats can be found in the bovine leukemia virus long terminal repeat, which are crucial for the LTR directed gene expression by the trans activator protein Tax. Previous studies demonstrated that the major target of the Tax directed activation are the CRE-like elements in the center of these repeats. In this work we report that another motif of the 21bp repeats is also required for the Tax activation. Gel retardation--with the wild type or mutant 21bp repeats--revealed that cellular factors from HeLa cells were specifically bound to the center (CRE-like element) and the 3'' region of the repeats, which contains a CAGCTG consensus AP-4 binding site. In vivo analysis using the synthetic 21bp repeats indicated that beyond the consensus CRE-like motif, the AP-4 site is also essential for Tax activation. To determine the role of AP-4 in BLV Tax trans activation, we used the AP-4 cDNA in antisense transient assays. In the in vivo experiments the antisense AP-4 RNA resulted in strongly decreased Tax activation. On the basis of these results we conclude that AP-4 is a good candidate of cellular factors involved in BLV Tax trans activation.     

Phenylalanine promotes interaction of transmembrane domains via GxxxG motifs

Interactions of transmembrane helices play a crucial role in the folding and oligomerisation of integral membrane proteins. In order to uncover novel sequence motifs mediating these interactions, we randomised one face of a transmembrane helix with a set of non-polar or moderately polar amino acids. Those sequences capable of self-interaction upon integration into bacterial inner membranes were selected by means of the ToxR/POSSYCCAT system. A comparison between low/medium-affinity and high-affinity sequences reveals that high-affinity sequences are strongly enriched in phenylalanine residues that are frequently observed at the − 3 position of GxxxG motifs, thus yielding FxxGxxxG motifs. Mutation of Phe or GxxxG in selected sequences significantly reduces self-interaction of the transmembrane domains without affecting their efficiency of membrane integration. Conversely, grafting FxxGxxxG onto unrelated transmembrane domains strongly enhances their interaction. Further, we find that FxxGxxxG is significantly over-represented in transmembrane domains of bitopic membrane proteins. The same motif contributes to self-interaction of the vesicular stomatitis virus G protein transmembrane domain. We conclude that Phe stabilises membrane-spanning GxxxG motifs. This is one example of how the role of certain side-chains in helix-helix interfaces is modulated by sequence context.     

Polyclonal bovine sera but not virus-neutralizing monoclonal antibodies block bovine leukemia virus (BLV) gp51 binding to recombinant BLV receptor BLVRcp1.

Bovine leukemia virus (BLV), a transactivating lymphotropic retrovirus, is the etiologic agent of enzootic lymphosarcoma or leukemia in cattle. Sera from BLV-infected animals possess high BLV-neutralizing antibody titres. The availability of the recombinant BLV receptor candidate, BLVRcp1, allowed us to determine a mechanism of virus neutralization by polyclonal sera and monoclonal antibodies (MAbs). Bovine sera from animals naturally infected with BLV blocked gp51 binding to recombinant BLVRcp1. In contrast, virus-neutralizing MAbs specific for gp51 F, G, and H epitopes did not prevent gp51-receptor attachment. Furthermore, gp51 neutralization epitopes F, G, and H were accessible to antibodies following gp51 attachment to BLVRcp1. This finding implies that virus neutralization by MAbs to defined BLV gp51 epitopes can occur subsequent to virus engagement of the receptor while polyclonal sera can specifically block virus attachment to the receptor. In conclusion, these data suggest that cell infection by BLV is a multistep process requiring receptor binding (inhibited by polyclonal sera) followed by a second, postbinding event(s) at the cell membrane (inhibited by anti-gp51 MAbs).     

Augmentation of bovine leukemia virus (BLV)-specific lymphocyte proliferation responses in ruminants by inoculation with BLV env-recombinant vaccinia virus: their role in the suppression of BLV replication.

Lymphocyte proliferation responses were investigated in sheep and cattle, in which the replication of bovine leukemia virus (BLV) had been known to be suppressed by inoculation with recombinant vaccinia virus (rVV) expressing BLV envelope glycoprotein (gp60). Enhanced lymphocyte proliferation responses were observed in animals inoculated with rVV, regardless of whether they were naive or BLV carriers. These responses were roughly inversely correlated to the growth of BLV in the peripheral blood leukocytes. In contrast, there was no apparent correlation between humoral immune response and BLV growth. Based on these results, it was suggested that rVV rendered its suppressive effect of BLV replication primarily via augmentation of cell-mediated immunity.     

Mutational analysis of the conserved motifs of influenza A virus polymerase basic protein 1.

Influenza virus polymerase complex is a heterotrimer consisting of polymerase basic protein 1 (PB1), polymerase basic protein 2 (PB2), and polymerase acidic protein (PA). Of these, only PB1, which has been implicated in RNA chain elongation, possesses the four conserved motifs (motifs I, II, III, and IV) and the four invariant amino acids (one in each motif) found among all viral RNA-dependent RNA or RNA-dependent DNA polymerases. We have modified an assay system developed by Huang et al. (T.-J. Huang, P. Palese, and M. Krystal, J. Virol. 64:5669-5673, 1990) to reconstitute the functional polymerase activity in vivo. Using this assay, we have examined the requirement of each of these motifs of PB1 in polymerase activity. We find that each of these invariant amino acids is critical for PB1 activity and that mutation in any one of these residues renders the protein nonfunctional. We also find that in motif III, which contains the SSDD sequence, the signature sequence of influenza virus RNA polymerase, SDD is essentially invariant and cannot accommodate sequences found in other RNA viral polymerases. However, conserved changes in the flanking sequences of SDD can be partially tolerated. These results provide the experimental evidence that influenza virus PB1 possesses a similar polymerase module as has been proposed for other RNA viruses and that the core SDD sequence of influenza virus PB1 represents a sequence variant of the GDN in negative-stranded nonsegmented RNA viruses, GDD in positive-stranded RNA virus and double-stranded RNA viruses, or MDD in retroviruses.