Analysis of Epstein-Barr virus glycoprotein B functional domains via linker insertion mutagenesis

Epstein-Barr Virus (EBV) glycoprotein B (gB) is essential for viral fusion events with epithelial and B cells. This glycoprotein has been studied extensively in other herpesvirus family members, but functional domains outside of the cytoplasmic tail have not been characterized in EBV gB. In this study, a total of 28 linker insertion mutations were generated throughout the length of gB. In general, the linker insertions did not disrupt intracellular expression and variably altered cell surface expression. Oligomerization was disrupted by insertions located between residues 561 and 620, indicating the location of a potential site of oligomer contacts between EBV gB monomers. In addition, a novel N-glycosylated form of wild-type gB was identified under nonreducing Western blot conditions that likely represents a mature form of the protein. Fusion activity was abolished in all but three variants containing mutations in the N-terminal region (gB30), within the ectodomain (gB421), and in the intracellular C-terminal domain (gB832) of the protein. Fusion activity with variants gB421 and gB832 was comparable to that of the wild type with epithelial and B cells, and only these two mutants, but not gB30, were able to complement gB-null virus and subsequently function in virus entry. The mutant gB30 exhibited a low level of fusion activity with B cells and was unable to complement gB-null virus. The mutations generated here indicate important structural domains, as well as regions important for function in fusion, within EBV gB.     

Characterization of a 170,000-dalton polyprotein encoded by the McDonough strain of feline sarcoma virus.

In this study, we demonstrated the expression of a 170,000-Mr polyprotein in each of several McDonough feline sarcoma virus (FeSV)-transformed mink cell clones and one McDonough FeSV-transformed rat clone. This polyprotein designated McDonough FeSV P170, contained feline leukemia virus (FeLV) p15, p12, and p30 immunological determinants and shared two of its five [35S]methionine-labeled tryptic peptides with FeLV Pr180gag-pol. Both of these peptides were shown to be specific to the p30 component of Pr180gag-pol. The remaining McDonough FeSV P170 methionine-containing peptides were not represented within either FeLV Pr180gag-pol or Pr82env. Of interest, of the three peptides specific to the nonstructural component of McDonough FeSV P170, one was also represented in the 115,000-Mr polyproteins encoded by the Gardner and Snyder-Theilen strains of FeSV. These findings raise the possibility that the nonstructural components of polyproteins encoded by each of the three independently derived feline transforming viruses contained both common and unique regions. Moreover, if the sequences encoding these components are involved in transformation, as appears to be the case, our findings establish that the position of their insertion within the gag-pol region of the FeLV genome can vary among individual isolates.     

Identification of functional regions in the transforming protein of Fujinami sarcoma virus by in-phase insertion mutagenesis

A novel mutagenesis procedure based on the insertion of a hexameric nucleotide sequence into Rsa I restriction sites of cloned DNA has been applied to a copy of the Fujinami sarcoma virus (FSV) genome with the aim of identifying functional regions in the transforming protein. Mutations specifying peptide insertions in both the NH2- and the COOH-terminal fps-specific portions of the transforming protein reduce or abolish the capacity of the genome to induce transformed foci in rat-2 cells. Insertion of multiple copies of the hexamer into one central position in the oncogene results in dislocation of the NH2- and COOH-terminal regions in the primary structure, but has no inhibitory effect on focus induction. Taken together, the results imply that both the NH2- and COOH-terminal fps-specific portions of the FSV oncogene product possess determinants which function in fibroblast transformation, and that cooperation of these two regions is not sensitive to their separation in the primary structure.     

Analysis of the role of brome mosaic virus 1a protein domains in RNA replication, using linker insertion mutagenesis

Brome mosaic virus (BMV) belongs to a "superfamily" of plant and animal positive-strand RNA viruses that share, among other features, three large domains of conserved sequence in nonstructural proteins involved in RNA replication. Two of these domains reside in the 109-kDa BMV 1a protein. To examine the role of 1a, we used biologically active cDNA clones of BMV RNA1 to construct a series of linker insertion mutants bearing two-codon insertions dispersed throughout the 1a gene. The majority of these mutations blocked BMV RNA replication in protoplasts, indicating that both intervirally conserved domains function in RNA replication. Coinoculation tests with a large number of mutant combinations failed to reveal detectable complementation between mutations in the N- and C-terminal conserved domains, implying that these two domains either function in some directly interdependent fashion or must be present in the same protein. Four widely spaced mutations with temperature-sensitive (ts) defects in RNA replication were identified, including a strongly ts insertion near the nucleotide-binding consensus of the helicaselike C-terminal domain. Temperature shift experiments with this mutant show that 1a protein is required for continued accumulation of all classes of viral RNA (positive strand, negative strand, and subgenomic) and is required for at least the first 10 h of infection. ts mutations were also identified in the 3' noncoding region of RNA1, 5' to conserved sequences previously implicated in cis for replication. Under nonpermissive conditions, the cis-acting partial inhibition of RNA1 accumulation caused by these noncoding mutations was also associated with reduced levels of the other BMV genomic RNAs. Comparison with previous BMV mutant results suggests that RNA replication is more sensitive to reductions in expression of 1a than of 2a, the other BMV-encoded protein involved in replication.     

gp140v-fms molecules expressed at the surface of cells transformed by the McDonough strain of feline sarcoma virus are phosphorylated in tyrosine and serine.

Cells transformed by the McDonough strain of feline sarcoma virus express at their surface a v-fms-specific transmembrane glycoprotein designated gp140v-fms. By labeling with 32Pi, gp140v-fms was shown to be phosphorylated 30-fold more in serine residues than were the cytosolic v-fms polypeptides gp180gag-fms and gp120v-fms. By using the phosphotyrosine phosphatase-specific inhibitor sodium orthovanadate, an additional tyrosine phosphorylation was observed in vivo, again involving predominantly gp140v-fms. In vitro studies showed that the v-fms proteins were phosphorylated by protein kinase C in a calcium- and phosphatidylserine-dependent manner.     

Isolation of a transformation-defective mutant of the McDonough strain of feline sarcoma virus exhibiting tyrosine kinase activity in vitro but not in vivo.

NRK cells transformed by the McDonough strain of feline sarcoma virus (SM-FeSV) were mutagenized by the use of 5'-azacytidine. Four cell lines expressing different transformation-defective phenotypes were isolated. Superinfection of these cell lines with simian sarcoma-associated virus (SSAV) led in three instances to the recovery of transforming virus particles carrying an intact fms gene. A nonconditional transformation-defective virus, designated td26-SM-FeSV (SSAV), was isolated from one of the cell lines. NRK cells infected with this mutant contained actin cables and fibronectin networks and exhibited normal cell morphology. Such cells formed only small colonies in soft agar and exhibited a mitogenic activity similar to that of noninfected cells. Cells infected with td26-SM-FeSV (SSAV) synthesized a gag-fms fusion glycoprotein (gp180gag-fms). This polypeptide was processed in the normal manner into the intracellular gp120v-fms and a transformation-defective gp140td-v-fms which was expressed at the surface of infected cells. This species had an increased electrophoretic mobility on polyacrylamide gels compared with the molecule from wild-type virus.gp140td-v-fms had endo-beta-N-acetylglucosaminidase H-resistant carbohydrate side chains. No tyrosine kinase activity was detectable in vivo in td26-SM-FeSV (SSAV)-infected cells even when the cells were treated with sodium orthovanadate. In vitro, fms molecules from td26-SM-FeSV (SSAV)-infected cells exhibited tyrosine kinase activity as determined by autophosphorylation and phosphorylation of exogenous (poly)Glu-Tyr. At low ATP concentrations (less than 5 microM) this in vitro tyrosine kinase activity was significantly reduced compared with that of the wild-type counterpart.     

Structure-function studies of human cholesteryl ester transfer protein by linker insertion scanning mutagenesis

Human plasma cholesteryl ester transfer protein (CETP) enhances transfer and exchange of cholesteryl ester (CE) and triglyceride (TG) between high-density lipoprotein and other lipoproteins. To define regions responsible for the neutral lipid transfer activities at the molecular level, a total of 27 linker insertion mutants at 18 different sites along the CETP molecule were prepared and transiently expressed in a mammalian cell line (COS). The inserted linkers were small (usually 6 bp) and did not interrupt the translational reading frame of the CETP cDNA. Although secretion of each mutant protein was less than that of wild-type CETP, the majority of the mutants had normal cholesteryl ester transfer activity (transfer activity per nanogram of CETP in media). However, insertional alterations in three regions severely impaired CE transfer activity: (1) in the region of amino acids 48-53; (2) at amino acid 165; and (3) in the region of amino acids 373-379. Although the impaired activities could also be a result of globally incorrect folding of these CETP mutants, hydrophobicity analysis and secondary structure predictions tended to exclude this possibility for most of the insertion sites at which insertions resulted in inactivation. The insertion at amino acid 379 occurs immediately after a triplet of lysine residues, suggesting that this region might be involved in an essential step in the mechanism of CE and TG transfer, such as the binding of CETP to phosphatidylcholine molecules in the lipoprotein surface. Effects on TG transfer activity were generally similar to those on CE transfer activity, suggesting a similar structural requirement for both neutral lipid transfer activities.     

Alanine scanning mutagenesis of a plant virus movement protein identifies three functional domains.

Alanine scanning mutagenesis was performed on the red clover necrotic mosaic virus (RCNMV) movement protein (MP), and 12 mutants were assayed in vitro for RNA binding characteristics and in vivo for their ability to potentiate RCNMV cell-to-cell movement. The mutant phenotypes that were identified in vitro and in vivo suggest both that cooperative RNA binding is not necessary for cell-to-cell movement in vivo and that only a fraction of the wild-type RNA binding may be required. The MP mutants defined at least three distinct functional regions in the MP: an RNA binding domain, a cooperative RNA binding domain, and a third domain that is necessary for cell-to-cell movement in vivo. This third domain may be required for targeting the MP to cell walls and plasmodesmata, interacting with host proteins, folding, or possibly binding RNA into a functional ribonucleoprotein complex capable of cell-to-cell movement.     

Site-directed and linker insertion mutagenesis of herpes simplex virus type 1 glycoprotein H.

The gH-gL complex of herpes simplex virus type 1 (HSV-1) is essential for virion infectivity and virus-induced cell fusion, but functional domains of the gH molecule remain to be defined. We have addressed this question by mutagenesis. A set of linker insertion mutants in HSV-1 gH was generated and tested in transient assays for their ability to complement a gH-negative virus. Insertions at three sites in the C-terminal third of the external domain affected the ability of gH to function in cell-cell fusion and virus entry, while insertions at six sites in the N-terminal half of the external domain induced conformational changes in gH such that it was not recognized by monoclonal antibody LP11, although expression at the cell surface was unchanged. A recombinant virus in which a potential integrin-binding motif, RGD, in gH was changed to the triplet RGE entered cells as efficiently as the wild type, indicating that HSV-1 entry is not mediated by means of the gH-RGD motif binding to cell surface integrins. Furthermore, mutagenesis of the glycosylation site which is positionally conserved in all herpesvirus gH sequences in close proximity to the transmembrane domain generated a recombinant virus that grew in vitro with wild-type single-step kinetics.     

Correspondence between immunological and functional domains in the transforming protein of Fujinami sarcoma virus.

Monoclonal antibodies reactive with either gag or fps portions of the wild-type Fujinami sarcoma virus transforming protein have been used to probe the structure of proteins encoded by mutant genomes constructed in vitro. The pattern of immunoreactivity suggests that the functional domain defined in genetic studies (Stone et al., Cell 37:549-558, 1984) corresponds to a discrete immunological domain in the native, wild-type Fujinami sarcoma virus protein. At least one mutation affecting both the structure and function of the proposed NH2-terminal fps-specific domain encodes a product with high specific activities in kinase assays. Furthermore, a cell line expressing high levels of this mutant protein is only moderately transformed. The striking correspondence between the immunological domain defined here and the functional domain inferred from the results of transfection experiments suggests that this non-kinase-specifying region constitutes a discrete structural as well as functional component of the viral protein.     

Biochemical and immunological characterization of polyproteins coded for by the McDonough, Gardner-Arnstein, and Snyder-Theilen strains of feline sarcoma virus.

The McDonough (SM), Gardner-Arnstein (GA), and Snyder-Theilen (ST) strains of feline sarcoma virus (FeSV) code for high-molecular-weight polyproteins that contain varying amounts of the amino-terminal region of the FeLV gag gene-coded precursor protein and a polypeptide(s) of an as yet undetermined nature. The SM-FeSV primary translational product is a 180,000-dalton polyprotein which is immediately processed into a highly unstable 60,000-dalton molecule containing the p15-p12-p30 fragment of the FeLV gag gene-coded precursor protein and a 120,000-dalton FeSV-specific polypeptide. The GA- and ST-FeSV genomes code for polyproteins of 95,000 and 85,000 daltons, respectively, which in addition to the amino-terminal moiety (p15-12 and a portion of p30) of the FeLV gag gene-coded precursor protein also contain FeSV-specific polypeptides. However, the GA- and ST-FeSV polyproteins appear to be relatively stable molecules (half-lives of around 16 h) and are not significantly processed into smaller polypeptides. Immunological and biochemical analysis of each of the above FeSV translational products revealed that the sarcoma-specific regions of the GA- and ST-FeSV polyproteins are antigenically cross-reactive and exhibit common methionine-containing peptides. These findings favor the concept that these sarcoma-specific polypeptides are coded for by the similar subsets of cellular sequences incorporated into the GA- and ST-FeSV genomes during the generation of these transforming agents.     

Preparation of rat monoclonal antibodies to epitopes encoded by the viral oncogene (v-fms) of McDonough feline sarcoma virus

The McDonough strain of feline sarcoma virus (SM-FeSV) contains a viral oncogene, v-fms, transduced from cat cellular genetic sequences designated c-fms. Monoclonal antibodies reactive to antigenic determinants encoded by v-fms were prepared by immunizing rats with live, syngeneic SM-FeSV-transformed cells, and fusing splenic lymphocytes from a tumor-bearing animal with cultured rat myeloma cells. Culture supernatants from hybrids producing antibodies to epitopes encoded by v-fms were identified by immunoprecipitation of radiolabeled polypeptides from SM-FeSV-transformed mink cells. Four positive hybrids were cloned twice in soft agar, established as stable lines, and grown in defined serum-free medium to facilitate purification of homogeneous antibodies. The monoclonal antibodies were used to assay SM-FeSV-specific products by "immunoblotting" of electrophoretically separated proteins, and by fixed-cell immunofluorescence.     

Biochemical characteristics of functional domains using feline foamy virus integrase mutants

We constructed deletion mutants and seven point mutants by polymerase chain reaction to investigate the specificity of feline foamy virus integrase functional domains. Complementation reactions were performed for three enzymatic activities such as 3’-end processing, strand transfer, and disintegration. The complementation reactions with deletion mutants showed several activities for 3’-end processing and strand transfer. The conserved central domain and the combination of the N-terminal or C-terminal domains increased disintegration activity significantly. In the complementation reactions between deletion and point mutants, the combination between D107V and deletion mutants revealed 3’-end processing activities, but the combination with others did not have any activity, including strand transfer activities. Disintegration activity increased evenly,except the combination with glutamic acid 200. These results suggest that an intact central domain mediates enzymatic activities but fails to show these activities in the absence of theN-terminal or C-terminal domains. [BMB Reports 2013; 46(1):53-58]     

Transformation-defective mutants of Snyder-Theilen feline sarcoma virus lack tyrosine-specific protein kinase activity.

Four phenotypically normal mink cell clones, each containing a transformation-defective provirus of the Snyder-Theilen strain of feline sarcoma virus (ST-FeSV), synthesized an 85,000-dalton viral polyprotein (P85) indistinguishable in size and antigenic complexity from that encoded by wild-type transforming ST-FeSV. An additional transformation-defective, ST-FeSV-containing flat cell clone produced a polyprotein of 88,000 daltons (P88). The viral polyproteins immunoprecipitated from cytoplasmic extracts of these cells lacked the tyrosine-specific protein kinase activity associated with the wild-type ST-FeSV gene product. In addition, the products encoded by representative transformation-defective ST-FeSV genomes were poorly phosphorylated in vivo and lacked detectable phosphotyrosine residues. Whereas proteins of ST-FeSV transformants contained elevated levels of phosphotyrosine, those of mink cells containing transformation-defective ST-FeSV exhibited phosphotyrosine levels no higher than those found in uninfected cells. These findings provide genetic evidence that the tyrosine-specific protein kinase activity associated with ST-FeSV P85 is required for virus-induced transformation.     

Distinct functional domains in the cowpea mosaic virus movement protein.

Cell-to-cell movement of cowpea mosaic virus particles in plants takes place with the help of tubules that penetrate presumably modified plasmodesmata. These tubules, which are built up by the virus-encoded 48-kDa movement protein (MP), are also formed on single protoplast cells. To determine whether the MP contains different functional domains, the effect of mutations in its coding region was studied. Mutations between amino acids 1 and 313 led to complete abolishment of the tubule-forming capacity, while a deletion in the C-terminal region resulted in tubules that could not take up virus particles. From these observations, it is concluded that the MP contains at least two distinct domains, one that is involved in tubule formation and that spans amino acids 1 and 313 and a second that is probably involved in the incorporation of virus particles in the tubule and that is located in the C terminus between amino acids 314 and 331.     

Dissection of functional domains of the yeast proton-pumping ATPase by directed mutagenesis.

Cation-pumping ATPases characterized by a phosphorylated intermediate have been proposed to contain kinase, phosphatase and transduction domains. Evidence is provided for this model by mutagenesis of critical residues in the proposed domains. The Glu233-Gln mutation blocks the turnover of the intermediate and serves to define the phosphatase domain. Mutations in aspartate residues 534, 560 and 638 alter the nucleotide specificity of the enzyme. These amino acids are therefore part of the ATP binding site. Lys474 seems to be essential for activity in this kinase domain. Finally, mutations in Asp378, the amino acid forming the phosphorylated intermediate, indicate that the formation of a phosphorylated intermediate is not an obligatory step in ATP hydrolysis but is required for coupling this process with proton pumping.     

Mapping the domains of CD134 as a functional receptor for feline immunodeficiency virus

The feline homologue of CD134 is the primary binding receptor for feline immunodeficiency virus (FIV), targeting the virus preferentially to activated CD4+ helper T cells. However, strains of FIV differ in utilization of CD134; the prototypic strain PPR requires a minimal determinant in the first cysteine-rich domain (CRD1) of feline CD134 to confer near-optimal receptor function, while strains such as GL8 require additional determinants in the CD134 CRD2. We map this determinant to a loop in CRD2 governing the interaction between the receptor and its ligand; the amino acid substitutions S78N-S79Y-K80E restored full viral receptor activity to the CDR2 of human CD134 in the context of feline CD134, with tyrosine-79 appearing to be the critical residue for restoration of receptor function.