Biomedical subjects

E G Niles

Publications and source records attributed to E G Niles.

At least 37 records · Page 2Linked to original sources

PubMed1995

Assembly of vaccinia virus: incorporation of p14 and p32 into the membrane of the intracellular mature virus.

The cytoplasmic assembly of vaccinia virus begins with the transformation of a two-membraned cisterna derived from the intermediate compartment between the endoplasmic reticulum and the Golgi complex. This cisterna develops into a viral crescent which eventually forms a spherical immature virus (IV) that matures into the intracellular mature virus (IMV). Using immunoelectron microscopy, we determined the subcellular localization of p32 and p14, two membrane-associated proteins of vaccinia virus. p32 was associated with vaccinia virus membranes at all stages of virion assembly, starting with the viral crescents, as well as with the membranes which accumulated during the inhibition of assembly by rifampin. There was also low but significant labelling of membranes of some cellular compartments, especially those in the vicinity of the Golgi complex. In contrast, anti-p14 labelled neither the crescents nor the IV but gave strong labelling of an intermediate form between IV and IMV and was then associated with all later viral forms. This protein was also not significantly detected on identifiable cellular membranes. Both p32 and p14 were abundantly expressed on the surface of intact IMV. Our data are consistent with a model whereby p32 would become inserted into cellular membranes before being incorporated into the crescents whereas p14 would be posttranslationally associated with the viral outer membrane at a specific later stage of the viral life cycle.

Animals↗

PubMed1994

Direct photolinkage of GTP to the vaccinia virus mRNA (guanine-7-) methyltransferase GTP methyl acceptor site.

Direct UV photolinkage of [alpha-32P]GTP to the methyl acceptor site of the vaccinia virus (guanine-7-) methyltransferase was attempted in order to identify the GTP binding region of this enzyme. Low-efficiency photolinkage of GTP to the carboxyl terminal domain of the large subunit, D1R498-844, was achieved and shown to be specific by several criteria. The half-saturation value for GTP was determined to be 35 microM which is equivalent to the catalytic Km for the substrate. GTP photolinkage was shown to be inhibited by GpppA, a substrate for the methyltransferase reaction, better than GMepppA, the reaction product. The addition of MgCl2, known to prevent GTP from serving as a methyl group acceptor in this reaction, was found to eliminate GTP photolinkage. Finally, AdoHcy, a potent product inhibitor of AdoMet binding, failed to inhibit GTP photolinkage, demonstrating that GTP was not linked to the AdoMet binding site. Chemical cleavage of the GTP-labeled enzyme permitted the identification of multiple radioactive peptides, demonstrating the existence of multiple interaction sites in the carboxyl terminal domain of the D1R subunit. The addition of the small D12L subunit has been shown to activate the (guanine-7-) methyltransferase activity in D1R498-844 30-50-fold. The efficiency of GTP photolinkage to the isolated D1R498-844 domain, however, was found to be only marginally effected by the addition of the D12L subunit, demonstrating that this enhancement of mRNA (guanine-7-) methyltransferase activity mediated by D12L was not achieved by altering the strength of GTP binding.

Binding Sites↗

PubMed1994

The mRNA (guanine-7-)methyltransferase domain of the vaccinia virus mRNA capping enzyme. Expression in Escherichia coli and structural and kinetic comparison to the intact capping enzyme.

The mRNA (guanine-7-)methyltransferase active site of the heterodimeric vaccinia virus mRNA capping enzyme was previously localized to the carboxyl-terminal third of the large subunit, D1R, associated with the small subunit, D12L (Cong, P., and Shuman, S. (1992) J. Biol. Chem. 267, 16424-16429; Higman, M. A., Bourgeois, N., and Niles, E. G. (1992) J. Biol. Chem. 267, 16430-16437). A plasmid was constructed which directs the coexpression of the carboxyl terminus of the D1R subunit from amino acids 498 to 844 and the D12L subunit in Escherichia coli. The mRNA (guanine-7-)methyltransferase catalytic activity in the isolated domain was found to be kinetically equivalent to that present in the intact enzyme. Through mobility shift and ultraviolet photolinkage analyses, both domains were shown to bind RNA in a saturable fashion. RNA binding was localized predominantly to the large subunit, but a low level of linkage of RNA to D12L was also observed. A low, but reproducible, level of mRNA (guanine-7-)methyltransferase activity was detected in the isolated D1R498-844 subunit demonstrating that the active site resides solely within the large subunit of the capping enzyme. This activity is enhanced 30- to 50-fold by the association of the D12L subunit.

Base Sequence↗

PubMed1994

Location of the S-adenosyl-L-methionine binding region of the vaccinia virus mRNA (guanine-7-)methyltransferase.

The mRNA (guanine-7-)methyltransferase activity of the heterodimeric vaccinia virus mRNA capping enzyme was previously mapped to the carboxyl-terminal 396 amino acids of the large subunit, D1R. This activity is enhanced 30- to 50-fold by the association of the the small subunit, D12L (Higman, M. A., Christen, L. A., and Niles, E. G. (1994) J. Biol. Chem. 269, 14974-14981). Irradiation with ultraviolet light specifically photolinks S-[methyl-3H]S-adenosyl-L-methionine to the large subunit of both the intact capping enzyme and a methyltransferase subdomain, which consists of the D12L subunit associated with the carboxyl-terminal 346 amino acids of the D1R subunit. The extent of linkage was shown to be dependent on the length of incubation, intensity of ultraviolet light, and the concentration of both active enzyme and substrate. The covalent modification was inhibited by S-adenosyl-L-homocysteine, a known competitive inhibitor of the mRNA (guanine-7-)methyltransferase activity, demonstrating specific photolabeling of the active site. Sequence-specific chemical cleavage of the photolinked methyltransferase domain with mild acid or cyanogen bromide revealed linkage of S-adenosyl-L-methionine to two regions of the large subunit. Analysis of the products of cyanogen bromide and hydroxylamine cleavage mapped the photolinked fragments to amino acids 499 to 579 and 806 to 844 of the D1R subunit. Photolinkage of AdoMet to D1R498-844 was shown to be unaffected by the association of the small subunit.

Binding Sites↗

PubMed1993

Identification of the vaccinia virus mRNA guanyltransferase active site lysine.

The vaccinia virus mRNA capping enzyme is a heterodimeric protein containing subunits of 97 and 33 kDa, the products of genes D1R and D12L, respectively. The enzyme catalyzes the first three reactions in the mRNA cap formation pathway: mRNA triphosphatase, guanyltransferase and (guanine-7-)methyltransferase. The guanyltransferase reaction proceeds by way of a covalent enzyme GMP (E-GMP) intermediate (Shuman, S. and Hurwitz, J. (1981) Proc. Natl. Acad. Sci. U.S.A. 78, 187-191) in which the GMP is linked to the large subunit through a lysine residue (Toyama, R., Mizumoto, K., Nakahara, Y., Tatsuno, T., and Kaziro, Y. (1983) Eur. J. Biochem. 2, 2195-2201; Roth, M. J., and Hurwitz, J. (1984) J. Biol Chem. 259, 13488-13494). In order to identify the map position of the guanyltransferase active site lysine residue, high specific activity [32P]E-GMP was prepared. Digestion of the E-GMP with hydroxylamine at pH 9.5 yielded a 31-kDa radioactive fragment derived from amino acids 1-273. Cleavage of E-GMP with cyanogen bromide produced a radioactive peptide of 14 kDa corresponding to amino acids 242-365. Lysine residues are found at positions 244 and 260. Staphylococcus aureus V8 protease digestion of cyanogen bromide-cleaved E-GMP yields a radioactive product of about 5 kDa in molecular mass corresponding to the peptide generated by cleavage at glutamic acid residues 253 and 297, demonstrating that lysine 260 is the site of linkage of GMP.

Amino Acid Sequence↗

PubMed1993

A poxvirus-encoded uracil DNA glycosylase is essential for virus viability.

Infection of cultured mammalian cells with the Leporipoxvirus Shope fibroma virus (SFV) causes the induction of a novel uracil DNA glycosylase activity in the cytoplasms of the infected cells. The induction of this activity, early in infection, correlates with the early expression of the SFV BamHI D6R open reading frame which possesses significant protein sequence similarity to eukaryotic and prokaryotic uracil DNA glycosylases. The SFV BamHI D6R open reading frame and the homologous HindIII D4R open reading frame from the Orthopoxvirus vaccinia virus were cloned under the regulation of a phage T7 promoter and expressed in Escherichia coli as insoluble high-molecular-weight aggregates. During electrophoresis on sodium dodecyl sulfate-polyacrylamide gels, the E. coli-expressed proteins migrate with an apparent molecular mass of 25 kDa. The insoluble protein aggregate generated by expression in E. coli was solubilized in urea and, following a subsequent refolding step, displayed the ability to excise uracil residues from double-stranded plasmid DNA substrates, with the subsequent formation of apyrimidinic sites. The viral enzyme, like all other characterized uracil DNA glycosylases, is active in the presence of high concentrations of EDTA, is substrate inhibited by uracil, and does not display any endonuclease activity. Attempts to inactivate the HindIII D4R gene of vaccinia virus by targeted insertion of a dominant xanthine-guanine phosphoribosyltransferase selection marker or direct insertion of a frame-shifted oligonucleotide were uniformly unsuccessful demonstrating that, unlike the uracil DNA glycosylase described for herpesviruses, the poxvirus enzyme is essential for virus viability.

Animals↗

PubMed1992

The vaccinia virus mRNA (guanine-N7-)-methyltransferase requires both subunits of the mRNA capping enzyme for activity.

Plasmid vectors capable of expressing the large and small subunits of the vaccinia virus mRNA capping enzyme were constructed and used to transform Escherichia coli. Conditions for the induction of the dimeric enzyme or the individual subunits in a soluble form were identified, and the capping enzyme was purified to near homogeneity. Proteolysis of the capping enzyme in bacteria yields a 60-kDa product shown previously to possess the mRNA triphosphatase and guanyltransferase activities (Shuman, S. (1990) J. Biol. Chem. 265, 11960-11966) was isolated and shown by amino acid sequence analysis to be derived from the NH2 terminus of D1R. The individual subunits lacked methyltransferase activity when assayed alone. However, mixing the D1R and D12L subunits permitted reconstitution of the methyltransferase activity, and this appearance in activity accompanied the association of the subunits. In contrast, mixing the D12L subunit with the D1R-60K proteolytic fragment failed to yield methyltransferase activity or result in a physical association of the two proteins. These results demonstrate that the methyltransferase active site requires the presence of the D12L subunit with the carboxyl-terminal portion of the D1R subunit. Furthermore, since the mRNA triphosphatase and guanyltransferase active sites reside in the NH2-terminal domain of the D1R subunit, and the methyltransferase activity is found in the carboxyl-terminal portion of this subunit and D12L, there must be at least two separate active sites in this enzyme.

Chromatography↗

PubMed1992

Phenotypic characterization of three temperature-sensitive mutations in the vaccinia virus early gene transcription initiation factor.

Vaccinia virus gene D6R encodes the small subunit of the virion early gene transcription initiation factor. Three temperature-sensitive mutations have been mapped to this gene. The biochemical phenotype exhibited by each mutation was examined. All mutants displayed altered viral protein synthesis in pulse-labelling analyses at both the permissive and non-permissive temperatures. The onset of early protein synthesis was delayed, and the rate of early protein synthesis was reduced in each case. Furthermore the shut-off of both host and early protein synthesis was delayed. In pulse-chase experiments, the stability of the D6R protein in E93- or C46-infected cells was shown to be reduced at 40 degrees C relative to that at 31 degrees C. Early mRNA was quantified in cells at 2 h post-infection and shown to be reduced substantially. The ability of each mutant virus to support transcription in vitro was examined at both temperatures and, of the three mutants, only S4 transcription was shown to exhibit reversible temperature sensitivity.

Gene Expression Regulation, Viral↗

PubMed1991

Isolation of a cDNA encoding a mammalian multiubiquitinating enzyme (E225K) and overexpression of the functional enzyme in Escherichia coli.

The ubiquitin (Ub)-conjugating enzyme E2(25K) catalyzes the synthesis of multi-Ub chains in which successive Ub units are linked by an isopeptide bond involving the epsilon-amino group of Lys-48 of Ubn, and the COOH-terminal Gly residue of Ubn+1 (Chen, Z., and Pickart, C. M. (1990) J. Biol. Chem., 265, 21835-21842). We now describe the polymerase chain reaction (PCR)-based cloning of an E2(25K)-encoding cDNA from a bovine thymus library, using degenerate oligonucleotide primers based on the sequences of two E2(25K) peptides. The cDNA encodes a 200-residue protein whose sequence bears similarities of 66 and 59%, respectively, to the sequences of the Ub-conjugating enzymes encoded by the UBC1 and UBC4/UBC5 genes of the yeast Saccharomyces cerevisiae. These three yeast E2s play key roles in Ub-dependent proteolysis (Seufert, W., McGrath, J. P., and Jentsch, S. (1990) EMBO J. 9, 4535-4541). Comparison of the amino acid sequence of E2(25K) with other known E2 sequences strongly suggests that Cys-92, one of two E2(25K) Cys residues, forms the Ub thiol ester adduct that is an intermediate in E2-catalyzed multiubiquitination. The E2(25K)-encoding cDNA was overexpressed in Escherichia coli, and the recombinant E2(25K) protein was purified to electrophoretic homogeneity; enzymatic assays showed that its multiubiquitinating activity was quantitatively identical with that of the native protein. The availability of a cloned cDNA will allow us to assess the physiological role of E2(25K).

Amino Acid Sequence↗

PubMed1991

Genetic and biochemical characterization of vaccinia virus genes D2L and D3R which encode virion structural proteins.

Polyclonal antisera raised against fusion proteins containing portions of the vaccinia virus D2L and D3R proteins were prepared. Immunoprecipitation of pulse-labeled infected cell extracts and Western blot analysis demonstrated that genes D2L and D3R encode 16.9- and 27-kDa proteins, respectively. Both are synthesized late during infection and there is no evidence for proteolytic processing of either protein. Western blots of purified virus and subvirion fractions showed that D2L and D3R are virion components, residing in a detergent-insoluble fraction, containing viral core structural proteins. Trypsin sensitivity experiments suggest that each is found in an equivalent position within the virus core. Pulse-chase analysis showed that both proteins exhibit biphasic stability in which an unstable nascent component is replaced by a stable form. This observation suggests that the stable component results from the insertion of D2L and D3R into an immature core structure. The DNA sequence of four ts mutants previously mapped to genes D2L and D3R is reported. Analysis of the ability of each mutant to synthesize and process viral proteins showed that protein synthetic patterns were indistinguishable from wild type, however, three of the four mutants were defective in the processing of the major virion structural precursor, p4a. Unlike the biphasic stability observed in wild-type infected cells, D2L and D3R were totally degraded in cells infected at 40 degrees with any of the four ts mutants. Stability of the D2L and D3R proteins, in cells treated with rifampicin, is unaffected which demonstrates that a block in morphogenesis is not directly responsible for the observed instability of the mutant proteins.

Base Sequence↗

PubMed1991

Intragenic and intergenic recombination between temperature-sensitive mutants of vaccinia virus.

The frequency of recombination for a complete set of two-factor crosses between vaccinia virus mutations separated by distances of between 54 and 10692 bp was determined. The results show that in intragenic crosses there is a linear relationship between the recombination frequency observed and distances between the mutations of up to 700 bp. However, no length dependence of the recombination frequency in intergenic crosses with a distance between mutations of 328 to greater than 10000 bp is observed. We attribute this lack of dependence to the high rate of viral DNA interchange, which makes some step other than the cross-over event rate-limiting. Furthermore, we believe that the observed difference in recombination frequency between inter- and intragenic recombination is due to complementation between temperature-sensitive mutants at the permissive temperature.

Cell Line↗

PubMed1990

Orthopoxvirus genetics.

Genetic analysis of orthopoxviruses has contributed substantially to our understanding of the functional organization of the poxvirus genome, and individual mutants provide invaluable tools for future studies of poxvirus biology. Deletion and transposition mutants, localized primarily in the termini of the genome, may be particularly useful for studying virus host range and pathogenicity. Numerous drug resistant and dependent mutants provide keys to understanding a wide variety of virus genes. A large number of well-characterized ts mutants, clustered in the center of the virus genome, are taking on an increasingly important role in research on the function of essential poxvirus genes. Genetic characterization of orthopoxviruses has progressed rapidly during the past decade, and one can reasonably anticipate a time when mutants will be available for the study of any poxvirus gene. Considerable progress toward this goal can be achieved through organized attempts to integrate and further characterize existing mutant collections and through the continued isolation and characterization of deletion, drug resistant, and ts mutants using established techniques. The most exciting possibility is that soon techniques will be available for directed mutagenesis to conditional lethality of any essential poxvirus gene.

Poxviridae↗

PubMed1990

Superinfection exclusion of vaccinia virus in virus-infected cell cultures.

Vaccinia virus-infected BSC 40 cells do not permit the replication of superinfecting vaccinia virus. The extent of superinfecting virus propagation depends on the time of superinfection; there is 90% exclusion by 4 hr after the initial infection, and more than 99% by 6 hr. When superinfection is attempted at 6 hr after infection, the superinfecting virus is incapable of carrying out DNA replication or early gene transcription, demonstrating that an early event in the virus life cycle is inhibited. The rate of adsorption of the superinfecting virus is unaltered which shows that exclusion is affected at a point between adsorption and early gene transcription. In order to exclude superinfection, the primary infecting virus does not require replication of its DNA or expression of its late genes but it must express one or more early genes.

Adsorption↗

PubMed1989

Vaccinia virus gene D12L encodes the small subunit of the viral mRNA capping enzyme.

Vaccinia virus gene D12L, which lies between nucleotides 14,350 and 13,487 in the HindIII D fragment, is transcribed at early times in infection and is capable of encoding a protein 287 amino acids in length with a predicted molecular mass of 33,331. A polyclonal antiserum was raised in rabbits to a fusion protein containing 279 amino acids of the D12L protein, and this serum was used to investigate both the time of synthesis and the function of the D12L protein. A combination of Western blot analysis and immunoprecipitation from pulse-labeled and pulse-chased cell extracts demonstrated that the synthesis of a 31-kDa protein begins early in infection, that it reaches a plateau by about 4 hr, and that it is stable in the infected cell. The D12L protein was localized by Western blot analysis of detergent-solubilized virions to the sodium deoxycholate soluble fraction which suggested that it may be a virion core-associated enzyme. Due to the similarity in apparent molecular weight between the D12L protein and the small subunit of the vaccinia mRNA capping complex the anti-D12L antiserum was employed in Western blot analysis of fractions generated during the purification of the virion mRNA capping enzyme. The 31-kDa D12L protein copurified with the virus capping enzyme through chromatography on heparin-agarose and phosphocellulose and also cosedimented with the capping enzyme through a glycerol density gradient. In addition, the anti-D12L antiserum coprecipitated the large subunit of the capping enzyme, confirming that gene D12L encodes the small subunit of the viral mRNA capping enzyme. An insertion mutation which destroys the gene D12L coding sequence was constructed in a plasmid containing a portion of both genes D11L and D12L and this plasmid was used to rescue a ts mutation, in a single step, in the adjacent gene D11L. Southern blot analysis of the re-plaque-purified virus permitted the identification of the mutant virus only when the mutant was propagated in the presence of wild-type helper virus. We concluded from these data that gene D12L is essential for virus propagation in tissue culture.

Base Sequence↗

PubMed1988

Transcription and translation mapping of the 13 genes in the vaccinia virus HindIII D fragment.

The vaccinia virus HindIII D fragment is 160,060 bp in length and encodes 13 complete open reading frames [Niles et al. (1986) Virology 153, 96-112; S. L. Weinrich and D. E. Hruby (1986). Nucleic Acids Res. 14, 3003-3016]. We have employed a two-step Northern hybridization protocol using single-stranded DNA probes from M13 recombinants in order to identify the mRNA products from the 13 genes. Six of these genes are expressed only at early times after infection; six are transcribed only at late times; one gene is expressed at both early and late times after virus infection. The D11 gene is transcribed into two late mRNA species, one full-length and the other derived from the 3' one-third of the coding sequence. Translation of hybrid-selected mRNA was carried out in an attempt to identify the protein products encoded by each mRNA. Protein products were found for each early gene but translation was successful for only two of the eight late mRNAs. With the completion of the physical map it is apparent that the early and late genes in the HindIII D fragment are arranged in order to minimize potential interference caused by the expression of closely packed viral genes.

DNA Restriction Enzymes↗

PubMed1988

Structure of the transcription initiation and termination sequences of seven early genes in the vaccinia virus HindIII D fragment.

The vaccinia virus HindIII D fragment is 16,060 bp in length and encodes 13 complete genes [E.G. Niles et al. (1986). Virology 153, 96-112; S. L. Weinrich and D. E. Hruby (1986). Nucleic Acids Res. 14, 3003-3016]. Six of these genes are expressed only at early times after infection and one gene is expressed at both early and late times [G. -J. Lee-Chen and E. G. Niles (1988). Virology 163, 52-63]. Transcript mapping by S1 nuclease protection studies was carried out and compared to the results of primer extension analyses, in order to locate map positions of the 5' termini of each early mRNA. The lengths of the products of in vitro transcription, from DNA templates which possess the transcription start regions of each of the early genes, were determined and compared to the lengths of DNA products generated by S1 nuclease protection and primer extension, in order to demonstrate that the 5' termini identified by S1 mapping and primer extension are due to transcription initiation and not to mRNA processing. For each of the early genes in the HindIII D fragment, transcription starts within 25 nucleotides of the translation initiation codon. The precise location of the 3' termini of each early transcript was identified by S1 nuclease mapping. In all but one case, the 3' ends map within 75 nucleotides of the putative transcription termination signal TTTTTNT [G. Rohrmann, L. Yuen, and B. Moss (1986).

Base Sequence↗

PubMed1988

Map positions of the 5' ends of eight mRNAs synthesized from the late genes in the vaccinia virus HindIII D fragment.

The map positions of the 5' ends of eight late mRNAs from the vaccinia virus HindIII D fragment were determined by a combination of S1 nuclease-protection studies and by primer extension analysis. For genes D2, D8, D10, and D11, a single set of 5' ends can be observed by S1 nuclease analysis which maps just upstream from the translation start site for each gene. For genes D3, D6, and D11a the situation is more complex. In addition to the ATG proximal family of protected DNA fragments, multiple larger protected DNA fragments map to sites up to several hundred base pairs upstream from the coding region. When primer extension mapping is carried out, large extended products are observed in all cases but that of gene D10. For genes D2, D8, and D13, these large DNA products are heterogenous in length and much longer than the S1 nuclease-protected DNA fragments. This has been observed previously for the late mRNA from the 11K gene by Bertholet et al. [(1987). Cell 50, 153]. In the case of mRNA from genes D3 and D6, however, the lengths of the extended DNA primers agree with the lengths of the S1 nuclease-protected DNA fragments. Therefore, for genes D3 and D6, the 5' regions of the mRNA must be derived from transcription of the DNA sequences upstream from the coding region of each gene. Since the structures at the 5' ends of the late mRNA assume more than one form, there may be multiple pathways for generating late mRNA.

Base Sequence↗

PubMed1988

Vaccinia virus gene D8 encodes a virion transmembrane protein.

Transcription mapping studies and DNA sequence analysis of the vaccinia virus HindIII D fragment predict that gene D8 encodes a protein 304 amino acids in length, with a molecular mass of 35,426 daltons, that is expressed at late times in infection. In order to determine whether the native D8 protein is required for virus propagation, we constructed a frameshift mutation in the D8 coding sequence. Virus containing this mutation were isolated and shown to replicate in a single-step growth experiment with wild type virus growth kinetics, demonstrating that the normal-length D8 protein is not essential for virus propagation in tissue culture. In order to investigate the synthesis of the wild-type and the mutant D8 proteins in virus-infected cells, we raised polyclonal antisera to a fusion protein consisting of a portion of the D8 coding sequence linked to the Escherichia coli trpE gene. Western blot (immunoblot) analysis of the time course of D8 protein synthesis in cells infected with either wild-type or mutant virus demonstrated that D8 protein was synthesized late in infection in each case and accumulated throughout the experiment. To determine whether the D8 protein was incorporated into the mutant or wild-type virus, purified virions were fractionated into Nonidet P-40-soluble, deoxycholate-soluble, and detergent-insoluble fractions. In both the wild-type and the mutant viruses, the D8 protein was an integral viral protein. The wild-type protein partitioned into the Nonidet P-40-soluble fraction, suggesting that it was a viral membrane protein. The mutant protein fractionated into the detergent-insoluble component, demonstrating that although the altered protein was incorporated into the virus, it was found in a abnormal location. In order to determine whether the D8 protein was present on the virion surface, the susceptibility of the D8 protein to proteolysis was tested by analyzing the products of incubation of the wild-type and mutant viruses with either chymotrypsin or trypsin. These studies demonstrated that the wild-type D8 protein was a transmembrane protein with a major extraviral domain that was released largely intact from the virus by trypsin. The mutant D8 protein was relatively refractory to proteolysis, confirming the hypothesis that although it is associated with the virus, it is in a conformation different from that of the wild-type protein. Tryptic digestion of the wild-type virus increased plaque formation severalfold, concomitant with the removal of the extraviral domain of the D8 protein.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗