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The stability, potency, and preservative effectiveness of two dilutions of epoetin alfa containing a bacteriostatic diluent were studied. Epoetin alfa 10,000 units/mL in single-use vials was diluted 1:1 and 1:1.5 with bacteriostatic 0.9% sodium chloride injection. USP tests of preservative effectiveness were performed on samples from two batches each of the 1:1 and the 1:1.5 dilutions. Appearance assessment, Western blot analysis, radioimmunoassay, and bioassay were used to determine the stability or potency of samples from three batches of each dilution that were stored at 5 degrees C or at 30 degrees C for 12 weeks. Both batches of the 1:1.5 dilution (epoetin alfa 4,000 units/mL with 0.54% benzyl alcohol) met the USP criteria for preserved solutions, while one batch of the 1:1 dilution (epoetin alfa 5,000 units/mL with 0.45% benzyl alcohol) did not. Epoetin alfa 10,000 units/mL diluted either 1:1 or 1:1.5 with bacteriostatic 0.9% sodium chloride injection and stored at 5 degrees C or at 30 degrees C remained stable and potent for 12 weeks. The addition of 1.5 mL of bacteriostatic 0.9% sodium chloride injection to a vial containing 1 mL of epoetin alfa 10,000 units/mL makes a solution of epoetin alfa 4,000 units/mL that meets the USP criteria for preservative effectiveness and remains stable and potent for 12 weeks at 5 degrees C.

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A portion of the antigen I/II (spaA, B, P1) gene of Streptococcus sobrinus 6715, containing the coding sequence for the amino terminal 684 amino acids of the protein, was cloned in bacteriophage lambda GT10. Selection was by immunological detection using a polyclonal antiserum to the antigen I/II from Strep. mutans. From the amino acid sequence, peptides were synthesized, 15 amino acids in length, that covered the entire sequence. In total, 260 synthetic peptides were synthesized and evaluated for their immunogenicity in Balb/C mice. Thirty-nine peptides were immunogenic, without carrier, and the antisera generated were tested for their ability to bind cells of Strep. mutans and Strep. sobrinus in a solid-phase assay. Antisera corresponding to peptides from five regions on the I/II molecule bound cells of both bacterial species. These peptides were then evaluated for their ability to stimulate in vitro murine lymphocyte proliferation, after in vivo immunization with Strep. sobrinus cells. Two of the peptides were capable of stimulating proliferation, as determined by incorporation of [3H]-thymidine into murine lymph node cells. The sequences of these 5 peptides were then compared to sequences found in the antigen I/II from Strep. mutans (Kelly et al., 1989). As expected, there was considerable homology between the cross-reactive peptides synthesized and the analogous region from Strep. mutans. This homology was not usually contiguous and suggests that the antibodies bind a face of antigen I/II that is in an alpha-helical conformation.(ABSTRACT TRUNCATED AT 250 WORDS)

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Detection of antibodies to human immunodeficiency virus (HIV) by enzyme-linked immunosorbent assay (ELISA) is the accepted method to screen blood products at risk to transmit infection. The presence of antibodies to HIV in 565 serum specimens from 274 patients with acquired immunodeficiency syndrome (AIDS) or AIDS-related complex, symptomatic and asymptomatic subjects at risk for AIDS, and controls was determined with an ELISA that incorporates synthetic peptides (designated E32/E34) representing sequences in the envelope glycoprotein gp41. Of 105 specimens from patients with AIDS or AIDS-related complex, 3 specimens that were negative by commercially licensed ELISA and immunoblot test were similarly unreactive in the E32/E34 ELISA. For homosexual men with generalized lymphadenopathy, 186 specimens were positive by the E32/E34 ELISA and 63 specimens were negative. In comparison, with the licensed ELISA, 184 of these samples were positive and 65 samples were negative. The two samples that were positive in the E32/E34 ELISA but not the commercial kit were also positive by immunoblotting. Sequential sera from one individual who apparently underwent seroconversion according to the commercial assays were all positive by E32/E34 ELISA and immunoblotting. Thus, the ELISA with synthetic peptides is an extremely sensitive and specific test of antibody response to HIV and has not yet yielded a negative result with a Western blot (immunoblot)-confirmed antibody-positive serum.

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Certain temperature-sensitive (ts) mutants of murine leukemia virus (MuLV) were observed to be defective in virus assembly. These mutants also accumulated intracellular core protein precursor, Pr65gag, at 39 degrees, the nonpermissive temperature. At 39 degrees, virions released from cells infected with the various ts mutants also contained elevated levels of Pr65gag relative to virions released at 33 degrees, the permissive temperature. Detergent extraction of pulse-labeled cells with Nonidet P-40 (NP-40) generated an NP-40-insoluble cytoskeleton-enriched fraction. Reextraction of this fraction with deoxycholate followed by gel electrophoresis of solubilized, immunoprecipitated viral proteins showed that in Moloney MuLV (Mo-MuLV) ts3-infected cells, and in Rauscher MuLV (R-MuLV) ts17- and ts24-infected cells, increased amounts of intracellular viral Pr65gag rapidly become associated with the cytoskeleton-enriched fraction during pulse labeling at nonpermissive temperature. Furthermore, examination of cell extracts from chase-incubated cells infected with these ts mutants revealed that Pr65gag accumulated in the cytoskeleton-enriched fraction at 39 degrees but not at 33 degrees. During steady-state labeling, as much as half of the intracellular Pr65gag becomes associated with the cytoskeleton-enriched fraction (i.e., is not solubilized by NP-40) at 39 degrees. At permissive temperature only 10-15% of the intracellular Pr65gag is cytoskeleton associated. In contrast, cells infected with R-MuLV ts25 or ts26 showed little or no preferential localization of Pr65gag in the cytoskeleton-enriched cell fraction during a short pulse at 39 degrees, but Pr65gag accumulated in both the NP-40-soluble and -insoluble fractions during a chase incubation relative to the condition at 33 degrees. Based upon these and previous results (Edbauer and Naso, 1983), models for retrovirus assembly are described in which the association of Pr65gag with the cell membrane and cytoskeleton plays a critical role in virus assembly, budding, and postbudding maturation.

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Two-dimensional gel electrophoresis, using either silver staining or pulse labeling with 35S-L-methionine and autoradiography, was employed to determine changes in the synthesis of proteins that may be involved in the antiproliferative effects of recombinant alpha interferon (IFNrA) on Burkitt's lymphoma Daudi cells. IFNrA initiated and/or augmented the synthesis of at least 13 proteins that were distinct in molecular weights and isoelectric properties. Synthesis of several of these IFN-enhanced proteins was inhibited by actinomycin-D, an inhibitor of mRNA synthesis. Although IFN-induced antiproliferative effects were observed at 48 h, an increase in the synthesis of several proteins was observed as early as 3 h. The levels of these IFN-enhanced proteins in treated cells continued to increase through 24 h. At least two proteins of approximately 17 kD were observed to be synthesized in IFN-treated cells but not in control cells. Neither inhibition of synthesis of any particular protein nor post-synthetic modification of proteins in response to IFNrA was observed with these methods. The results of this study are compared and contrasted to those of several other laboratories.

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Our studies have shown a rapid and specific association of Rauscher murine leukemia virus (R-MuLV) precursor polyprotein Pr65gag with cytoskeletal elements in infected mouse fibroblasts. The Pr65gag associated with Nonidet P-40 (NP-40)-insoluble cytoskeletal structures appears to be subphosphorylated in comparison to NP-40-soluble Pr65gag. The association of Pr65gag with skeletal elements can be disrupted by extraction of the cytoskeleton with sodium deoxycholate, an ionic detergent, or with buffers of high ionic strength. Both the skeleton-associated Pr65gag and its NP-40-soluble counterpart can be labeled with [3H]palmitate, indicating their probable association with lipids presumably in the plasma membrane. Pr65gag molecules bound to skeletal elements in the infected cell appear to be more stable to proteolytic processing than NP-40-soluble Pr65gag. While the association of Pr65gag with cytoskeleton elements in the cell is neither increased nor decreased by blocking virus assembly and release with interferon, Pr65gag appears to accumulate in the cytoskeleton-enriched fraction of cells chronically infected with a temperature sensitive mutant of R-MuLV (ts 17) when such cells are grown at the nonpermissive temperature. Based on these and other results, we have proposed a model for the active role of cytoskeleton associated Pr65gag in retrovirus assembly.

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A glycosylated 45,000-Mr protein containing Rauscher murine leukemia virus p15 and p12 antigenic sites and tryptic peptides was identified in Rauscher murine leukemia virus-infected cells. This glycoprotein, termed gP45gag, was also shown to contain a single tryptic peptide also present in gPr80gag and its unglycosylated apoprotein precursor Pr75gag, but lacking in Pr65gag or Pr40gag. The presence of this peptide only in viral precursor proteins containing the so-called leader (L) sequence strongly suggests that gPr45gag is an N-terminal fragment of larger glycosylated gag polyproteins, composed of L sequences in addition to p15 and p12. The kinetics of appearance of radiolabeled gPr45gag and its disappearance during chase-incubation is suggestive of a precursor-like role for this intermediate gene product. An observed 27,000-Mr glycosylated polypeptide, termed gP27gag and containing p15 but not p12, p30, or p10 antigenic determinants, is a candidate cleavage product derived from gPr45gag. These observations suggest that gPr45gag and its putative cleavage product gP27gag represent an authentic pathway for intracellular processing of glycosylated core proteins.

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The intracellular precursor polyproteins of simian sarcoma-simian-associated virus [SiSV(SiAV)] were compared to the intracellular proteins of the human retrovirus isolates. HL23V, HEL12V and A1476V, by radioimmunoprecipitation followed by SDS-polyacrylamide gel electrophoresis and tryptic peptide analysis. Cells infected with SiSV(SiAV) were characterized by polyproteins Pr200gag-pol, gPr80env, Pr80gag, Pr60gag and Pr40gag. Identical intracellular precursor polyprotein profiles were obtained from cells infected with HL23V, HEL12V and A1476V. Tryptic digest mapping of peptides containing [3H]leucine showed the structural composition of Pr60gag to be the virus core proteins, p28, p15/p12 and p10. The SiSV(SiAV) envelope precursor, gPr80env, contained the structural determinants of mature viral gp70 and a non-glycosylated protein termed p15E. The homology of the human isolate viruses, HL23V, HEL12V and A1476V, to the SiSV(SiAV)/GaLV (gibbon ape leukaemia virus) family of viruses was confirmed by mapping studies. Both gPr80env and Pr60gag of SiAV were identical by tryptic peptide mapping to the respective proteins from the three human retrovirus isolates examined. The potential significance of these results to considerations of the origins of SiAV and the SiAV-like human isolates is discussed.

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Interferon (IFN) treatment of NIH Swiss mouse embryo cells chronically infected with Rauscher murine leukemia virus (R-MuLV) drastically reduced the release of virus particles from the cells. The characterization of intracellular and extracellular viral specific proteins and polyproteins immunologically with various antisera, and structurally by tryptic digest mapping experiments, indicated that the antiretroviral action of IFN was not due to an IFN-induced alteration in the synthesis of any viral protein. Steady state labeling experiments, however, showed that the processing of three viral specific precursor polyproteins, namely gPr90env, Pr40gag, and Pr25gag, were perceptively slowed in IFN-treated cells. This effect was apparently not related to the ability of these proteins to be modified by phosphorylation or glycosylation after translation since these processes occurred normally in the IFN-treated cells. The treatment of cells with IFN also caused the accumulation of a small amount of a fucosylated viral glycoprotein precursor, termed gP93env, in virus. With the exception of this minor protein, virus released from IFN-treated cells were normal in their content of viral proteins. These virus particles were only slightly less infectious, particle for particle, than virus released from control cultures. Based on these results, we suggest that IFN causes an as yet unelucidated alteration in cell membrane structure of function, or both, which prevents either the insertion of viral core precursor molecules into membrane or the recruitment or clustering of such viral polyproteins into virus assembly centers in the membrane. This suggested mechanism of IFN action is discussed in detail.

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Intracellular precursor polyproteins of three baboon endogenous retrovirus (BaEV) isolates, m7, 455K, and BILN, were compared with the intracellular proteins of the type C human isolated HL23V by radioimmunoprecipitation, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and tryptic peptide analysis. Human and canine cells infected with m7-BaEV and canine thymus cells infected with BILN-BaEV were characterized by identical precursor polyproteins Pr85gag, Pr70-71gag, Pr65gag, and gPr85env. Canine cells infected with 455K-BaEV consistently showed a slightly different pattern of precursor polyproteins. These included Pr85gag, Pr70gag, Pr67gag, and gPR85env. By tryptic digest mapping of peptides containing [3H]leucine, m7-BaEV and 455K-BaEV were shown to be highly related. By comparison, mapping studies showed that BILN-BaEV was less highly related to m7-BaEV than ws 455K-BaEV. Differences in these related BaEV isolates presumably reflected virus-specific differential cleavage of core protein precursors or alterations in polyprotein primary structure or both. Chase-incubated cells infected with BaEV also contained a stable, p28-related polyprotein termed P72gag. This polyprotein migrated upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis slightly slower than the major core protein precursor Pr70-71gag and appeared to arise by posttranslational modification of Pr70-71gag. Immunoprecipitation of extracts of HL23V-infected cells with antisera to simian sarcoma-simian-associated virus proteins and BaEV proteins confirmed that these cells contained two unrelated viral components, one that was similar to m7-BaEV or BILN-BaEV and a second that was related to simian sarcoma-simian-associated virus. Tryptic digest mapping of BaEV and HL23V prcursor polyproteins suggested that the BaEV-like component of HL23V weas more closely related to m7-BaEV than to 455K-BaEV or BILN-BaEV.

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A glycoprotein of molecular weight 130,000 (gP130) has been precipitated from the cytoplasm of GR-strain mouse mammary tumor (GR-MMT) cells by a rabbit antiserum (anti-MMTV) to GR-strain mouse mammary tumor virus (GR-MMTV). This protein was not precipitated by antisera specific for detergent-disrupted C3H-strain MMTV (C3H-MMTV); C3H-MMTV glycoproteins; C3H-MMTV nonglycosylated proteins; GR-MMTV p25 or p12; RIII strain (milk) MMTV proteins; or Rauscher murine leukemia virus (R-MuLV) proteins; nor was it precipitated by normal rabbit serum. Two-dimensional thin layer analysis of 35S-methionine-containing tryptic peptides revealed that five of nine gp33 peptides and one of seven gp55 peptides are shared by gP130 and gPr76env. The envelope protein precursor, gPr76env, contains all of the gp33 peptides and six of seven gp55 peptides. One peptide in gPr76env, possibly a gp55-gp33 junction peptide, is also apparently present in gP130. Six of ten p25 peptides and four more gag-related peptides are shared by PR78gag and gP130. Protein gP130 also contains several tryptic peptides not found in gPr76env or in the core protein precursors Pr78gag, Pr110gag or Pr180gag-pol. Radioimmunoprecipitation experiments showed that gP130 could be precipitated from extracts of GR-MMTV cells with anti-MMTV serum even after antibodies to the known MMTV structural proteins had been removed from the serum by absorption. Both gP130 and a second protein, p30, were found in immunoprecipitates of detergent-disrupted isotopically labeled GR-MMTV treated with the absorbed anti-MMTV serum. These results suggest that antibodies to gP130 in the anti-MMTV serum are capable of recognizing those protein sequences unique to gP130; that is, those protein sequences which are not related to viral structural proteins. In light of these data and data published previously, gP130 is apparently a polyprotein containing juxtaposed components translated from the 5' and 3' end of the MMTV genome and protein components not previously identified as virus-specific.

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Mouse mammary tumor virus (MMTV) derived from the culture medium of GR cells contained seven proteins, identified as gp55, gp33, p25, pp20, p16, p12, and p10. The major viral phosphoprotein was the 20,000-molecular-weight protein, pp20. Immunoprecipitation of cytoplasmic extracts from pulse-labeled GR cells identified three MMTV gag-specific proteins, termed Pr78(gag), Pr110(gag), and Pr180(gag+). These intracellular polyproteins were precipitable from cytoplasmic extracts by antisera to virions p25 and p12 but not by antisera to gp55. The major intracellular gag-specific precursor polyprotein, Pr78(gag), contained antigenic determinants and tryptic peptides characteristic of p25, p12, p10, and presumably pp20. This precursor is presumably derived from nascent chain cleavage or rapid posttranslational cleavage of the larger intracellular precursor-like protein, designated Pr110(gag). Pr110(gag) contained all but one of the leucine-containing tryptic peptides of Pr78(gag), plus several additional peptides. In addition to Pr78(gag) and Pr110(gag), monospecific antisera to virion p12 and p25 were also capable of precipitating from pulse-labeled cells a small amount of a 180,000-molecular-weight precursor-like protein, designated Pr180(gag+). This large polyprotein contained nearly all of the leucine-containing tryptic peptides of Pr78(gag) and Pr110(gag) plus several additional peptides. By analogy to type C viral systems, Pr180(gag+) is presumed to represent a gag-pol common precursor which is the major pathway for synthesis of MMTV polymerase. Immunoprecipitation of cytoplasmic extracts from pulse-labeled cells with antisera to gp55 identified two env-specific proteins, designated gPr76(env) and gP79(env). The major env precursor, gPr76(env), could be labeled with radioactive glucosamine and was shown to contain antigenic determinants and tryptic peptides characteristic of gp55 and gp33. A minor glycoprotein, gP79(env), contained both fucose and glucosamine and was precipitable from cytoplasmic extracts with monospecific serum to gp55. It is suggested that gP79(env) represents fucosylated gPr76(env) which is transiently synthesized and cleaved rapidly into gp55 and gp33.

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Under steady-state labeling conditions, Rauscher murine leukemia virus-infected NIH Swiss mouse cells contain at least three major polyproteins derived from the viral gag gene. They have molecular weights of 65,000, 40,000, and 25,000. They have been termed pPr65gag, Pr40gag, and pPr25gag. pPr65gag has been shown by a number of laboratories to be composed of all four core proteins (p15, pp12, p30, and p10). In this paper, Pr40gag was found to contain p30 and p10 antigenic determinants and peptide sequences, whereas pPr25gag was found to contain p15 and pp12. Pr40gag and pPr25gag are rapidly labeled precursor proteins that were detectable early in pulse-chase experiments. Both precursors disappeared during the later stages of the chase period concurrent with the appearance of the mature viral core proteins. pPr65gag and pPr25gag were found to be phosphorylated, pPr25 having a higher specific activity of 32P than pPr65. In spite of this, peptide mapping studies, as well as the identification of the phosphorylated amino acid residues of pPr65, and pPr25, and pp12, indicated that the same sites are phosphorylated regardless of whether the precursors or the mature pp12 are examined.

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Rauscher murine leukemia virus glycoprotein gp69/71 and non-glycosylated p15(E) are synthesized by way of a 90,000-dalton precursor glycoprotein, termed Pr2a+b. Peptide mapping experiments showed that Pr2a+b contains all the tyrosine-containing tryptic peptides of gp69/71. Two additional tyrosine-containing tryptic peptides in Pr2a+b that are not detected in gp69/71 are found in p15(E). Thus, gp69/71 and p15(E) peptide sequences account for all the tyrosine tryptic peptides of Pr2a+b. The gene order of the two proteins was determined by pulse-labeling infected cells in the presence and absence of pactamycin at concentrations of the inhibitor that prevent initiation of translation, but not elongation. The gene order was found to be: (2)HN-gp69/71-p15(E)-COOH. A newly identified major viral protein, termed p12(E), migrates in sodium dodecyl sulfate-polyacrylamide gels in the "p12" region. It is related to p15(E) as determined by tryptic mapping experiments. p15(E) and p12(E) are not phosphorylated, and both can be separated from phosphoprotein p12 by guanidine hydrochloride-agarose chromatography. p12(E) and p15(E) elute in the void volume fraction, whereas phosphoprotein p12 elutes between p15 and p10. The two p12 proteins can also be separated from each other by two-dimensional gel electrophoresis involving isoelectric focusing in the first dimension and sodium dodecyl sulfate-gel electrophoresis in the second dimension.

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It was previously demonstrated that the 60,000 dalton (p60) precursor-like polyprotein containing murine p30 was a constituent of the feline leukemia virus pseudotype of Moloney sarcoma virus [m1MSV(FeLV)]. It is now shown that p60 is detected in cells of five mammalian species transformed by m1MSV, indicating that p60 is specified by this genome. Moreover, little or no murine p30 is detected in the m1MSV-transformed cells, suggesting that the murine group p30 antigenic reactivity of S + L- cells is ude to p60. Pulse-chase studies in cells producing m1MSV(FeLV) show that p60 is the largest polypeptide detectable during the pulse, and that intracellular p60 is not cleaved into smaller (for example, p30) polypeptides during chase periods of up to 10 hr. The lack of cleavage of p60 is in contrast to the properties of p30 precursors detected in cells containing replicating avian or mammalian RNA tumor viruses. The inefficient cleavage of intracellular p60 and the kinetics of appearance of murine p30 in extracellular m1MSV(FeLV) suggest that p60 cleavage to p30 occurs in cells shortly before virus release. While only p60 was detected in the m1MSV-transformed cells, p60 and p70 were detected in m3MSV-transformed cells, and no immunoprecipitable polypeptides were detected in HT-1 MSV-transformed cells. The observed differences in the intracellular polypeptide expression by each of the strains of MSV suggests differences in genetic content.

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The cleavage of an intracellular 67,000- to 70,000-dalton precursor, termed Pr4 to Rauscher leukemia virus (RLV) p30 protein proceeded at a slower rate when virus-producing cells were treated with actinomycin D (AMD). Treatment with AMD also caused a slight accumulation of Pr4 in purified early virus particles produced by a cell line which usually produces virions that contain little Pr4. The cleavage of other intracellular viral precursor polypeptides was not affected by treatment with AMD. Treatment of infected cells with cycloheximide, on the other hand, allowed the cleavage of Pr4 to proceed at the usual rate for a short period of time before further cleavage was drastically slowed or prevented. The cleavage of several other viral precursor polypeptides was also inhibited by treatment with cycloheximide. Different lines of evidence suggest that the mechanism of action of AMD is not due to a possible indirect effect on protein synthesis. Thus, the rate of cleavage of Pr4 was not affected by the length of pretreatment with AMD between 1 to 8 h. In addition, the combined effect of AMD and cycloheximide, at their maximal inhibitory concentrations, was greater than the effect of either drug alone, indicating the involvement of two at least partially different mechanisms in the action of AMD and cycloheximide. Furthermore, AMD did not affect the pulse labeling of viral precursor polypeptides. These results suggest that the interaction with viral RNA, whose production is inhibited by AMD, accelerates the cleavage of Pr4 to p30 during virus assembly. A hypothetical model is presented to illustrate th possible advantages of having a step in virus assembly in which genomic RNA interacts with a precursor to capsid proteins before the cleavage of that precursor.

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Rauscher leukemia virus glycoprotein gp69/71 is synthesized in virus-infected cells by way of a 90,000 dalton glycoprotein precursor, termed Pr2a+b. This precursor could be labeled with radioactive glucosamine and methionine but not with fucose; whereas gp69/71 could be detected by labeling with radioactive glucosamine, fucose, or a mixture of amino acids but seemed to be deficient in methionine relative to Pr2a+b. Pr2a+b and gp69/71, were specifically precipitated by an antiserum prepared against phosphocellulose purified Rauscher gp69/71. Other virus-specific precursors, in addition to Pr2a+b, could be precipitated by antiserum prepared against detergent disrupted virus. Neither Pr2a+b nor gp69/71 was precipitated from cell extracts by antisera to Rauscher p30. Tryptic maps of Pr2a+b and gp69/71 showed that these glycoproteins share many tryptic peptides. Pulse-chase experiments with 14C-labeled amino acids indicated that gp69/71 was not radio-labeled during the pulse-labeling period but slowly appeared during the chase incubations. Pr2a+b, however, was rapidly labeled and tended to disappear during long chases. Furthermore, two nonglycosylated viral proteins, termed p15E and p12E, are structurally related to Pr2a+b. Viral p15E and p12E contained the same methionine-containing tryptic peptide fraction as Pr2a+b as determined by ion-exchange chromatography. These results provide evidence that Pr2a+b is a precursor to gp69/71 and establish a structural and possible precursor-product relationship between Pr2a+b, p15E, and p12E.

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