Presence of circulating antibodies against gag-gene MuLV proteins in patients with autoimmune connective tissue disorders

An immunoblotting procedure using viral proteins from purified murine sarcoma virus or MSV-(MLV) has been developed to characterize antiviral antibodies in sera from patients with autoimmune connective tissue disorders. Fifty-eight sera with anti-Sm, anti-RNP, anti-SS-B (La), and other undefined specificities were found to react with several major viral polypeptide bands. Most of them corresponded to gag-gene-encoded products: pr65^gag, p40^gag, p30, p15, p12 and p10. Other bands with molecular weights averaging 90K, 60K, 45K, and 28K were recognized by a few sera. Immunological specificity of the reaction was assessed by reproducing the tests with IgG purified from sera and from corresponding F(ab′)2 fragments. Moreover, the specificity of the reaction with gag proteins was confirmed by repeating the tests with p30 and p15 prior purified by immunoprecipitation with anti-p30 and anti-p15 goat sera. Furthermore, the gag polypeptides were recognized by human sera by replacing MSV-(MLV) by three other murine retroviruses of different origin. An indirect confirmation of these results was obtained by applying this method to sera of MRL lpr/lpr mice which develop an autoimmune syndrome comparable to that of human systemic lupus erythematosus. In agreement with previously published results (C. Rordorf, C. Gambke, and J. Gordon (1983), J. Immunol. Methods59, 105–112), we found that anti-gag-gene antibodies were present in the sera of individual mice. Patterns of reactivity were found to vary with the age of the animals. No retroviral polypeptide was significantly detected in the great majority (80%) of sera from normal donors. However, 5 out of 25 sera showed faint bands although to a lesser extent than pathological sera. These five sera also reacted with HeLa cell purified HnRNPs, suggesting that their normal status should be reconsidered.     

Detection and characterization of simian retroviruses homologous to human T-cell leukemia virus type I

Lymphoid cell lines were established from five different species of monkeys which were positive in antibodies cross-reactive with human T-cell leukemia virus type I (HTLV-I) and were shown to contain provirus sequences homologous to HTLV-I. Gene-specific probes of HTLV-I, gag, pol, env, pX, and LTR, hybridized efficiently with monkey DNAs from these cell lines under stringent conditions, indicating that the proviruses are very similar to HTLV-I along with whole viral genomes. However, the preliminary restriction mapping turned out the difference between simian retroviruses and HTLV-I and also among simian retroviruses. These findings suggest a common ancestor of simian and human retroviruses, but exclude the recent interspecies transmission between monkeys and humans.     

Purification and chemical and immunological characterization of avian reticuloendotheliosis virus gag-gene-encoded structural proteins

Five gag-gene-encoded structural proteins, designated p12, pp18, pp20, p30, and p10 were purified from replication-competent avian reticuloendotheliosis-associated virus (REV-A) by high-performance liquid chromatography complemented with chloroform-methanol extraction and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Based on amino acid composition and NH2- and COOH-terminal sequence analysis p12, pp18, p30, and p10 are distinct from one another, whereas pp20 is likely identical to pp18 in primary structure. The p12 was resistant to Edman degradation and was found to be myristylated at the NH2-terminal amino group. Sequence comparisons among the retrovirus family show that pp18/pp20 and p10 are, respectively, homologs of phospho-proteins and nucleic acid-binding proteins. A comparison of terminal sequences with the nucleotide sequence of spleen necrosis virus (SNV) revealed that the gag genes of SNV and REV-A are highly conserved; together with the identification of REV-A gag-precursor polyprotein, Pr60gag in immunoprecipitates of radiolabeled cell lysates, this comparison also led to the establishment of the organization of Pr60gag, viz., NH2-p12-pp18-p30-p10-OH. Sequence comparisons show that REV-A/SNV is related to mammalian type C viruses: the pp18-p30 region is most homologous to the macaque/colobus group and least to simian sarcoma virus (SSV), whereas both the 5'- and 3'-gag regions (i.e., p12 and p10) are clostest to SSV. Immunological studies using monospecific antisera and Western-blot analysis showed that antigenic determinants of REV-A p30 are conserved in most of mammalian type C and type D viruses, but those of REV-A p12 are shared only with simian sarcoma-associated virus (SSAV) and endogenous viruses of macaques.     

Human T-cell leukemia virus type II: primary structure analysis of the major internal protein, p24 and the nucleic acid binding protein, p15

The 24,000-molecular-weight major internal protein (p24) and the 15,000-molecular-weight nucleic acid binding protein (p15) of human T-cell leukemia virus type II (HTLV-II) were subjected to amino acid composition and amino-terminal amino acid sequence analysis. A comparison of amino acid composition of p24 and p15 of HTLV-II with those of the analogous proteins of HTLV-I revealed that these two proteins share overall similarity. Further, alignment of the amino-terminal amino acid sequence for the first 27 residues of p24 and 34 residues of p15 from HTLV-II showed extensive sequence homology with analogous proteins of HTLV-I. These data suggest that although disease associated with HTLV-I is malignant T-cell leukemia and that associated with HTLV-II is a relatively benign variant of hairy-cell leukemia, HTLV-I and HTLV-II are closely related to each other, at least in their gag-gene-encoded sequences.     

Studies on human parainfluenza virus 3: characterization of the structural proteins and in vitro synthesized proteins coded by mRNAs isolated from infected cells

The structural proteins of human parainfluenza virus 3, a member of the paramyxovirus family, were characterized by SDS-polyacrylamide gel electrophoresis of radiolabeled virus. The purified virion contains at least eight structural proteins, with estimated molecular weights of 251K, 90K, 71K, 68K, 65K, 51K, 35K, and 21K, respectively. Three of the polypeptides (71K, 65K, and 51K) were identified as glycoproteins based on their incorporation of [3H]glucosamine. Disruption of the virus by Triton X-100 in the presence of increasing salt concentrations indicated that the polypeptides of molecular weights 251K, 90K, 68K, and 21K were components of the nucleocapsid. In parainfluenza virus 3 infected BS-C-1 cells, seven virus structural polypeptides were identified. Six structural proteins (90K, 71K, 68K, 51K, 35K, and 21K) were detected in the cell lysate at 7 hr after infection, while at 10 hr an additional polypeptide (251K) was also observed. At least two nonstructural polypeptides of molecular weights 30K and 25K were also detected in infected cells. mRNAs isolated from virus-infected cells were translated in a cell-free protein-synthesizing system. The in vitro translation products were identical to the authentic virion polypeptides as determined by partial digestion with staphylococcal V8 protease.     

Characterization of human p53 antigens employing primate specific monoclonal antibodies

p53 is a cellular protein whose levels are some 1500-2000 times higher in adenovirus and SV40-transformed human cell lines than in homologous nontransformed cells. Monoclonal antibodies have been produced that detect p53 of primate origin but not of rodent origin. These monoclonal antibodies have been employed to study the properties of p53 antigens from human cell lines. Human p53 proteins of at least five different apparent molecular-weight classes in SDS-polyacrylamide gels have been detected. In some cell lines, at least two distinct molecular-weight species are expressed and these two forms have similar or identical partial peptide maps. Both molecular-weight forms can be resolved into seven or eight species upon isoelectric focusing in a two-dimensional gel system. There is also some indication of differences in the partial peptide maps of human p53 antigens derived from different human transformed cell lines. A radioimmunometric assay was employed to study the steady-state levels of oligomeric p53 in normal and transformed cell lines. Antibody affinity chromatography has been employed to purify p53 protein which was then used to quantitate the steady-state levels of p53 in different human cell lines. Normal cells had little or no detectable p53 antigen. Transformed cells or tumor-derived cell lines varied between no detectable p53 protein and 450 micrograms of p53 protein/g of cellular protein (in SV80 cells). There was a great diversity in the levels of p53 antigen in human cells. SV40- and adenovirus-transformed cells had by far the highest levels of p53 antigen. These are the viruses whose tumor antigens have been shown to be associated in an oligomeric complex with p53 in transformed cells. Eleven out of fifteen human tumor derived or transformed cell lines contained greater than five-fold higher levels of p53 antigen than normal human cells.     

Characterization of the nuclear polyhedrosis virus DNA of Adoxophyes orana and of Barathra brassicae

Circular double-stranded DNA was isolated from nuclear polyhedrosis virus (NPV) of Adoxophyes orana (virus particles singly embedded in the polyhedral matrix) and NPV of Barathra brassicae (virus particles multiply embedded in the polyhedral matrix), and some of their physical properties were determined. The molecular weights of A. orana and B. brassicae NPV-DNA, 6.7 x 10⁷ and 8.9 x 10⁷, respectively, were determined by electron microscopy and by renaturation kinetics analysis. The latter analysis also showed that both genomes do not contain repetitive sequences. Absence of homology between DNA of these two viruses was shown by competition hybridization of A. orana NPV-DNA with B. brassicae NPV-DNA. Analysis of these DNAs with the restriction endonuclease EcoRI confirmed that they are different. The buoyant densities in CsCl of A. orana NPV-DNA and of B. brassicae NPV-DNA, 1.694 and 1.696 g/cm³, respectively, are consistent with (G + C) contents of 34.5 and 37%, respectively, as determined by thermal denaturation.     

Avian erythroblastosis virus E26: Nucleotide sequence of the tripartite onc gene and of the LTR,and analysis of the cellular prototype of the viral ets sequence

An intact 5.7-kb provirus of the avian erythroblastosis virus E26 has been molecularly cloned for comparisons with avian myeloblastosis virus (AMV) and other avian tumor viruses. E26 and AMV transform hemopoietic cells exclusively. Both cause myeloblastosis, but E26 also causes erythroblastosis. Sequence analysis of the proviral DNA showed that: (i) The tripartite transforming gene of E26 forms a contiguous reading frame of 1046 codons, including 272 gag, 283 myb^E, and 491 ets codons. No subgenomic ets-specific mRNA was detected in E26-infected cells. By contrast, the onc gene of AMV consists almost entirely of a myb^A sequence expressed via subgenomic mRNA that extends over the 5′ and 3′ ends of myb^E. (ii) myb^E is only slightly diverged from the myb^A homolog of AMV and even less from the cellular proto-myb sequence with no characteristic mutation that sets apart the two viruses from proto-myb. (iii) The U5 region of the long terminal repeat (LTR) of E26 and AMV are colinear and differ only in scattered point mutations. The U3 region of the E26 LTR is different from that of AMV but is colinear and closely related with that of avian carcinoma virus MH2 and also with that of Prague Rous sarcoma virus (RSV), except for an unexpected 16-nucleotide substitution of 22 RSV nucleotides. Upstream of the 3′ LTR, the c region of E26 appears to be the same as that of RSV for 70 nucleotides and very similar to those of AMV and MH2 for about 20 to 30 nucleotides. Since the U3s of E26, MH2 and RSV are very closely related and neither MH2 nor RSV show a particular erythroblast tropism, it is possible that the U3 does not play a critical role in the erythroblast tropism of E26. Electrophoretic size analyses of chicken DNA digested with restriction enzymes indicate that DNA fragments totaling over 50 kb hybridize with viral ets DNA.     

Sequence homology of the simian retrovirus genome with human T-cell leukemia virus type I

A retrovirus highly related to human T-cell leukemia virus type I (HTLV-I) was isolated from a T-cell line established from a seropositive pig-tailed monkey and the provirus genome was molecularly cloned using HTLV-I as a probe. The monkey virus (STLV) had the genomic structure of the LTR-gag-pol-env-pX-LTR. Analysis of the env-pX-LTR region revealed the 90% homology of the nucleotide sequence with that of HTLV-I in each region. This high homology of the sequence indicates that STLV is a member of the HTLV family, but apparently different from HTLV-I. This suggests that the possibility of recent interspecies transmission from monkeys to humans in the endemic area is very small. From its similarity to HTLV, STLV should be useful as an animal model in studies on natural HTLV infection and leukemogenesis of HTLV in humans.     

Involvement of the htpR gene product of Escherichia coli in phage λ development

Growth of phage λ at high temperature requires a functional htpR host gene. The stages of the phage growth cycle shown to be dependent on htpR gene function include prophage excision and particle morphogenesis. Two types of morphogenetic abnormalities have been detected. One is a defect in phage tail assembly that results from a deficiency in tail fibers even though gpJ is produced. The severity of this defect is phage-strain specific. The second morphogenetic defect is less clearly defined, but results in formation of aberrant phage head structures. These abnormalities in λ reproduction are presumed to be caused by the absence in htpR mutant host cells at high temperature of one or more of the heat-shock proteins of Escherichia coli whose synthesis is known to be regulated by the htpR gene.     

Genetic relatedness between A/Swine/Iowa/15/30(H1N1) and human influenza viruses

The nucleotide sequences of the M and NS1 genes of influenza virus A/Swine/Iowa/15/30 (A/SW/IW/30)(H1N1) were determined with cloned DNAs and compared with reported sequences of human and avian influenza viruses. A/SW/IW/30 virus was found to be closely similar to A/PR/8/34(H1N1) virus in the nucleotide sequences of the M and NS1 genes, the base differences between the two strains being 64 out of 1027 nucleotides in the M gene and 52 out of 740 in the NS1 gene. Based on the assumptions that these two viruses were derived from a common ancestor and that the rate of base changes per year was the same in man and in swine, it was estimated that the progenitor virus was in circulation during the period from 1915 to 1920. This estimation was compatible with the epidemiological findings suggesting that the progenitor of the swine influenza virus was the agent of the 1918 influenza pandemic. Furthermore, the M and NS1 gene sequences of A/FPV/Rostock/34(H7N6) virus were much closer to those of A/SW/IW/30 and A/PR/8/34 viruses than to A/duck/Alberta/60/76(H12N5) virus, but not as close as the A/SW/IW/30 virus was to A/PR/8/34 virus.     

Antigenic structure of the hemagglutinin of influenza virus B/Hong Kong/8/73 as determined from gene sequence analysis of variants selected with monoclonal antibodies

Antigenic variation among influenza B viruses is different from that of influenza A in several ways. Antigenic shift has not been observed, distinct antigenic variants of influenza B cocirculate, and antigenically similar viruses have been isolated many years apart. To study the mechanism of antigenic drift in influenza B viruses, monoclonal antibodies were used to select antigenic variants of B/Hong Kong/8/73 virus hemagglutinin (HA). Analyses of the nucleotide sequences of the HA gene of B/Hong Kong/8/73 and the eight variants identified specific regions of the influenza B HA molecule involved in antigenicity, and enabled antigenic mapping data to be correlated with the structure of the protein. The altered amino acids in the variants, when compared to the HA of A/Aichi/2/68, were found in two of the four antigenic regions previously identified for type A viruses. In addition, four of the eight variants showed multiple nucleotide changes some of which gave rise to double amino acid changes. In addition, in the present study monoclonal antibodies which belong to the same antigenic group recognize amino acid changes in regions corresponding to antigenic sites A and B of the H3 HA. These results are in contrast to those obtained with HA variants of A/Memphis/1/71 virus. In the influenza A studies only single amino acid changes were found and these correlated well with the three-dimensional structure as determined by D. C. Wiley, I. A. Wilson, and J. J. Skehel, (1981, Nature (London) 289, 366-373); monoclonal antibodies which recognized one region did not recognize any of the other antigenic sites. Our results suggest that although the basic three-dimensional structure of the influenza B HA may be similar to that of A viruses, the B HA molecule may be folded in a more compact manner so that antigenic sites A and B are in closer proximity to each other than in the H3 structure.     

Identification of gag and env gene products of human T-cell leukemia virus (HTLV)

The gag and env gene products of human T-cell leukemia virus (HTLV) were identified with rabbit antisera against the synthetic peptides and a polypeptide produced in Escherichia coli, which corresponded to parts of the proteins predicted from the nucleotide sequence of HTLV [M. Seiki, S. Hattori, Y. Hirayama, and M. Yoshida (1983). Proc. Natl. Acad. Sci. USA 80, 3618-3622]. Viral proteins were detected by immunoprecipitation in two HTLV-producing cell lines. The precursor of gag products was a protein with an apparent molecular weight of 53,000 (Pr53), and was shown to be processed into three mature gag proteins, p19, p24, and p15, in this order, from the 5' end of the gag gene. The processing sites were confirmed to be the same as those predicted from the nucleotide sequence. The env gene product was identified as a glycoprotein of 62,000 Da (gp62), which was processed into gp46 and p20E. All the viral antigens described above were also detected with sera from ATL patients, indicating that all these proteins are expressed in the patients.     

Heterogeneity of p15(E)-related polypeptides expressed by MuLV-infected cells

The p15(E)-related polypeptides of radiation leukemia virus (RadLV)-derived viruses and of cells infected with prototype MuLV were analyzed by immunoprecipitation, SDS-PAGE, and immunofluorescence analyses. It was found that the p15(E)-related molecules of ecotropic and xenotropic viruses derived from RadLV lymphoma cell lines were distinguishable by reactivity with monoclonal anti-p15(E) antibodies and by SDS-PAGE profile. Ecotropic MuLV of RadLV origin encoded the p15(E)a antigen and produced a Pr15(E) of 20K MW. In contrast, xenotropic virus derived from RadLV did not express the p15(E)a antigen and by SDS-PAGE its Pr15(E) migrated at 21K. A previously undescribed, p15(E)-related molecule of 18.5K MW was associated with xenotropic RadLV. These differences were also reproduced by the prototype ecotropic, xenotropic, and dualtropic viruses.     

The B/C gene of simian virus 40.

Temperature-sensitive B, C, and BC mutants of Simian virus 40 (SV40) map in the late region of the viral genome inHin fragments K, F, J, and G, a DNA segment of about 1200 nucleotide pairs (Lai, C.-J., and Nathans, D. (1975)Virology 66, 70–81). To define the B/C region further, mutants of SV40 with deletions in this genomic segment were constructed by enzymatic excision of DNA from the viral genome, followed by cloning in the presence of a complementing tsA mutant of SV40. After localization of deleted genome segments by analysis of endo R fragments and electron microscopic heteroduplex mapping, selected deletion mutants were tested for complementation by ts mutants and were screened for their ability to produce new viral proteins in infected cells. Complementation tests indicated that B/C deletion mutations are in a cistron distinct from that of tsA and tsD mutations and that the junction between the B/C and D genes is within Hin-K. Two of the deletion mutants produced new proteins detectable in infected cells. More detailed analysis of one of these proteins (of molecular weight 25,000) indicated that it precipitated with antiserum against dissociated SV40 capsids, and that all but one of its lysine-containing tryptic peptides cochromatographed with SV40 VP1 tryptic peptides. We conclude that the B/C gene, containing approximately 1200 nucleotide pairs, codes for VPl. Since deletion mutants lacking Hin-E do not complement B mutants, we suggest that the Hin-E DNA segment has a signal required for expression of the B/C gene.     

The 126,000 molecular weight protein of tobacco mosaic virus is associated with host chromatin in mosaic-diseased tobacco plants

The infection-related polypeptide of apparent molecular weight (MW) 116,000, which previously was shown to be associated with host chromatin from mosaic-diseased leaves of tobacco mosaic virus (TMV)-infected tobacco, comigrated during electrophoresis in polyacrylamide gels with the 126,000 molecular weight protein translated from TMV RNA in vitro. Limited proteolysis of the 116,000 MW polypeptide and of the 126,000 MW translational product with protease V8 generated the same peptides, indicating that the proteins were very similar or identical. Thus, the virally coded 126,000 MW protein is associated with host chromatin. The possibility that viruses may perturb host metabolism at the genome level is discussed.     

Identification of HTLV p19 specific natural human antibodies by competition with monoclonal antibody

A competitive binding assay using a monoclonal antibody to the human T-cell lymphoma/leukemia virus (HTLV) p19 was developed for use in detecting natural antibodies to the protein in human sera. The specificity of the assay for HTLV p19 was demonstrated using a variety of antisera. While sera known to contain antibodies to HTLV p19 competed in the assay, antisera prepared against purified HTLV p24, the major core protein of the virus, or against other disrupted type-C retroviruses did not. Sera of Japanese patients with adult T-cell leukemia and similar T-cell malignant lymphomas were examined by this technique for the presence of antibodies to HTLV p19. The results were compared with those obtained by a solid-phase radioimmunoassay (RIA) against disrupted HTLV. The majority of Japanese ATL patients possess natural antibodies to HTLV as shown by solid-phase RIA (88%) and also specifically to HTLV p19 (77%). Similarly, 50% of Japanese patients with similar T-cell malignant lymphomas possess HTLV antibodies by solid-phase RIA and nearly as many (42%) possess anti-p19 reactivity. Twelve and eight percent, respectively, of normal Japanese donors from the ATL endemic region possessed HTLV-specific antibody by the solid-phase RIA or competitive binding assay. Normal donors from nonendemic areas lacked antibodies to HTLV. These results extend our previous findings of natural antibodies to HTLV in Japanese patients with ATL. The finding of p19-specific antibodies in these Japanese sera, together with previous reports of natural antibodies to HTLV p24 in sera from this same geographic cluster, strengthens the association of HTLV with Japanese ATL.