Structure of the human C7 gene and comparison with the C6, C8A, C8B, and C9 genes.

The seventh component of complement is a single chain plasma glycoprotein that is involved in the cytolytic phase of complement activation. We have determined the structure of the C7 gene, which is encoded by 18 exons whose sizes vary from 56 to 244 bp. For the most part, the exons do not correspond to the protein homology units. However, two intron/exon boundaries occur at junctions between different functional parts of the protein. The first is at a site between the end of the C9 homology unit and the carboxyl-terminal extension which is also a feature of C6. The second of these boundaries occurs between the regions encoding two pairs of cysteine-rich modules (the short consensus repeats and the factor I modules) located in the carboxyl-terminal part of C7. In contrast to the exons, the introns range considerably in size from 0.5 to 8.5 kbp. The complete analysis indicates that the gene encoding C7 is approximately 80 kbp in length. We show here that the C7 gene is highly homologous to that for C6, and also to C8A, C8B, and C9, confirming and extending the published data. With the exception of exon 1, all intron/exon boundaries are preserved with respect to phase when compared with C6.

Molecular basis of subtotal complement C6 deficiency. A carboxy-terminally truncated but functionally active C6.

Individuals with subtotal complement C6 deficiency possess a C6 molecule that is 14% shorter than normal C6 and present in low but detectable concentrations (1-2% of the normal mean). We now show that this dysmorphic C6 is bactericidally active and lacks an epitope that was mapped to the most carboxy-terminal part of C6 using C6 cDNA fragments expressed as fusion proteins in the pUEX expression system. We thus predicted that the abnormal C6 molecule might be carboxy-terminally truncated and sought a mutation in an area approximately 14% from the carboxy-terminal end of the coding sequence. By sequencing PCR-amplified products from this region, we found, in three individuals from two families, a mutation that might plausibly be responsible for the defect. All three have an abnormal 5' splice donor site of intron 15, which would probably prevent splicing. An in-frame stop codon is found 17 codons downstream from the intron boundary, which would lead to a truncated polypeptide 13.5% smaller than normal C6. This result was unexpected, as earlier studies mapped the C5b binding site, or a putative enzymatic region, to this part of C6. Interestingly, all three subjects were probably heterozygous for both subtotal C6 and complete C6 deficiency.

Structure of the human C6 gene.

The terminal components of the complement system (C6-C9) are related proteins, differing in size and complexity. They seem to be typical mosaic proteins, composed of modules which are homologous with parts of other proteins. Individual elements in a mosaic protein are often bounded by introns in the gene, and where they are duplicated within a polypeptide, partial gene duplication within the gene is responsible. It is often found in such genes that the intron/exon boundaries are of the class 1 type. We have examined the boundaries of 17 of the 18 exons of C6 and five of C7. When considered with published data for C9, only one of the protein elements appears to follow the conventional pattern. These data suggest a more complex evolutionary history for the genes of the terminal complement components than had been anticipated and challenge the notions both that discovery of a recognized protein module is of predictive value in relation to gene structure and that these genes evolved from the simple to the complex.

C6 epitope expression by an unrelated antisense cDNA clone: an inadvertent surface-simulation mimotope.

A cDNA clone which directs the expression of a fusion protein reacting with anti-C6 antibodies has been isolated and sequenced. A synthetic peptide corresponding to the 14 C-terminal residues of the expressed protein elicited the production of antibodies which are specific for native C6, confirming the presence of a C6 epitope on the expressed protein. However, analysis of the intron-exon boundaries of a corresponding genomic clone revealed that the expression clone is in antisense orientation, and is therefore not C6 cDNA. Comparison of the sequences of the expression clone and expressed protein with those for C6 have not demonstrated any significant sequence homology. It is therefore apparent that what has been cloned is a mimotope for C6 which includes in its continuous sequence an epitope that is conformational in C6 and not represented as a continuous sequence in the C6 molecule. Although this was not the purpose of the investigation, these results confirm that screening random expression libraries with antibodies may be an alternative to the synthetic peptide approach to obtain mimotopes reacting with particular antibodies.

Alpha interferon suppresses virion but not soluble human immunodeficiency virus antigen production in chronically infected T-lymphocytic cells.

Alpha interferon (IFN-alpha) is effective in preventing the release of human immunodeficiency virus (HIV) from chronically infected T-lymphocytic (ACH-2) and promonocytic (U1) cell lines stimulated with the phorbol ester phorbol-12-myristate-13 acetate (PMA). In the present study, we observed that together with particle production, shedding of HIV antigen (p24gag) occurs in the T-cell line ACH-2 both constitutively and after stimulation with PMA. IFN-alpha, although effective in suppressing the release of HIV particles, did not inhibit shedding of p24gag into the culture supernatants of either unstimulated or PMA-stimulated cells. These observations may be of relevance in the evaluation of the in vivo efficacy of IFN-alpha treatment of HIV-infected individuals as determined by levels of p24 antigen in plasma.

Frequency of indeterminate western blot tests in healthy adults at low risk for human immunodeficiency virus infection. The NIAID AIDS Vaccine Clinical Trails Network.

As part of a phase 1 trial of a candidate AIDS vaccine, blood specimens were collected from 168 healthy adult volunteers at minimal or no risk for becoming infected with human immunodeficiency virus type 1 (HIV-1). These specimens were screened for evidence of HIV-1 infection by enzyme immunoassay (EIA) and the Biotech/Du Pont Western blot (n = 168), culture (n = 122), and polymerase chain reaction assay (n = 20). None of the subjects had a positive test result by any of these assays, but 32% had indeterminate Western blot tests, most of which demonstrated a single band of low intensity. The most common bands were p24 (47%), p55 (34%), and p66 (36%); envelope bands were unusual (gp41, 2%; gp120, 2%). No serum specimen collected after 2-11 months from individuals with indeterminate Western blot results was positive by EIA or Western blot. There was 91% agreement in the test results of the first and second serum samples when the same lot of Western blot kit was used but only 36% agreement when different lots were used. The Biotech/Du Pont Western blot kit thus frequently yields indeterminate test results in the absence of HIV-1 infection, the reproducibility of which is subject to lot-to-lot variability.

Characterization of serum antibody responses to recombinant HIV-1 gp160 vaccine by enzyme immunoassay. NIAID AIDS Vaccine Clinical Trials Network.

An enzyme immunoassay (EIA) was developed to measure serum antibody responses of healthy adult volunteers vaccinated with 40 or 80 micrograms of human immunodeficiency virus type 1 (HIV-1) recombinant gp160 (rgp160) vaccine at 0, 1, 6, and 18 months. This assay, which used purified rgp160 as antigen, was compared with the Biotech/Du Pont HIV-1 Western blot and the Abbott HIV-1 EIA. Of 33 volunteers who received three doses of rgp160 vaccine, seroresponses were detected in 91% by rgp160 EIA, 97% by Western blot, and 30% by HIV-1 EIA. The level of IgG rgp160 EIA antibody (mainly IgG1) peaked after the third immunization; 64% of 33 vaccinees still had detectable antibody by 12 months. The fourth immunization induced anamnestic IgG EIA antibody in 23 of 24 vaccinees, with titers ranging from 1:200 to 1:25,600. Neutralizing antibody was not detected in postvaccination sera by microtiter syncytium formation inhibition assay. Additional testing of sera by EIA indicated that the immune response to the vaccine was directed toward epitopes on both gp120 and gp41. Seroresponses to the immunodominant epitopes on gp41 were infrequent and none were detected to the neutralization epitope in the V3 region of gp120. This highly sensitive EIA is useful for characterizing HIV-1-specific antibody responses induced by an HIV-1 gp160 subunit vaccine.

Viral antigen stimulation of the production of human monokines capable of regulating HIV1 expression.

We have previously described model systems for cytokine-induced regulation of chronically HIV-infected promonocyte and T cell clones. Using these systems, we have shown that monokines contained in supernatants from LPS-stimulated human monocyte/macrophages (MO) up-regulate HIV expression, reflected by an increase in reverse transcriptase activity, viral RNA levels, and expressed viral proteins. Current studies were designed to determine whether viral Ag can interact with MO and secondarily affect HIV1 expression by stimulating monokine production. We found that certain herpes-group viruses, including CMV and EBV, augment HIV1 expression by inducing monokine production, whereas others, such as HSV1, HSV2, varicella-zoster virus, and human herpes virus 6 were unable to function in this capacity. The HSV1 and HSV2 Ag which failed to stimulate monokine production did not interfere with MO stimulation by CMV Ag, suggesting that failure to induce HIV expression was not attributable to MO suppression. When nonherpes group viruses were tested, we found that human adenovirus, hepatitis B virus, and vaccinia virus all failed to stimulate the production of monokines capable of activating HIV in the chronically infected cell lines. In contrast, HIV1 can augment its own expression by inducing the secretion of monokines which up-regulate HIV expression in the infected cells. The viral Ag-induced MO supernatants capable of up-regulating HIV expression did so in a dose-dependent manner, whereas viral Ag alone produced no significant change. Monokine production mediated by viral Ag was not attributable to contaminating endotoxin. These studies provide a model to determine whether other opportunistic infections may induce the expression of HIV by indirect mechanisms, such as the stimulation of cytokine production.

Identification of epitopes on respiratory syncytial virus proteins by competitive binding immunoassay.

To characterize the interrelationship of monoclonal antibodies (MAbs) against respiratory syncytial virus (RSV) and their respective epitopes, we developed a competitive binding assay based on the biotin-avidin system and a tissue culture enzyme-linked immunosorbent assay. The competitive binding assay clearly distinguished between competing and noncompeting MAbs. Eight MAbs against the fusion protein (F protein) demonstrated two blocking patterns consistent with two antigenic sites. MAbs reacting at one site neutralized the virus, while those reacting at the other site did not. Eight MAbs against the large glycoprotein (G protein) demonstrated five blocking patterns consistent with three antigenic sites, one with three epitopes and the other two with one each. None of the MAbs against G protein neutralized the virus. The reaction pattern of the MAbs against three strains of RSV identified three additional epitopes on the F protein and no additional epitopes on the G protein. The epitopes on G protein showed the greatest antigenic diversity among the three strains. These results help us better understand the functional and antigenic structure of the two surface glycoproteins of RSV.

Analysis of the respiratory syncytial virus fusion protein using monoclonal and polyclonal antibodies.

At least four distinct epitopes are described on the respiratory syncytial virus fusion protein (VP70) using 13 monoclonal antibodies in solid-phase competitive binding studies. Two, and possibly three, fusion-inhibiting epitopes, one non-fusion-inhibiting neutralizing epitope, and one non-neutralizing epitope are described. All but the latter site demonstrated partial overlap, suggesting possible topographical proximity of these epitopes. Polyclonal rabbit sera to VP70, which neutralized virus but did not inhibit fusion of infected cells, blocked the binding of all fusion-inhibiting monoclonal antibodies to VP70 in the solid-phase assay but did not inhibit their effect in vitro. Western blot analysis of these monoclonal antibodies demonstrates that one fusion-inhibiting epitope is localized on the 48K fragment of VP70 and is resistant to denaturation by heat, 2-mercaptoethanol and SDS.

Kinetics of synthesis of respiratory syncytial virus glycoproteins.

The synthesis of the two respiratory syncytial (RS) virus glycoproteins (VP66 and VP84) was examined under standard conditions and after treatment with tunicamycin and monensin. The protein backbone for VP66, the fusion protein (F1,2) is cotranslationally glycosylated to form F0, which is cleaved to form F1,2 by 20 min of chase. Monensin treatment inhibited the cleavage of F0 over an 80 min chase period, indicating that this occurred late in the transit of F0 through the Golgi apparatus or after exit from the Golgi apparatus. Tunicamycin treatment resulted in the synthesis of a 50K to 55K unglycosylated F0 which is cleaved to a 40K protein. VP84, the large glycoprotein, contains a protein backbone of only 26K to 30K which is modified by N-linked and probable O-linked glycosylation. Tunicamycin treatment results in the synthesis of a 70K protein (p70) which incorporates [3H]glucosamine and [3H]fucose but not [3H]mannose. Glycosylated precursors varying in mol. wt. from 29K to 45K (p45) are found in infected cells at regular 2K to 3K intervals, producing a 'ladder' effect. The step from p45 to VP84 is severely delayed by monensin treatment thereby enhancing the 'ladder' effect of the precursors.

Quantification of respiratory syncytial virus polypeptides in nasal secretions by monoclonal antibodies.

An indirect enzyme-linked immunosorbent assay (ELISA) which uses monoclonal antibody as solid-phase immunosorbent was developed to measure specific polypeptides of respiratory syncytial virus (RSV). The assay was used to examine 43 nasopharyngeal (NP) aspirates from RSV-positive infants that had been examined previously for RSV by culture, direct immunofluorescence, and polyclonal antibody ELISA. Frozen NP aspirates were serially diluted and examined for the 66K mol. wt. fusion glycoprotein (F), the 84K large surface glycoprotein (G) and the 41K nucleoprotein (N) by monoclonal capture ELISA. F protein was detected in all 43 specimens, G protein was detectable in 20 (47%) and N protein in 22 (51%) of 43 NP aspirates. In specimens with detectable G and N proteins, F was detected by endpoint titration at approximately tenfold greater dilutions than either G or N. In 19 sequential NP aspirates from five patients with RSV infection, F was present in higher titre throughout infection. In 20 cases, matching cell culture isolates were examined by immunofluorescence with strain-specific monoclonal antibodies. Three of 20 isolates showed strain-specific differences by their lack of reaction with anti-G monoclonal antibody. Titration of the 20 cell culture isolates by monoclonal antibody capture ELISA showed the relative amount of F and N proteins to be equal in all cases, whereas levels of G protein tended to be slightly lower. Reconstruction experiments with NP aspirates demonstrated that degradation of F and N proteins did not occur in NP aspirates, but that G protein antigenicity appeared to be affected by nasal secretions. When compared with cell culture-grown material, nasal secretions contained abundant F protein but a surprisingly low concentration of N protein.

Antigenic characterization of respiratory syncytial virus strains with monoclonal antibodies.

To study the antigenic characteristics of respiratory syncytial virus (RSV), we developed and evaluated monoclonal antibodies (MAbs) to three strains of RSV: 11 to Long, 4 to 18537, and 9 to A2. Six of these MAbs immunoprecipitated the nucleoprotein, six the large glycoprotein, and 11 the fusion protein. By the pattern of the reactions of these MAbs to 16 strains of RSV in an indirect immunofluorescence assay or enzyme-linked immunosorbent assay, we were able to distinguish three subgroups. With a panel of 10 of these 24 MAbs, we tested 26 strains isolated between 1979 and 1982 in Boston and found that 22 belonged to group 1, 4 to group 2, and none to group 3. The pattern of the reactions of the MAbs against representative strains from the three groups identified nine epitopes by indirect immunofluorescence assay: three of each on the nucleoprotein, the large glycoprotein, and the fusion protein. These results, along with those of previous studies, suggest that groups 1 and 3 are antigenically similar and group 2 is antigenically more distinct.

A viral antigen-bearing cell line derived from culture of Paget's bone cells.

This study documents the characteristics of a viral antigen-bearing cell line derived from co-culture of the bone from a 71-year-old woman with Paget's disease of bone and HEp-2 cells. The cell line has survived in continuous culture for 3 1/2 years and 185 subcultures. The cells are epithelioid in appearance, produce alkaline and acid phosphatase, increase alkaline phosphatase activity in response to 1,25-(OH)2-D3 and contain receptors for 1,25-(OH)2-D3. Immunofluorescent studies utilizing antisera to respiratory syncytial virus and measles virus reveal antigens of both viruses in the cells. These cells do not produce bone in culture and the adenylate cyclase activity found in their plasma membrane does not increase significantly in response to parathyroid hormone or calcitonin. The cells are not contact inhibited and form spherical colonies in agarose. They are aneuploid and have a modal number of 62-74 as well as HeLa markers. When injected into athymic mice, osteosarcomas are produced. These tumors continue to bear viral antigens. The availability of this cell line should aid in further studies of the viral antigens associated with Paget's disease of bone.

Immunochemical identification of viral and nonviral proteins of the respiratory syncytial virus virion.

We identified by immunochemical methods 13 polypeptides associated with the infectious respiratory syncytial virus virion. Eight of these polypeptides (VP200, VP84, VP66, VP43, VP40, VP37, VP28, and VP19) were identified as virus specific. Two other polypeptides, (VP) 22 and (VP) 12, are provisionally considered to be of viral origin. Three nonviral proteins are also intimately associated with the infectious virion. These nonviral proteins were identified as cellular actin and two proteins with bovine serum albumin immunospecificity. VP40 was identified as the major ribonucleoprotein. Based on biochemical and biophysical similarities with paramyxovirus proteins, other respiratory syncytial virus proteins are believed to have these specific viral functions: VP84, "hemagglutinin"; VP66, undissociated fusion protein, F1,2; VP43, F1; and VP19, F2, VP66 contains a major determinant involved in viral infectivity since all neutralizing antibodies tested, including a monoclone, precipitated this protein.

Effect of interferon on mouse leukaemia virus (MuLV). V. Abnormal proteins in virions of Rauscher MuLV produced in the presence of interferon.

Interferon treatment of JLSV-6 cells chronically infected with Rauscher MuLV leads to the formation of non-infectious particles ('interferon' virions) containing the structural proteins coded by the env and gag genes as well as additional virus polypeptides. The major glycoprotein detected in the control virions is gp71, but 'interferon' virions contain in addition an 85K mol. wt. (gp85) glucosamine-containing, fucose-deficient glycoprotein. This is recognized by antiserum to MuLV and may be related to env pr85. Surface iodination of intact virions indicates that gp71 and gp85 are the two major components of the external envelope. However, whereas in control virions gp71 associates with p15E (gp90), this complex was not detected in 'interferon' virions. Analysis of radio-labelled (3H-amino acids or iodinated) proteins from disrupted 'interferon' virions revealed the presence of 65K, 55K, 40K, 20K and 12K mol. wt. polypeptides which could be precipitated with antiserum against MuLV. There was a distinct difference in the patterns of incorporation of pulse-labelled 3H-amino acid polypeptides into virions in the presence and absence of interferon. Those polypeptides labelled in the presence of interferon and recovered in the extracellular virions in a chase with interferon appeared to have substantially fewer copies of p30 and more of gag pr55 polypeptide than the controls. These results indicate that in the presence of interferon there are changes in the proteolytic cleavage associated with virion assembly.

Effect of interferon on murine leukaemia virus infection. IV. Formation of non-infectious virus in chronically infected cells.

Interferon (150 units/ml) was used to treat SC-1 and AKR-2B cells which were chronically infected with murine leukaemia virus (MuLV). This led to a 100-fold decrease in the amount of infectious virus released into the medium and a 10-fold decrease in the number of virus particles measured by the virion-associated reverse transcriptase assay. However, there was little change in the amount of cell-associated infectious virus, though nearly twice as many cell-associated virions were counted in electron micrographs. With both types of cells, interferon blocked MuLV replication at the post-budding stage, but it did not change the morphology of the particles produced or their content of virion 70S RNA. Infectious virus assembled on the cell membranes of interferon-treated cells was less stable at 37 degrees C than that grown in the absence of interferon. Release of infectious virus from interferon-treated cells was not inhibited by actinomycin D or cycloheximide, though both agents inhibited virus production in controls. These results show that interferon inhibits MuLV replication through effects on virion assembly; these lead both to the formation of non-infectious particles and of fewer virions. Kinetic analysis further shows that interferon affects MuLV assembly rapidly and induction of an antiviral protein may not be required.

The errors in assembly of MuLV in interferon treated cells.

Interferon treatment of JLSV-6 cells chronically infected with Rauscher MuLV leads to the formation of noninfectious particles (interferon virions) containing the structural proteins of env and gag genes as well as additional viral polypeptides. In the control virions the major glycoprotein detected is gp71, interferon virions contain in addition to gp71 and 85k dalton (gp85) glucosamine-containing, fucose-deficient glycoprotein which is recognized by antiserum to MuLV but not by the gp71 antiserum. The surface iodination of the intact virions indicates that both gp71 and gp85 are the major components of the external virions envelope. However, unlike the control virions in which gp71 associates with p15E (gp90), the gp71-p15E complex was not detected in interferon virions. The analysis of the iodinated proteins of the disrupted interferon virions revealed the presence of 85k and 65k dalton polypeptides preciptable with antiserum against MuLV, which are not present in the control virions. The difference in the polypeptide pattern of virions produced in the presence of interferon does not seem to be a consequence of the slowdown in the synthesis of viral proteins or their processing in the interferon-treated cells. Both the structural proteins of env and gag genes seem to be synthesized and processed at a comparable rate in the interferon-treated and -untreated cells. These results indicate an alteration of virus assembly in the presence of interferon.