Biomedical subjects
Publications and source records attributed to R B Luftig.
A surprisingly large number of animal viruses interact with cytoskeletal elements inside infected cells at different stages of replication. For example, a viral protease is activated during assembly of murine leukemia viruses at the cell surface, which causes both the morphological conversion of 'immature' to 'mature' particles and a drastic reduction of the actin stress fiber network.
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We have characterized the structure and infectivity of an avian retrovirus, myeloblastosis associated virus (MAV), containing a genetically altered proteinase (PR). A site-directed mutant of MAV-PR that shows an increased proteolytic activity in vitro (about 20 times higher kcat/Km) as a consequence of substituting five amino acids from the substrate-binding pocket with those corresponding to the HIV-1 PR was cloned into a full-sized MAV plasmid. In particular, the wild-type MAV-PR gene was replaced with the mutant one. Despite encoding for an enzyme with increased PR activity, mutant plasmid-transfected turkey fibroblasts displayed an unimpaired virus production in cell cultures. Further, the mutant progeny virus was infectious and its pattern of gag processing products appeared identical to that of wild-type virus. However, by electron microscopy we found that the predominant morphology of mutant viral particles was altered. Instead of a centrally collapsed avian retroviral core, a more diffuse core was visualized for wild-type mutant virions, similar to that observed in mammalian C-type retroviruses.
Monoclonal antibodies (mAbs) against HIV-1 protease (HIV PR), the essential protease of human immunodeficiency virus type 1 (HIV-1), were produced in Armenian hamsters. Studies of direct binding to synthetic peptides and inhibition of binding to intact protease by peptide competition showed that five mAbs recognized an epitope that includes the sequence LPGRWKPK (residues 38-45), which lies near the region of the protease called the flap. All of the mAbs react specifically with HIV PR in Western blots. Because of structural conservation of the epitope in the proteases of many HIV-1 isolates, mAbs to this epitope are likely to be useful for detection of HIV PR in field isolates of HIV-1. Also, mAbs specific for this epitope, which lies close to the flap of HIV PR, may be useful for functional studies of HIV PR and possibly for the design of inhibitors of protease activity that bind outside the enzyme's catalytic site.
HIV-1 replication requires limited proteolysis of gag and gag-pol encoded precursor proteins by a specific viral proteinase (PR). Sequences of 20 different HIV-1 strains were compared in order to determine regions of conservation and variability within the PR gene. Viral strains included: (a) five new ones derived from New Orleans patient isolates, (b) four established ones grown in our laboratory, (c) eight, whose sequences were published in the Los Alamos Data Base (1990), (d) one Ugandan, and (e) two Brazilian isolates. In the first two groups, HIV proviral DNA extracted from infected lymphocytes was grown in tissue culture and directly amplified by PCR using specific primers flanking the PR gene. Amplified DNA was directly sequenced using a modified di-deoxy sequencing procedure. Sequence data showed a 25% variation among the 20 different HIV strains studied at the amino acid level, including 8% nonconservative changes and 17% conservative changes. Moreover, five noncontiguous regions were able to be delineated in which the PR showed no amino acid changes. These areas included amino acids (I) 1-9 (amino terminal sequence); (II) 21-32 (sequence around the active site); (III) 47-56 (top of the flap); (IV) 78-88; and (V) 94-99 (carboxy terminal sequence). Our results are consistent with those obtained from X-ray crystallography studies as well as single site mutational analysis.
Although maximal synthesis of HSP70 is induced early (6-12 hr) after adenovirus type 5 (Ad5) infection of HEp-2 or HeLa cells, the total amount of HSP70 appears to be increased at late times of infection (18-24 hr). Since virion structural proteins also accumulate at these times, we investigated the possible interaction between Ad5 structural proteins and HSP70 by immunoprecipitation of infected cell extracts with antibodies to either ATP-affinity-purified HSP70 or to CsCl-gradient-purified Ad5 virions. We found that HSP70 and a 62-kDa Ad-specific protein coimmunoprecipitated from infected cell extracts. Antibody which recognizes one of these two proteins does not cross-react with the other. Thus, the association between HSP70 and the 62-kDa protein appears specific. Using different antisera to specific adenovirus structural proteins, we have identified the 62-kDa protein as the Ad5 fiber protein.
Properties of human enteric adenovirus type 41 (Ad41) binding to its receptor on the surface of HEp-2 cells were investigated. The binding was found to be temperature-dependent, saturable, and specific. Analysis of the binding data showed a single class of 4.3 x 10(4) receptor sites per cell, an equilibrium dissociation constant of 21.0 nM, and no cooperativity among receptor sites. Trypsin-treated HEp-2 cells subsequently grown in the presence of tunicamycin or 2-deoxyglucose recovered full Ad41 binding activity, but could not if subsequently grown in the presence of cycloheximide. These data indicate that a single type of virus receptor, likely protein in nature, is present on the surface of HEp-2 cells to specifically bind Ad41.
Human cells (HEp-2) infected with adenovirus type 5 (Ad5) at early times (5-7 h) after infection exhibit stabilization of the filamentous actin network against disruption by latrunculin (300-2000 ng), a potent microfilament toxin. This protection is abrogated by pretreatment of infected cells with cycloheximide, suggesting that it is due to a protein induced early after Ad5 infection. Support for a role of HSP70 (heat shock protein of Mr = 70 kDa) in actin stabilization is based on several findings; (i) HSP70 is induced at early times post-infection in Ad5-infected HEp-2 cells, (ii) heat shock treatment (42 degrees C) of uninfected HEp-2 or HeLa cells results in a rearrangement of actin filaments around the nucleus, that is resistant to disruption by latrunculin, (iii) using a DNase I inhibition assay, the percentage of filamentous actin increases from 50 to 65% of total following heat shock of uninfected cells, and (iv) HSP70 induces actin polymerization from monomers in vitro.
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Differing reports exist about the replication of human enteric adenoviruses (EnAds) in various cell lines. There was a suggestion that EnAds are defective, do not grow on primary human diploid cells, and behave like Ad host-range mutants, i.e., they require early gene products from other Ad types for efficient growth. Thus, initially the Graham-293 cell line, which contains the E1 region (E1A, E1B) of Ad5, was thought to be an ideal host for EnAds, because it provided the needed functions. Our findings, however, question this contention and show that Ad41 strain TAK, cultured on 293 cells, rapidly loses its infectivity (greater than 90% on the first and 100% by the second passage). In contrast to the results with 293 cells, we found that Ad41 strain TAK can be serially grown to high titers on several continuous cell lines: namely, HeLa, HI407, or HEp-2 cells. In order to investigate the basis for the rapid loss of the Ad41 infectivity upon passaging in 293 cells, Ad41 virions were purified from 293, as well as from HEp-2 cells, and their composition was analyzed. When structural proteins of the complete virions (rho = 1.34 g/cm3) were compared by Western blot analysis using polyclonal antibodies to HEp-2-grown virus, only traces of protein V (Mr 46,000 Da) could be detected in particles from 293 cells. In contrast, Ad41 particles obtained from HEp-2 cells exhibited a strong band at the position of protein V. Further, if polyclonal antibodies to 293-grown Ad41 were used in the Western blot, no protein V band was detected in HEp-2-grown virus. Finally, we note that new protein bands (Mr 25,000-35,000 Da) could be observed upon Western blot analysis of 293-derived complete and incomplete Ad41 particles. All of these observations taken together suggest that the low infectivity of Ad41 particles, prepared from 293 cells, could be due to a defect in assembly.
It had been postulated that due to lack of growth of enteric adenovirus 41 (Ad41) on human primary cells and its growth on Graham-293 cells there was a defect in the Ad41 E1A region. However, we found as a result of careful evaluation of Ad41 growth on several primary cell lines (HEK, WI-38, or Detroit 551) that efficient viral multiplication is possible if the serum concentration in the medium used postinfection (p.i.) is kept between 0.2 and 1%. In contrast, only slight growth of Ad41 occurs in infected cells maintained in 5% serum and virtually no viral replication is found in infected cells cultivated in medium with 10% serum. The serum inhibitory effect appears limited to primary cells because no difference in Ad41 replication, as assayed by accumulation of Ad41 DNA, was found in infected continuous cell lines (HEp-2, 293) cultivated p.i. in either 1 or 10% FBS. Also, this effect appears specific for enteric adenoviruses, such as Ad41, since conventional adenoviruses, such as Ad5, grow well in both 1 and 10% FBS. The above results show that Ad41 can grow in a variety of primary cell lines, under specific culture conditions. In addition, we found that Ad41-infected primary cells grown in medium containing 0.2% serum had an increase in synthesis of the 70-kDa heat shock protein (HSP70) at about 6 hr p.i. and also Ad41 was able to complement the Ad5 E1A deletion mutant dl312. These results show that the E1A function of Ad41 is not impaired in infected cells.
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HIV protease is a virally coded enzyme that cleaves gag as well as gag-pol precursor polyproteins into functional products needed for virus assembly. A pUC plasmid containing an HIV insert starting at the 5' end of the pol gene and ending just inside the intergrase coding sequence was expressed in E. coli. It provided an 11 kD gene product (protease) that specifically cleaved the Gazdar MuLV Pr65gag precursor into Pr40gag (p30 + p10) and Pr27gag (p15 + p12) intermediates, as well as lower molecular weight gag-encoded products. These were detected by immunoblotting with either MuLV anti-p30 or p12 sera. Using cleavage of MuLV Pr65gag as an assay system, pepstatin A, fusidic acid, and cerulenin were observed to inhibit HIV protease cleavage by greater than 50% at concentrations of 0.1, 0.2-0.5, and 0.5 mM, respectively.
We have found that preparations of rate-zonal purified Moloney murine leukemia virus originally obtained from the National Cancer Institute Resources Program, when separated by SDS-PAGE in the absence of mercaptoethanol (beta-MSH), exhibited a doublet envelope glycoprotein band of approximately 69/67 kD. When the same samples were run in the presence of beta-MSH, a single band at 70 kD (gp70) was observed. Western blot analysis with polyclonal antiserum identified both the 69- and 67-kD bands as envelope gene products. Tryptic peptide mapping of each of the gp67 and gp69 bands confirmed the serological data, with each showing conserved as well as unique peptides. These results imply that the Moloney murine leukemia virus samples examined above contain two structurally different envelope gene products. Western blot analysis using ecotropic and dualtropic specific sera suggest that gp69 is derived from an ecotropic virus, while gp67 is from a dualtropic virus. This is consistent with the results of an earlier study which showed that the majority of the cysteines (4/5) in dualtropic gp70 are lost by a single deletion relative to the ecotropic gp70 species. This would account for the difference in mobility observed in the SDS-PAGE profile in the absence of beta-MSH. It would indicate that the cysteines play an important role in defining structural differences that separate the ecotropic and dualtropic gp70s.
We detected phosphorylation of the major Moloney murine leukemia virus (M-MuLV) capsid polypeptide, p30, by using 32Pi-labeled virions. This was observed both on two-dimensional polyacrylamide gels directly or on one-dimensional gels of viral lysates that had been immunoprecipitated with monospecific goat anti-p30 serum. The phosphorylation event had been difficult to detect because pp12 the major virion phosphoprotein incorporates almost all of the 32P label added to infected cells (Y. Yoshinaka and R. B. Luftig, Virology 116:181-195, 1982). When immunoprecipitates from M-MuLV lysates labeled with 32Pi were compared with those labeled with [35S]methionine, it was calculated that the degree of phosphorylation at the p30 domain of Pr65gag was only 0.22 to 0.54% relative to phosphorylation at the p12 domain. Two-dimensional gel electrophoresis of the 32P-labeled p30 immunoprecipitates showed that there were three phosphorylated p30 forms with isoelectric points (pIs) of 5.7, 5.8, and 6.0. These forms were generally more acidic than the [35S]methionine-labeled p30 forms, which had pIs of 6.0, 6.1, 6.3 (the major constituent with greater than 80% of the label), and 6.6. The predominant phosphoamino acid of the major phosphorylated p30 form (pI 5.8) was phosphoserine. Further, tryptic peptide analysis of this p30 form showed that only one peptide was predominantly phosphorylated. Based on a comparison of specific labeling of p30 tryptic peptides with [14C]serine, [35S]methionine, and 32Pi, we tentatively assigned the phosphorylation site to a 2.4-kilodalton NH2-terminal peptide containing triple tandem serines spanning the region from amino acids 4 to 24.