Biomedical subjects
R Trauger
Publications and source records attributed to R Trauger.
Association of ERK2 mitogen-activated protein kinase with human immunodeficiency virus particles.
Here we report the presence of a protein kinase activity associated with human immunodeficiency virus type 1 (HIV-1) particles. We observed phosphorylation of five major proteins by the endogenous protein kinase activity. Phosphoamino acid analysis revealed phosphorylated serine and threonine residues. In addition, we observed autophosphorylation of two proteins in the presence of gamma-ATP in an in-gel phosphorylation assay. These two proteins are not linked by a disulfide bond, suggesting that two different protein kinases are associated with HIV-1 virions. Our results indicate the presence of ERK2 mitogen-activated protein kinase and of a 53,000-molecular-weight protein kinase associated with virions. Moreover, the use of different HIV strains derived from T cells and promonocytic cells, as well as the use of human T-cell leukemia virus type 1 particles, demonstrates that ERK2 is strongly associated with retrovirus particles in a cell-independent manner. Exogenous substrates, such as histone proteins, and a viral substrate, such as Gag protein, are phosphorylated by virus-associated protein kinases.
Inactivated HIV-1 Immunogen: impact on markers of disease progression.
The pursuit of valid markers of disease progression in human immunodeficiency virus type 1 (HIV-1) infection is especially relevant considering the potential treatment alternatives that presently are under evaluation. Because HIV-1 infection results in a virally induced immune suppression characterized by the loss of cell-mediated immunity (CMI), depletion of CD4+ cells, loss of core antibody, and an increase in viral burden, these markers seemed to be appropriate to monitor in a controlled study. We monitored a number of virologic, immunologic, and cytologic markers of disease progression in 103 subjects who were enrolled in a 12-month, double-blind, randomized, adjuvant-controlled study of the HIV-1 inactivated Immunogen. The markers included HIV-1 DNA, HIV-1 RNA, CD4 percent, p24 antibody, and lymphocyte proliferation. Analysis of HIV-1 DNA with a quantitatively polymerase chain reaction (PCR) assay indicated a treatment effect on viral burden in the HIV-1 Immunogen-treated group. Analysis of HIV-1 RNA revealed a similar trend favoring the Immunogen-treated group. In addition, a significant effect was shown on CD4 percent and CMI in the Immunogen-treated group. An analysis of CMI that used stimulation indices underrepresented the immunogenicity of the Immunogen. Further examination revealed that the lymphocytes of the HIV-1 Immunogen-treated patients were proliferating in vitro without exogenous antigen. Although the clinical significance of this phenomenon currently is unknown, it may be a relevant prognostic marker for assessment of HIV-1 therapy. The data presented here support the concept that immunotherapy with the HIV-1 Immunogen may slow disease progression.(ABSTRACT TRUNCATED AT 250 WORDS)
Reduction-modifiable properties of Moloney murine leukemia virus gp70 as an indicator of envelope glycoprotein heterogeneity.
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.
Characterization of a conformationally sensitive epitope on Moloney murine leukemia virus gp70.
We describe here several properties of a conformationally sensitive epitope common to the Moloney (M) and Rauscher (R) murine leukemia virus (MLV) gp70 family of glycoproteins, e.g., M-MLV gp70 and R-MLV gp71. This epitope was not detected by western blotting or by enzyme-linked immunosorbent assay experiments with three different lots of polyclonal R-MLV gp69/71 sera. However, it was detected when a specific monoclonal antibody, R47, was used in western blotting or immuno-dot-blotting experiments with the two viruses. R47 maps to a central 14-kd segment on R-MLV gp71 which spans an important structural domain, namely, that corresponding to the recombination site between ecotropic and endogenous envelope glycoprotein-coding sequences. Analysis of the western as well as dot blots developed with the R47 monoclonal antibody showed that about a 25-fold higher affinity for the epitope existed on R-MLV gp71, relative to M-MLV gp70. It thus appears that R-MLV and M-MLV gp70, although very closely related both structurally and serologically, are conformationally distinct in one domain, namely, the site of recombination between ecotropic and endogenous gp70-coding sequences.
Characterization of highly purified, inactivated HIV-1 particles isolated by anion exchange chromatography.
This report characterizes inactivated, gp120 depleted, HIV-1 particles purified by an anion exchange chromatography production process. This antigen formulated with incomplete Freund's adjuvant constitutes Remune, which is being evaluated in a phase III clinical endpoint trial to determine the effect of this immune-based therapy on clinical progression of HIV-1 seropositive patients. Multiple production lots of the inactivated HIV-1 antigen strain HZ321, isolated by anion exchange chromatography, exhibit purity of > 95% by gel filtration. These findings are corroborated by thin section electron microscopy showing a homogenous field of intact particles. Analyses of the purified virus particles for protein, lipid, carbohydrate and RNA show structural retention of the envelope proteins, lipid bilayer and core components after large scale processing. The qualitative identification of at least 85% of total HIV-1 protein is determined by ELISA, Western blot, HPLC and amino acid sequencing analyses. Quantitative values are assigned to 50% of these proteins. The data confirm the presence of virally encoded proteins p6, p7, pI15, p17, p24, p32, pI39Gag, gp41, pp55Gag, p66/51, Vpr, Vif and Nef. Excellent consistency between production lots and equivalency to HIV-1 preparations purified by sucrose density gradient sedimentation has been established for protein and lipid composition, and overall purity. These findings further establish that non-viral encoded proteins and lipids are integral structural components of the intact virion and are not contaminants unique to a particular isolation method. The data confirm the presence of multicomponent antigens in the viral particles for stimulating a broad HIV-1 specific immune response. Finally, the work demonstrates that the two inactivation procedures (beta-propiolactone and gamma irradiation), which achieve efficient viral inactivation meeting US FDA guidelines, do not damage the protein antigens of the viral particles.
Safety and immunogenicity of REMUNE in HIV-infected Thai subjects.
The safety and immunogenicity of REMUNE, an HIV-specific immune based therapy for HIV infection, was evaluated in a cohort of 30 HIV infected subjects in Thailand. This therapy utilizes a gp120 depleted inactivated virus (HZ321), which exhibits a high degree of conservation with the core antigens of both type B' and E strains of HIV, the predominant Thailand isolates. The treatment was well tolerated, with no serious adverse events reported over the course of the 4-month trial. Treatment in which four doses were administered with REMUNE appeared to boost HIV-specific immune responses, with approximately 75% of the treated subjects demonstrating an increase in either the repertoire or the intensity of the serological response to HIV as measured by Western blot. CD4%, viral load, and weight remained stable over the course of the 4-month study relative to baseline values. Viral subtyping of this cohort revealed a predominance of type 'E'. These data suggest that REMUNE is safe and immunogenic in seropositive Thai subjects and supports further study of the therapeutic potential of REMUNE to treat HIV-1 infection.