Gerhard Jahn

A very strong enhancer is located upstream of an immediate early gene of human cytomegalovirus

A strong transcription enhancer was identified in the genomic DNA (235 kb) of human cytomegalovirus (HCMV), a ubiquitous and severe pathogen of the herpesvirus group. Cotransfection of enhancerless SV40 DNA with randomly fragmented HCMV DNA yielded two SV40-HCMV recombinant viruses that had incorporated overlapping segments of HCMV DNA to substitute for the missing SV40 enhancer. Within HCMV, these enhancer sequences are located upstream of the transcription initiation site of the major immediate-early gene, between nucleotides -118 and -524. Deletion studies with the HCMV enhancer, which harbors a variety of repeated sequence motifs, show that different subsets of this enhancer can substitute for the SV40 enhancer. The HCMV enhancer, which seems to have little cell type or species preference, is severalfold more active than the SV40 enhancer. It is the strongest enhancer we have analyzed so far, a property that makes it a useful component of eukaryotic expression vectors.

Alpha-lipoic acid is an effective inhibitor of human immuno-deficiency virus (HIV-1) replication

SummaryAlpha-lipoic acid, a naturally occuring disulfide-compound that acts as a cellular coenzyme, inhibits replication of HIV-1 in cultured lymphoid T-cells. Alpha-lipoic acid was added 16 hours after infection of the T-cell lines Jurkat, SupT1 and Molt-4 with HTLV IIIB and HIV-1 Wai (a wild type HIV-1 isolate). We observed a dose dependent inhibition of HIV-1-replication in CPE (Cytopathic effect) formation, reverse transcriptase activity and plaque formation on CD4-transformed HeLa-cells. An over 90% reduction of reverse transcriptase activity could be achieved with 70 μg alpha-lipoic acid/ml, a complete reduction of plaque-forming units at concentrations of ≥35 μg alpha-lipoic acid/ml. An augmentation of the antiviral activity was seen by combination of zidovudine and low dose of alpha-lipoic acid (7 μg/ ml). Trypan blue staining revealed no toxic effects of alpha-lipoic acids on peripheral blood mononuclear cells and T-cell lines even in concentrations of ≥70 μg/ml. Therefore, we propose the inclusion of alpha-lipoic acid into chemotherapy trials in combination with zidovudine.

The DNA-binding protein P52 of human cytomegalovirus reacts with monoclonal antibody CCH2 and associates with the nuclear membrane at late times after infection

Monoclonal antibody CCH2 is commonly used for the detection of human cytomegalovirus (HCMV) infected cells in tissue sections as well as in cultured cells. The specificity of CCH2 was determined by screening a recombinant lambda-gt11 cDNA gene bank from HCMV-infected fibroblasts. By sequencing a reactive clone, the antigen was identified to be the non-structural DNA binding protein p52 of HCMV (UL44 reading frame). The viral insert from the lambda clone was recloned in bacterial expression vectors. For this, a new vector, pRos-RS, was constructed. The resulting clones were tested in immunoblot analyses. They were reactive with CCH2 as well as with reconvalescent sera positive for antibodies against HCMV, by this proving the specificity of CCH2. Using this monoclonal antibody in confocal microscopy, the subcellular localization of p52 in infected cells was analyzed. In these analyses, p52 was found to be nuclear and to be associated with the nuclear membrane at late times after infection.

Inhibition of HIV-1 replication by a high-copy-number vector expressing antisense RNA for reverse transcriptase.

We report the construction of a high-copy-number (hcn) expression vector for human cells. Amplification of this vector occurs due to the presence of an element derived from the murine DNA encoding ribosomal RNA (rDNA). HIV-1 replication in Jurkat T lymphocytes is nearly abolished when antisense RNA directed against the gene encoding reverse transcriptase is expressed from this hcn vector. The replication of the virus is only slightly reduced by the plasmid control version lacking the murine amplification-promoting element. This kind of hcn vector may represent an important improvement for the genetic engineering of eukaryotic cells and may also provide some ideas for the future gene therapy of some human diseases.

Early diagnosis and successful treatment of acute cytomegalovirus encephalitis in a renal transplant recipient

SummaryWe report the case of a 40-year-old male HIV-negative renal transplant patient with allograft rejection and immunosuppressive therapy who presented with acute cytomegalovirus (CMV) encephalitis. CT and MRI of the brain were normal but EEG showed diffuse slowing and dysrhythmia. In cerebrospinal fluid (CSF) initially 81 cells/μl were found and immunocytochemistry showed a decreased CD4/CD8 ratio and increased values of activated lymphocytes, natural killer cells and immunoglobulin-containing cells. CMV-specific IgM antibodies in CSF and serum, immunostaining of CMV antigen in CSF cells and virus culture from CSF and urine were negative. During the first 3 weeks of illness no intrathecal production of immunoglobulins could be detected. Early diagnosis of CMV encephalitis was made by in situ hybridization (ISH) on CSF cell preparations and the polymerase chain reaction (PCR) which was positive in CSF and blood. On day 26 diagnosis was confirmed by detection of CMV-specific intrathecal IgG production. The patient was treated with ganciclovir, anti-CMV immunoglobulins and intrathecal beta interferon. He recovered completely after 2 months. Our data demonstrate the usefulness of ISH and PCR in the early diagnosis of CMV encephalitis and perhaps may encourage the use of intrathecal beta interferon in other patients with this disease.

Generation and application of a monoclonal antibody raised against a recombinant cytomegalovirus-specific polypeptide

SummaryProcedures for diagnostics of cytomegalovirus infections include histopathology, cell culture, serology, and direct detection of viral antigens or nucleic acids within infected cells or tissues. In order to develop a new diagnostic reagent for viral antigen detection, we generated a mouse monoclonal antibody. This antibody was raised against a recombinant antigen representing part of the large phosphorylated structural protein pp150 of human cytomegalovirus. The monoclonal antibody was shown to be useful for antigen detection by immunofluorescence and immunoenzymatic staining in infected cells from cell culture as well as from infected organs. The antibody proved to be reactive even in paraffin-embedded sections from tissue specimens.

Generation and effective enrichment of selectable human cytomegalovirus mutants using site-directed insertion of the neo gene

Studies on the biology and function of human cytomegalovirus (HCMV) genes have been hampered by the limited number of viral mutants available for genetic analyses. We have developed a simple procedure to generate and enrich for HCMV recombinants. By inserting the bacterial neo gene, encoding neomycin/kanamycin phosphotransferase, into the large HCMV DNA genome using homologous recombination, selectable mutants of this complex herpesvirus were isolated for the first time. The synthesis of Neo from the viral genome was used to effectively enrich for recombinant viruses (re-viruses) in permissive culture cells grown in the presence of Geneticin (G418). A quick assay for Neo activity in infected cells, based on phosphorylation of kanamycin (Km), was used to easily identify viral recombinants in the process of screening and isolation. This procedure, not used previously to identify re-viruses, proved to be very useful for screening of large numbers of HCMV recombinants. Analysis of re-virus by Southern blotting revealed that the insertion of the marker gene had resulted in the expected deletion of the open reading frames, TRL 13/14 and UL 1-5, of HCMV. Re-virus was stable and showed no differences in growth kinetics as compared to wild-type (wt) virus. The insertion of a selectable marker gene into the HCMV genome and identification of viral recombinants by the Km phosphorylation assay, as presented here, provides the rationale for effective generation, enrichment and stable propagation of HCMV mutants.

Proteins of Human Cytomegalovirus that Elicit Humoral Immunity

Several of the cytomegalovirus (CMV) genes encoding glycoproteins, structural proteins, and infected-cell proteins that elicit an immune response in human infection have been mapped. Human sera and monoclonal antibodies react with these viral polypeptides made as native molecules in CMV-infected cells, as genetically engineered proteins, as truncated derivatives expressed in eukaryotic cells, and as bacterial fusion proteins from portions of the reading frames cloned into prokaryotic expression vectors. Synthetic oligopeptides from immunodominant regions of these molecules have also been used as antibody targets. Studies on proteins encoded by reading frames UL55, UL32, and UL44, on glycoprotein B, and on phosphoproteins pp150 and pp65 have been particularly fruitful. Recent developments employing these and other immunogenic CMV proteins as antigens for serodiagnosis of primary, recurrent, and past CMV infections are discussed.

Expression of human immunodeficiency virus (HIV) in naturally infected peripheral blood mononuclear cells: comparison of a standard co-culture technique with a newly developed microculture method

Peripheral blood mononuclear cells (PBMCs) from 29 patients infected with human immunodeficiency virus (HIV) were cultured by two different methods. One was the standard co-culture technique, the other a newly developed microculture method. In this assay 10(6) PBMCs were cultivated in 250 microliters medium, no activating agents or allogeneic cells were present. P24 antigen production measured by this method was found in 7 out of 11 PBMC cultures of patients in the Walter Reed (WR) stage 1 or 2, whereas only 4 samples were positive by the co-culture procedure. Cultures from patients in the later stages of the disease (WR 5/6) showed a higher p24 production by the co-culture method than by the microculture assay. It is assumed that rapidly growing HIV strains can be better assessed by the co-culture method which may select for these strains. P24 expression can be more easily obtained by the microculture technique even in cases where slowly replicating strains may be present. In conclusion, results from the microculture procedure described may be a useful supplementation to findings observed by the co-culture method.

Recombinant Antigens in Viral Diagnosis

DNA-fragments coding for a variety of different viral antigens have been cloned and expressed in E. coli. Selected purified recombinant antigens were used for detection of specific antibodies by the means of ELISA technique. This approach has been used for the development of four different ELISAs for the detection of HIV- and EBV-specific antibodies.