Vpu as a human‐immunodeficiency‐virus‐type‐1‐encoded 81‐amino‐acid integral‐membrane protein was expressed in Escherichia coli using the inducible p trc promoter of an ATG fusion vector. Recombinant Vpu is associated with membranes of E. coli and could be partially solubilized by detergents. Recombinant Vpu was phosphorylated in vitro with purified porcine casein kinase II (CKII) as well as with a CKII‐related protein kinase found in cytoplasmic extracts of human and hamster cells. Recombinant Vpu associated with E. coli membranes has turned out to be the best substrate for in vitro phosphorylation with CKII. This reaction can be inhibited by heparin and the ATP analogue 5,6‐dichloro‐1‐(β‐D‐ribofuranosyl)benzimidazole (DRB), both known to be potent inhibitors of CKII. Radiolabelled γATP and γGTP were used as phosphate donors for in vitro phosphorylation of recombinant Vpu. In vivo phosphorylation of Vpu‐1‐infected H9 cells was also inhibited by DRB. We concluded therefrom that the Vpu protein is phosphorylated by the ubiquitous CKII in HIV‐1‐infected human host cells. Two seryl residues in the sequence of Vpu (position 52 and 56) correspond to the consensus s / T XX D / E for CKII. These potential phosphorylation sites are located within a well‐conserved dodecapeptide of Vpu (residues 47–58), which is found in different HIV‐1 strains as well as in a Vpu‐like protein of SIV CPZ. Monoclonal and polyclonal antibodies directed against two different epitopes of Vpu were used for immunoprecipitation of Vpu from HIV‐1‐infected cells and for detection of Vpu in Western blot analyses. Vpu from HIV‐1‐infected cells as well as recombinant Vpu expressed in E. coli were determined by SDS/PAGE using 6 M urea to be 9 kDa, which corresponds to the calculated molecular mass of Vpu.
Monoclonal antibodies (mabs) against Mycoplasma (M.) bovis were prepared for use in diagnosis of bovine mastitis. From the original 32 hybridomas actively secreting mabs against M. bovis, 6 stable lines were cloned. Two of them, Mb 5D8 and Mb 4F6, recognized M. bovis antigens of estimated molecular weights of 33 and 26 kDa, respectively. They showed no cross-reaction to other bovine mycoplasmas, thus rendering them useful for specific detection of this pathogen. All mabs investigated cross-reacted with M. agalactiae which is known to be closely related to M. bovis, but does not occur in cattle. Two other mabs, Mb 5D4 and Mb 1F6, exhibited further cross-reactions to a number of bovine mycoplasma species. Finally, mabs Mb 5D5 and Mb 2G5 reacted with all mycoplasmas tested. The possibility that they recognized constituents of the broth culture medium is discussed.
Mycoplasma bovis, the main causative agent of mycoplasmal mastitis, arthritis and pneumonia in cattle, causes considerable economic losses. Veterinary hygiene measures would be most effective if introduced at an early stage, especially the culling of cows shedding the pathogen for the control of mastitis. It is therefore crucial to ensure that diagnostic methods are available which can perform rapid and specific detection of the agent at acceptable costs. Six different detection methods have been compared and evaluated in terms of performance parameters and suitability for routine diagnosis. Conventional M. bovis isolation and identification from culture is the only technique used for routine diagnosis at present. However, this process is rather laborious and time-consuming, and final results are available only after several days. Enzyme-linked immunosorbent assay (ELISA) techniques can be used to screen for M. bovis antibodies or antigens in clinically-diseased animals. Detection of the agent in subclinical cases was accomplished in pre-incubated samples by an antigen capture ELISA involving a monoclonal antibody. Whole-cell protein patterns generated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis were used to identify and classify field isolates. Nucleic acid hybridizations using probes of defined specificity were conducted both as filter dot blot assay and in solution with ribosomal ribonucleic acid as the target. The latter was found to be potentially suitable for the screening of biological samples, although problems due to high background and reduced specificity remained. Finally, the presence of M. bovis cells in culture supernatant and in milk samples was demonstrated using the polymerase chain reaction. This procedure is potentially superior to all others currently available, due to its high sensitivity, specificity and speed. However, a number of practical problems must be solved prior to full-scale introduction of this technique for routine diagnosis.
Two distinct intranuclear locations were identified for alternatively spliced RNA transcripts expressed from the pNL4-3 infectious molecular clone of human immunodeficiency virus (HIV) type 1. Multiply spliced HIV RNA encoding tat was detected within the nucleus in large clusters; immunostaining and colocalization studies using laser-scanning confocal microscopy revealed that these structures contained the non-small nuclear ribonucleoprotein RNA processing factor, SC35. In contrast, unspliced gag RNA was detected in much smaller granules distributed throughout the nucleus, with little or no association with SC35-containing granules. Analyses of nuclear RNA expressed from recombinant plasmids encoding gag (pCMVgag-2) alone or tat (pCMVtat-2) alone revealed distributions corresponding to those obtained with pNL4-3, indicating that expression within the context of the HIV provirus was not required for the distinct RNA locations detected for these transcripts. The presence of unspliced gag RNA in small granules was confirmed in infections of H9 T-lymphocytic cells, indicating that gag localization was not restricted to transient expression systems. The intranuclear distribution of gag RNA was dependent on specific RNA sequences. Deletion of a portion of the gag gene of pCMVgag-2, containing a cis-repressing inhibitory region, resulted in redirection of unspliced gag RNA from small granules into large SC35-containing clusters. The addition of the Rev-responsive element, RRE, to the deleted pCMVgag-2 construct resulted in RNA transcripts which were no longer associated with SC35. We also identified a cellular intron, rabbit beta-globin-intervening sequence 2 (IVS-2) which, when introduced into pCMVgag-2, redirected unspliced gag RNA into SC35-containing granules and permitted rev-independent Gag expression. These findings suggest that redirecting intranuclear RNA localization may influence gene expression. Color micrographs from this article are available for view at http//128.231.216.2/lmmhome.htm.
The kinetics of human immunodeficiency virus type 1 (HIV-1)-induced cell death were investigated in cell-to-cell and cell-free models of virus transmission. Cocultivation of HIV-1 chronically infected H9 donor cells with uninfected H9 recipient cells resulted in rapid induction of programmed cell death. Within 8 h, apoptotic chromatin condensation was identified by histologic staining. In addition, many single cells with apoptotic nuclei were observed, indicating that stable cell fusion was not a requirement for apoptosis to occur. By 12 to 18 h of coculture, a DNA fragmentation ladder characteristic of apoptosis was detected by agarose gel electrophoresis. Quantitation of apoptosis by measurement of nuclear DNA content revealed that at least 20 to 30% of the nuclei were undergoing apoptosis by 24 h after cocultivation. The appearance of condensed nuclei and fragmented DNA occurred as HIV reverse transcription was completed, and it was not inhibited by zidovudine, suggesting that induction of apoptosis did not require new HIV replication. Soluble CD4 inhibited apoptosis, demonstrating that Env-CD4 interactions were required for apoptosis. In contrast to that in cell-to-cell transmission, apoptosis in cell-free HIV infections was markedly inefficient and was not observed until 70 to 90 h after infections were initiated. These findings indicate that HIV-1 induction of programmed destruction of the nucleus is initiated at the time of cell-cell cocultivation by a mechanism which requires CD4-Env interactions but not new HIV replication.
