Vladimir Ovod

Abundant expression of HIV Nef and Rev proteins in brain astrocytes in vivo is associated with dementia.

ObjectiveTo relate the expression of HIV regulatory proteins and HIV-specific mRNA in the brain cells of infected individuals with clinical neurological disease. DesignFormalin-fixed postmortem brain tissue from 14 HIV-infected adult patients, with previous repeated neurological and neuroradiological examinations, was studied by immunohistochemical and molecular biological methods. Samples from non-infected brains served as controls. MethodsImmunohistochemistry with monoclonal antibodies (MAb) was combined with in situ RNA hybridization. Target cells were identified with MAb to glial fibrillary acidic protein (GFAP; astrocytes), CD68 (activated macrophages) and Ricinus communis agglutinin (RCA-1; microglia, endothelial cells). For HIV, a panel of MAb against HIV Nef, Tat, Rev and Env proteins or probes specific for all classes of mRNA (net), for singly or non-spliced mRNA (env) and for non-spliced mRNA (gag/pot) were used. ResultsNef protein was detected in subcortical or subpial astrocytes in seven out of 14 samples, and in multinucleated giant cells in two cases. Gag/pol or env mRNA-expressing astrocytes were detected in four cases. In four out of five cases studied, HIV Rev, but not Tat, was also expressed in astrocytes. Six out of the seven patients with Nef-positive astrocytes had suffered from moderate to severe dementia. The patient with most rapidly progressing severe dementia showed extensive HIV mRNA expression together with Nef and Rev expression in astrocytes. ConclusionIn adult human brain, astrocytes are infected by HIV and preferentially express HIV Nef and Rev proteins but are also sometimes productively infected. Astrocyte infection is associated with moderate to severe dementia which agrees with recent knowledge on the housekeeping activities of astrocytes and their eventual role in learning and memory.

Expression kinetics and subcellular localization of HIV-1 regulatory proteins Nef, Tat and Rev in acutely and chronically infected lymphoid cell lines

SummaryInformation concerning the expression kinetics and subcellular localization of HIV regulatory proteins is of importance in understanding the viral pathogenesis and may be relevant for drug and vaccine development, as well. We have used combined immunocytochemistry and in situ hybridization to study firstly, the order of expression of regulatory HIV-1 proteins Nef, Rev and Tat in relation to non-spliced and spliced mRNA expression and secondly, the subcellular localization of these proteins in acutely and chronically infected human T-cell lines. We used monoclonal antibodies against HIV-1 Nef, Tat, Rev and gp160, and RNA probes reacting either with all mRNAs (nef) or only with the full-length mRNA (gag-pol). In acutely infected MT-4 and H9 cells, four distinct phases of infection could be defined. In the first phase lasting from 0 to 6 h post-infection, only incoming virus could be demonstrated by gp160 immunocytochemistry. During the second, regulatory phase (6–9 h), abundant cytoplasmic expression of Nef, Rev and Tat proteins and a positive in situ RNA hybridization with the nef probe was seen, while the in situ hybridization with full-length mRNA probe and immunohistochemistry for gp160 were still negative. The productive phase (12–48 h) was characterized by abundant expression of full-length mRNA and gp160, and by the nuclear localization of Nef and Tat proteins. In contrast, an antibody that recognized the RRE binding region of the Rev protein localized Rev in the cytoplasm both during the regulatory and productive phase. During the fourth, cytopathic phase, the expression of mRNA or viral proteins decreased and the regulatory proteins studied were again mainly localized in the cytoplasm. Based on the results, we speculate that HIV Nef may function as a nuclear factor, and that Tat is possibly bound by cellular proteins before its transport to the nucleus.

Cellular localization of Nef expressed in persistently HIV-1-infected low-producer astrocytes

ObjectivesThe characterization and localization of HIV-1 Nef highly expressed in permanently infected astrocytes (TH4–7-5) as a model for latent infection of human brain cells. DesignImmunochemical methods are an appropriate tool to investigate expression and localization of cellular proteins. MethodsNef expression was analysed by Western blot and immunoperoxidase staining using a panel of monoclonal and polyclonal antibodies. Cellular localization studies were performed by indirect immunofluorescence and subcellular fractionation of TH4–7-5 cells. Myristoylation of Nef was investigated by immunoprecipitation of [3H]myristic acid-labelled cell extract. TH4–7-5 nef gene was cloned and amplified by polymerase chain reaction and the nef nucleotide sequence analysed. ResultsReactivities of various Nef-specific antibodies with Nef antigen in TH4–7-5 cells were demonstrated by Western blot analysis. Immunofluorescence revealed cytoplasmic perinuclear staining of Nef with most antibodies. However, one monoclonal antibody against amino acids 168–175 of Nef showed intense homogeneous nuclear staining in TH4–7-5 cells. Reactivity of this Nef antibody was blocked with recombinant Nef derived from TH4–7-5 cells. After subcellular fractionation, Nef was detected in nuclear, membrane and cytosolic fractions of TH4–7-5 cells. No myristoylated Nef antigen was detectable, perhaps because of a serine residue at position 2 of the TH4–7-5 nef gene instead of the glycine residue required for myristoylation. ConclusionsChronically HIV-1-infected astrocytoma cells with restricted virus production express different antigenic forms of Nef, which can be distinguished by their subcellular localization. Variant subcellular targeting of Nef suggests the existence of multiple activities of Nef within HIV-infected cells.

Chicken avidin-related proteins show altered biotin-binding and physico-chemical properties as compared with avidin

Chicken avidin and bacterial streptavidin are proteins familiar from their use in various (strept)avidin-biotin technological applications. Avidin binds the vitamin biotin with the highest affinity known for non-covalent interactions found in nature. The gene encoding avidin (AVD) has homologues in chicken, named avidin-related genes (AVRs). In the present study we used the AVR genes to produce recombinant AVR proteins (AVRs 1, 2, 3, 4/5, 6 and 7) in insect cell cultures and characterized their biotin-binding affinity and biochemical properties. Amino acid sequence analysis and molecular modelling were also used to predict and explain the properties of the AVRs. We found that the AVR proteins are very similar to avidin, both structurally and functionally. Despite the numerous amino acid substitutions in the subunit interface regions, the AVRs form extremely stable tetramers similar to those of avidin. Differences were found in some physico-chemical properties of the AVRs as compared with avidin, including lowered pI, increased glycosylation and, most notably, reversible biotin binding for two AVRs (AVR1 and AVR2). Molecular modelling showed how the replacement Lys(111)-->isoleucine in AVR2 alters the shape of the biotin-binding pocket and thus results in reversible binding. Both modelling and biochemical analyses showed that disulphide bonds can form and link monomers in AVR4/5, a property not found in avidin. These, together with the other properties of the AVRs described in the present paper, may offer advantages over avidin and streptavidin, making the AVRs applicable for improved avidin-biotin technological applications.

Humoral and cellular immune responses to HIV-1 nef in mice DNA-immunised with non-replicating or self-replicating expression vectors.

OBJECTIVE HIV accessory protein Nef is expressed early in the infectious cycle of the virus and has been shown to be an effective immunogen in humoral and cellular immune responses. We have used two different self-replicating pBN vectors and one non-replicating pCGal2 derived (pCG) vector expressing HIV-1 Nef in DNA immunisation of mice in order to determine their efficiency in raising humoral and cellular immune responses. DESIGN AND METHODS The expression of Nef by the three plasmids was tested by transfections into COS-1 cells. Balb/c mice were immunised with the pBN-NEF and pCGE2-NEF constructs using gold particle bombardment. Immunoblotting and immunocytochemistry were used to detect in vitro expression of Nef. 51Cr release assay, ELISA and immunoblotting were used to detect cellular and humoral immune responses in immunised mice. RESULTS Efficient in vitro expression of Nef was detected in pBN and pCGE2-NEF transfected cells, in pBN-NEF transfected cells the expression lasting up to three weeks. Anti-Nef antibodies in sera of 13 of 16 pBN-NEF immunised mice were detected within four weeks after the last immunisation, whereas only 2 of 12 pCGE2-NEF immunised mice had very weak anti-Nef antibodies. Twelve of the pBN-NEF immunised mice (75%) and 6 the pCGE2-NEF immunised mice (50%) showed Nef-specific cytotoxic T lymphocyte (CTL) responses within four weeks. CONCLUSIONS We conclude that the three eukaryotic expression vectors tested are capable of inducing a cell mediated immune response towards HIV-1 Nef and should be considered as part of a genetic HIV vaccine.

Subcellular localization of pentachlorophenol 4-monooxygenase in Sphingobium chlorophenolicum ATCC 39723

We have studied the subcellular localization of pentachlorophenol 4-monooxygenase (PCP4MO) in Sphingobium chlorophenolicum ATCC 39723 during induction by pentachlorophenol (PCP). Using a monoclonal antibody CL6 specific to the native and recombinant PCP4MO, the enzyme was primarily found soluble as determined by immunoblot and ELISA analyses of cellular fractions. However, the enzyme was observed both in the soluble and membrane-bound forms during induction for 2-4 h, suggesting its translocation out from the cytoplasm. Electron microscopy confirmed that PCP4MO was predominantly present in the cytoplasm at 1 h, whereas at 4 h significant amount was detected also in the membrane and periplasm. After 6 h, the majority of PCP4MO was in the periplasm and only small amount was bound to the inner membrane or present in the cytoplasm. The results indicate that after biosynthesis PCP4MO in S. chlorophenolicum is exported via the inner membrane to the final location in the periplasm.

HIV-1 Nef co-localizes with the astrocyte-specific cytoskeleton protein GFAP in persistently nef-expressing human astrocytes.

In T-cells HIV-1 Nef exerts various functions and interacts with actin. In astrocytes interaction of Nef with cellular proteins is poorly understood. Therefore, human astrocytic cell clones stably transfected with nef-genes derived from HIV-1 Bru and its myristoylation-defective TH-variant were investigated by confocal laser scanning microscopy for expression of Nef and cytoskeleton proteins actin and GFAP, a marker for activated astrocytes. Myristoylated Nef was detected in cytoplasm, Golgi and plasmamembrane, while non-myristoylated Nef was exclusively cytoplasmic. Nef co-localised with GFAP in the perinuclear region of astrocytes. In contrast, Nef did not interact with actin filaments in human astrocytes. Nef/GFAP interaction could contribute to changes in morphology and activation state of astrocytes shown previously which are both critical for development of astrogliosis in HIV-1 infected brain.

Structure of the O-polysaccharide of Pseudomonas syringae pv. delphinii NCPPB 1879T Having Side Chains of 3-Acetamido-3,6-dideoxy-D-galactose Residues

AbstractThe O-polysaccharide (OPS) was obtained from the lipopolysaccharide of Pseudomonas syringae pv. delphinii NCPPB 1879T and studied by sugar and methylation analyses, Smith degradation, and 1H- and 13C-NMR spectroscopy. The OPS was found to contain residues of L-rhamnose (L-Rha) and 3-acetamido-3,6-dideoxy-D-galactose (D-Fuc3NAc), and the following structure of the major (n = 2) and minor (n = 3) heptasaccharide repeating units of the OPS was established:

Interactions of single and combined human immunodeficiency virus type 1 (HIV-1) DNA vaccines.

\begin{gathered} {\alpha} - {D} - {Fuc}p3{NAc} - (1 \to 2) - {\alpha} - {D} - {Fuc}p3{NAc} - (1 \to 2) - {\alpha} - {D} - {Fuc}p3{Nac} \hfill \\ { 1} \hfill \\ { } \downarrow \hfill \\ { 4} \hfill \\ { } \to {2} - {\alpha} - {L} - {Rha}p - (1 \to 3) - {\alpha} - {L} - {Rha}p - (1 \to 3) - {\alpha} - {L} - {Rha}p(1 \to n) - {\alpha} - {L} - {Rha}p - (1 \to \hfill \\ \hfill \\ \end{gathered}