Macrophages and CD4؉ lymphocytes are the principal target cells for human immunodeficiency virus type 1 (HIV-1) infection, but the molecular details of infection may differ between these cell types. During studies to identify cellular molecules that could be involved in macrophage infection, we observed inhibition of HIV-1 infection of macrophages by monoclonal antibody (MAb) to the tetraspan transmembrane glycoprotein CD63. Pretreatment of primary macrophages with anti-CD63 MAb, but not MAbs to other macrophage cell surface tetraspanins (CD9, CD81, and CD82), was shown to inhibit infection by several R5 and dualtropic strains, but not by X4 isolates. The block to productive infection was postfusion, as assessed by macrophage cell-cell fusion assays, but was prior to reverse transcription, as determined by quantitative PCR assay for new viral DNA formation. The inhibitory effects of anti-CD63 in primary macrophages could not be explained by changes in the levels of CD4, CCR5, or ␤-chemokines. Infections of peripheral blood lymphocytes and certain cell lines were unaffected by treatment with anti-CD63, suggesting that the role of CD63 in HIV-1 infection may be specific for macrophages.

Experimental evidence suggests that random, spontaneous (stochastic) fluctuations in gene expression have important biological consequences, including determination of cell fate and phenotypic variation within isogenic populations. We propose that fluctuations in gene expression represent a valuable tool to explore therapeutic strategies for patients who have suffered traumatic brain injury (TBI), for which there is no effective drug therapy. We have studied the effects of TBI on the hippocampus because TBI survivors commonly suffer cognitive problems that are associated with hippocampal damage. In our previous studies we separated dying and surviving hippocampal neurons by laser capture microdissection and observed unexplainable variations in post-TBI gene expression, even though dying and surviving neurons were adjacent and morphologically identical. We hypothesized that, in hippocampal neurons that subsequently are subjected to TBI, randomly increased pre-TBI expression of genes that are associated with neuroprotection predisposes neurons to survival; conversely, randomly decreased expression of these genes predisposes neurons to death. Thus, to identify genes that are associated with endogenous neuroprotection, we performed a comparative, high-resolution transcriptome analysis of dying and surviving hippocampal neurons in rats subjected to TBI. We found that surviving hippocampal neurons express a distinct molecular signature — increased expression of networks of genes that are associated with regeneration, cellular reprogramming, development, and synaptic plasticity. In dying neurons we found decreased expression of genes in those networks. Based on these data, we propose a hypothetical model in which hippocampal neuronal survival is determined by a rheostat that adds injury-induced genomic signals to expression of pro-survival genes, which pre-TBI varies randomly and spontaneously from neuron to neuron. We suggest that pharmacotherapeutic strategies that co-activate multiple survival signals and enhance self-repair mechanisms have the potential to shift the cell survival rheostat to favor survival and therefore improve functional outcome after TBI.

Despite the key role played by the RNase H of human immunodeficiency virus-1 reverse transcriptase (HIV-1 RT) in viral proliferation, only a few inhibitors of RNase H have been reported. Using in vitro combinatorial selection methods and the RNase H domain of the HIV RT, we have selected double-stranded DNA thioaptamers (aptamers with selected thiophosphate backbone substitutions) that inhibit RNase H activity and viral replication. The selected thioaptamer sequences had a very high proportion of G residues. The consensus sequence for the selected thioaptamers showed G clusters separated by single residues at the 5'-end of the sequence. Gel electrophoresis mobility shift assays and nuclear magnetic resonance spectroscopy showed that the selected thioaptamer binds to the isolated RNase H domain, but did not bind to a structurally similar RNase H from Escherichia coli. The lead thioaptamer, R12-2, showed specific binding to HIV-1 RT with a binding constant (K(d)) of 70 nM. The thioaptamer inhibited the RNase H activity of intact HIV-1 RT. In cell culture, transfection of thioaptamer R12-2 (0.5 microg/mL) markedly inhibited viral production and exhibited a dose response of inhibition with R12-2 concentrations ranging from 0.03 to 2.0 microg/mL (IC(50) < 100 nM). Inhibition was also seen across a wide range of virus inoculum, ranging from a multiplicity of infection (moi) of 0.0005 to 0.05, with a reduction of the level of virus production by more than 50% at high moi. Suppression of virus was comparable to that seen with AZT when moi

HIV infection typically involves interaction of Env with CD4 and a chemokine coreceptor, either CCR5 or CXCR4. Other cellular factors supporting HIV replication have also been characterized. We previously demonstrated a role for CD63 in early HIV infection events in macrophages via inhibition by anti-CD63 antibody pretreatment. To confirm the requirement for CD63 in HIV replication, we decreased CD63 expression using CD63-specific short interfering RNAs (siRNA), and showed inhibition of HIV replication in macrophages. Surprisingly, pretreatment with CD63 siRNA not only silenced CD63 expression by 90%, but also inhibited HIV-1 replication in a cultured cell line (U373-MAGI) which had been previously shown to be insensitive to CD63 monoclonal antibody inhibition. Although the anti-CD63 antibody was previously shown to inhibit early HIV infection events only in macrophages, we now show a potential role for CD63 in later HIV replication events in macrophages and cell lines. Further delineation of the role of CD63 in HIV replication may lead to development of novel therapeutic compounds.

The protein kinase casein kinase 2 (CK2) is ubiquitous in eukaryotic cells and is apparently involved in the control of cell division. The holoenzyme is a tetramer composed of two catalytic subunits ( a and/ or a') and regulatory subunits @). The a and a' subunits are encoded by different genes but are very similar in amino acid sequence, except that (I' is normally considerably shorter. There have been extensive biochemical studies with recombinant n and p subunits of many species, but only one previous description of the activity of an isolated recombinant a' subunit from human CK2 (Bodenbach, L., Fauss, J., Robitzki, A., Krehan, A., Lorenz, P., Lozeman, F. J. & Pyerin, W. (1994) Recombinant human casein kinase IT. A study with the complete set of subunits (a, a', and p), site-directed autophosphorylation mutants and a bicistronically expressed holoenzyme, Eur: J. Biochem. 220,[263][264][265][266][267][268][269][270][271][272][273]. In the present work, the isolation and bacterial expression of a cDNA coding for the a' subunit of zebrafish (Danio rerio) is reported. The clone covers the complete coding region that generates a protein of 348 amino acids that is 86% identical to the a' subunits of human and chicken, and 82% identical to the sequenced portion of the CK2a subunit of zebrafish. The recombinant a' subunit has apparent K,,, values for ATP (6 pM), GTP (20 pM), casein (2.0 mg/ml) and the model peptide RRRDDDSEDD (0.3 mM) which are very similar to those of the recombinant a subunit of Xenopus laevis. The a' subunit k,;,, was 7.2 min-' which is again similar to that of Xenopus laevis a subunit (7.5 min-'). The a' subunit also behaved similarly to CK2n with regard to optimal concentrations for Mg" or Mntz and to the inhibition by heparin and the poly(GluxOTyrZO) peptide. However a' kinase activity was less sensitive to poly(U) inhibition than a, it was more heat stable than n, and a' was slightly more sensitive to KC1 inhibition than a. The difference in salt sensitivity, however, was enhanced by the presence of the regulatory p subunit which shifted the optimal salt concentration of the phosphorylating activity. The aipZ holoenzyme was inhibited by KCI concentrations above 100 mM, while the a& enzyme was stimulted by KC1 concentrations up to 150 mM and required 180 mM for inhibition.Another important difference between a and a' is seen in the degree of the stimulation of casein phosphorylation activity in the presence of the regulatory subunit. When assayed at 100 mM KCI stoichiometric amounts of CK2p produced maximal stimulation of both a' (D. rerin) and n ( X . laevis), however the activity levels with a' were stimulated 20-fold by while the addition of [ I stimulated 1x (X. luevis) only 7 -8-fold. Abbreviations. CK2, protein lunase CK2 or casein kinase 2; CK2a, the a subunit of protein kinase CK2; CK2a', the a' subunit of protein kinase CK2; CK2P, the /I subunit of protein kinase CK2.Enzyme. Protein kinase CK2 (EC 2.7.1.37).Note. The novel sequence data published here have been submitted to the EMBL s...