Immunologic environment influences macrophage response to Porphyromonas gingivalis.

Macrophages adapt both phenotypically and functionally to the cytokine balance in host tissue microenvironments. Recent studies established that macrophages contribute an important yet poorly understood role in the development of infection-elicited oral bone loss. We hypothesized that macrophage adaptation to inflammatory signals encountered before pathogen interaction would significantly influence the subsequent immune response of these cells to the keystone oral pathobiont Porphyromonas gingivalis. Employing classically activated (M1) and alternatively activated (M2) murine bone-marrow-derived macrophage (BMDMø), we observed that immunologic activation of macrophages before P. gingivalis challenge dictated phenotype-specific changes in the expression of inflammation-associated molecules important to sensing and tuning host response to bacterial infection including Toll-like receptors 2 and 4, CD14, CD18 and CD11b (together comprising CR3), major histocompatibility complex class II, CD80, and CD86. M2 cells responded to P. gingivalis with higher expression of tumor necrosis factor-α, interleukin-6, monocyte chemoattractant protein-1, macrophage inflammatory protein-1α, regulated on activation normal T cell expressed and secreted, and KC than M1 cells. M1 BMDMø expressed higher levels of interleukin-10 to P. gingivalis than M2 BMDMø. Functionally, we observed that M2 BMDMø bound P. gingivalis more robustly than M1 BMDMø. These data describe an important contribution of macrophage skewing in the subsequent development of the cellular immune response to P. gingivalis.

Both PU.1 and nuclear factor-kappa B mediate lipopolysaccharide- induced HIV-1 long terminal repeat transcription in macrophages.

We recently reported that LPS stimulation of monocytic cells leads to the activation of PU.1, a member of the Ets family of transcription factors. Phosphorylation of PU.1 by protein kinase CK2 was found to up-regulate its trans-activation function, but not its DNA binding activity. Previous studies suggested that Ets proteins could bind to NF-kappa B motifs at the tetrameric core sequence TTCC. In macrophages, LPS-inducible HIV-1 gene expression is mediated in part by binding of NF-kappa B to identical tandem binding sites located within the long terminal repeat (LTR). Thus, we performed additional studies to determine whether PU.1 also played a role in regulating HIV-1 gene expression in macrophages. Our functional studies revealed that activation of the HIV-1 LTR in LPS-stimulated cells requires both NF-kappa B and PU.1. Extensive mutagenesis of the HIV-1 LTR revealed that PU.1-dependent activation requires the Ets motif within the upstream NF-kappa B site, whereas NF-kappa B itself binds to the downstream site. We also found that insertion of five additional nucleotides between the NF-kappa B sites abolished LPS inducibility, suggesting a direct interaction between factors that bind these sites. Lastly, we found that mutation of PU.1 at serine 148, which prevents its phosphorylation by CK2, blocked its ability to activate the HIV-1 LTR in response to LPS. These effects were promoter specific because PU.1 did not affect LPS-inducible activation of a distinct NF-kappa B-dependent promoter. While these data do not demonstrate direct binding of PU.1 to the HIV-1 LTR, they illustrate a novel role for PU.1 in activation of the HIV-1 LTR by LPS.

Retinoid-induced repression of human immunodeficiency virus type 1 core promoter activity inhibits virus replication.

The rates of mother-to-child transmission of human immunodeficiency virus type 1 (HIV-1), progression to AIDS following HIV-1 infection, and AIDS-associated mortality are all inversely correlated with serum vitamin A levels (R. D. Semba, W. T. Caiaffa, N. M. H. Graham, S. Cohn, and D. Vlahov, J. Infect. Dis. 171:1196-1202, 1995; R. D. Semba, N. M. H. Graham, W. T. Caiaffa, J. B. Margolik, L. Clement, and D. Vlahov, Arch. Intern. Med. 153:2149-2154, 1993; R. D. Semba, P. G. Miotti, J. D. Chiphangwi, A. J. Saah, J. K. Canner, G. A. Dallabetta, and D. R. Hoover, Lancet 343:1593-1596, 1994). Here we show that physiological concentrations of vitamin A, as retinol or as its metabolite, all-trans retinoic acid, repressed HIV-1Ba-L replication in monocyte-derived macrophages (MDMs). Repression required retinoid treatment of peripheral monocytes during their in vitro differentiation into MDMs. Retinoids had no repressive effect if they were added after virus infection. Retinol, as well as all-trans retinoic acid and 9-cis retinoic acid, also repressed HIV-1 long terminal repeat (LTR)-directed expression up to 200-fold in transfected THP-1 monocytes. Analysis of HIV-1 LTR deletion mutants demonstrated that retinoids were able to repress activation of HIV-1 expression by both NF-kappaB and Tat. A cis-acting sequence required for retinoid-mediated repression of HIV-1 transcription was localized between nucleotides -51 and +12 of the HIV-1 LTR within the core promoter. Protein-DNA cross-linking experiments identified four proteins specific to retinoid-treated cells that bound to the core promoter. We conclude that retinoids render macrophages resistant to virus replication by modulating the interaction of cellular transcription factors with the viral core promoter.

A lipidated anti-Tat antibody enters living cells and blocks HIV-1 viral replication.

We have developed a chemical modification of antibodies, lipidation, which enables their intracellular delivery into living cells. Intracellular localization of lipidated antibodies was demonstrated by confocal microscopy and by measuring cellular uptake of 125I-labeled lipidated antibodies. Functionally, a lipidated monoclonal antibody directed against the Tat protein from human immunodeficiency virus type 1 (HIV-1) inhibited viral replication of several HIV-1 isolates by approximately 85% as shown by increased viability of infected cells and decreased reverse transcriptase activity. The antibody in its native form had no such effect. These data show that lipidated antibodies can reach and functionally inhibit intracellular targets. Lipidation may help to facilitate the development of intracellular immunotherapy for AIDS.

A new approach to investigating the relationship between productive infection and cytopathicity in vivo.

We describe a novel experimental approach to analyzing virus-host relationships and potential mechanisms of cytopathicity in vivo in simian immunodeficiency virus (SIV) infections. Progressive destruction of lymphoid tissue in the course of infection by SIV or human immunodeficiency virus (HIV) accompanies the loss of CD4+ T lymphocytes and sets the stage for AIDS. Because one of the important early events in this pathological process is lysis of follicular dendritic cells (FDCs), we investigated the controversial role of productive SIV infection in the destruction of FDCs. To differentiate productive infections from the known association of virus with FDCs as immune complexes trapped on cell surfaces, we used detection of spliced viral mRNAs in cells as evidence of productive infection. We found that spliced and unspliced viral RNAs could be detected by in situ hybridization (ISH) with specific antisense oligonucleotide probes in lymphocytes and macrophages with sensitivities of fewer than ten copies of spliced viral RNA per cell. We detected only unspliced RNA in germinal centers where FDCs reside. Thus, no productive infection of these cells can be detected in vivo by this assay, and their destruction likely occurs by indirect mechanisms that have yet to be determined.

IL-16 represses HIV-1 promoter activity.

IL-16 is produced by CD8+ lymphocytes and has been reported to inhibit HIV-1 and SIV replication in infected PBMCs. CD4 serves as a receptor for the secreted form of IL-16, and IL-16 binding to CD4 induces signal transduction, which affects the activation state of the cell. We hypothesized, therefore, that the effect of IL-16 on HIV-1 replication might occur at the level of virus expression. In transient transfection studies with HIV-1 LTR-reporter gene constructs we found that pretreatment of CD4+ lymphoid cells with recombinant IL-16 repressed HIV-1 promoter activity up to 60-fold, preventing both PMA and Tat activation. This effect of IL-16 required sequences contained within the core enhancer, but was not simply due to the down-regulation of transcription factors binding to this element.

Synergistic activation of simian immunodeficiency virus and human immunodeficiency virus type 1 transcription by retinoic acid and phorbol ester through an NF-kappa B-independent mechanism.

The activation of human immunodeficiency virus type 1 (HIV-1) expression in latently infected cells by exogenous agents is believed to be important in the progression of AIDS. Most factors that are known to activate HIV-1 gene expression increase the binding of NF-kappa B or NF-kappa B-like transcription factors to the HIV-1 core enhancer region. In this report, we demonstrate that retinoic acid (RA) treatment of promonocytic U937 cells stimulates expression from the simian immunodeficiency virus (SIVmac) long terminal repeat (LTR). Furthermore, RA and phorbol 12-myristate 13-acetate (PMA) synergistically stimulated both SIVmac and HIV-1 LTRs to levels of expression comparable to that achieved by the viral transactivator Tat. The cis-acting elements required for a response to RA and PMA cotreatment are located between nucleotides -50 and +1 of SIVmac and between nucleotides -83 and +80 of HIV-1. Thus, the synergistic stimulation induced by RA and PMA is NF-kappa B independent. Analysis of deletion mutants of the SIVmac LTR demonstrates that RA and PMA stimulation cooperates with NF-kappa B and Sp1. An SIVmac LTR-reporter gene construct [pLTR(-50/+466)CAT] lacking NF-kappa B and Sp1 binding sites was not activated by Tat in untreated cells but was activated in cells that were cotreated with RA and PMA. Furthermore, gel retardation assays demonstrated that RA treatment causes a change in the pattern of a cellular factor(s) which binds to the -50 through +1 region of the SIVmac LTR. These data suggest that RA induces a PMA-activatable cellular factor that cooperates with NF-kappa B, Sp1, or Tat to stimulate LTR-directed transcription.

Feline leukemia virus subgroup C phenotype evolves through distinct alterations near the N terminus of the envelope surface glycoprotein.

Feline leukemia viruses (FeLVs) belonging to the C subgroup induce aplastic anemia in domestic cats and have the ability, unique among FeLV strains, to proliferate in guinea pig fibroblasts in tissue culture. Previous studies have shown that the pathogenic and host range specificity of a prototype molecular clone of FeLV-C [FeLV-Sarma-C (FSC)] colocalize to a region encoding the 3' 73 amino acids of the pol gene product and the N-terminal 241 amino acids of the envelope surface glycoprotein named SU. Here, we amplified, via PCR, cloned, and sequenced the SU coding sequence from three additional anemia-inducing subgroup C FeLV isolates. Chimeric viruses were constructed by replacement of fragments of FeLV-C envelope genes into the FeLV-A prototype virus 61E. Using a modified vesicular stomatitis virus-FeLV pseudotype assay, we demonstrated that the subgroup C receptor specificity for each virus was determined by changes within the N-terminal 87-92 amino acids of SU, in which most changes occurred within the 15- to 20-amino-acid first variable region (V1). Determinants for growth in guinea pig cells colocalized to this region. Despite the consistent localization of biological determinants, the only consistent features that distinguished the deduced FeLV-A and FeLV-C proteins was one lysine-to-arginine change and a structural prediction of an alpha-helix in FeLV-A proteins versus random coil in FeLV-C proteins within V1. However, arginine in equilibrium with lysine substitutions were not sufficient to convert the subgroup A virus to the subgroup C phenotype or vice versa. Thus, certain distinct structural changes within the N-terminal region of FeLV SU can result in convergent viral phenotypes.

Role of TAR RNA splicing in translational regulation of simian immunodeficiency virus from rhesus macaques.

The untranslated leader sequences of rhesus macaque simian immunodeficiency virus mRNAs form a stable secondary structure, TAR. This structure can be modified by RNA splicing. In this study, the role of TAR splicing in virus replication was investigated. The proportion of viral RNAs containing a spliced TAR structure is high early after infection and decreases at later times. Moreover, proviruses containing mutations which prevent TAR splicing are significantly delayed in replication. These mutant viruses require approximately 20 days to achieve half-maximal virus production, in contrast to wild-type viruses, which require approximately 8 days. We attribute this delay to the inefficient translation of unspliced-TAR-containing mRNAs. The molecular basis for this translational effect was examined in in vitro assays. We found that spliced-TAR-containing mRNAs were translated up to 8.5 times more efficiently than were similar mRNAs containing an unspliced TAR leader. Furthermore, these spliced-TAR-containing mRNAs were more efficiently associated with ribosomes. We postulate that the level of TAR splicing provides a balance for the optimal expression of both viral proteins and genomic RNA and therefore ultimately controls the production of infectious virions.

Simian immunodeficiency virus displays complex patterns of RNA splicing.

The human and simian immunodeficiency viruses encode at least six gene products that apparently serve regulatory functions. To evaluate the regulation of simian immunodeficiency virus gene expression at the level of RNA splicing, we used the polymerase chain reaction to amplify and clone cDNAs corresponding to a large array of mRNAs from infected cells. We identified mRNAs that used splice acceptor sites upstream of the initiator codons for tat, rev, vpr, nef, vif, and vpx, suggesting that these proteins may be expressed from different mRNAs. We also provide hybridization data suggesting that the same splice acceptor site may be used for both rev and env mRNAs. Furthermore, we isolated both tat and rev cDNAs that utilized three alternative splice acceptor sites at the start of coding exon 2, indicating that different versions of these proteins may be encoded. Finally, approximately 10 to 20% of simian immunodeficiency virus mRNAs spliced an intron from their untranslated 5' ends, and sequences contained within this intron constituted a portion of the tat-responsive TAR element. Thus, alternative pre-mRNA splicing adds a level of complexity to simian immunodeficiency virus expression, which may affect several levels of gene regulation.

Expression of human DNA topoisomerase I in yeast cells lacking yeast DNA topoisomerase I: restoration of sensitivity of the cells to the antitumor drug camptothecin.

Yeast Saccharomyces cerevisiae strains that are permeable to the antitumor alkaloid camptothecin are killed by the drug if they express DNA topoisomerase I, the cellular target of the drug (J. Nitiss and J.C. Wang, Proc. Natl. Acad. Sci. USA, 85: 7501-7505, 1988). We show that in a yeast strain permeable to camptothecin but lacking DNA topoisomerase I, sensitivity to the drug was restored upon expression of human DNA topoisomerase I from a plasmid-borne human complementary DNA clone. When the human enzyme was expressed from a galactose-inducible, glucose-repressible yeast promoter, PGAL1, sensitivity to camptothecin was observed in the presence of galactose but not in the presence of glucose. Expression of human DNA topoisomerase I in Escherichia coli was also demonstrated by the complementation of a conditional lethal E. coli DNA topoisomerase I mutant. These systems can be used in the study of human DNA topoisomerase I-camptothecin interactions and in the screening of additional therapeutics targeting the enzyme.

Alteration of hsp82 gene expression by the gypsy transposon and suppressor genes in Drosophila melanogaster.

Several mutations in Drosophila result from insertion of the gypsy retrotransposon. Gypsy insertion mutagenesis and its modulation by allele-specific modifier genes were investigated by inserting gypsy or fragments of it into the intron of the Drosophila hsp82 heat shock gene. With gypsy in the parallel orientation, nearly all transcripts in transfected cells and transformed pupae were truncated in the 5' long terminal repeat (LTR). Truncation also occurred in or near the 3' LTR. The 5' LTR polyadenylation signal was strongly potentiated by a downstream 326-bp internal gypsy segment in either orientation. Anti-parallel gypsy reduced the amount of normal transcript to a much smaller extent, and a low level of truncation occurred within gypsy. No evidence was found for effects of the gypsy insertions on the hsp82 promoter. Mutations in the allelespecific modifier genes su(f) and su(w alpha) had effects on the amounts of readthrough transcripts consistent with their genetic behavior, whereas the effects of mutations in su(Hw) were only partly in accord with genetic expectations.

Functional comparison of transactivation by simian immunodeficiency virus from rhesus macaques and human immunodeficiency virus type 1.

Simian immunodeficiency virus from rhesus macaques (SIVmac), like human immunodeficiency virus type 1 (HIV-1), encodes a transactivator (tat) which stimulates long terminal repeat (LTR)-directed gene expression. We performed cotransfection assays of SIVmac and HIV-1 tat constructs with LTR-CAT reporter plasmids. The primary effect of transactivation for both SIVmac and HIV-1 is an increase in LTR-directed mRNA accumulation. The SIVmac tat gene product partially transactivates an HIV-1 LTR, whereas the HIV-1 tat gene product fully transactivates an SIVmac LTR. Significant transactivation is achieved by the product of coding exon 1 of the HIV-1 tat gene; however, inclusion of coding exon 2 results in a further increase in mRNA accumulation. In contrast, coding exon 2 of the SIVmac tat gene is required for significant transactivation. These results imply that the tat proteins of SIVmac and HIV-1 are functionally similar but not interchangeable. In addition, an in vitro-generated mutation in SIVmac tat disrupts splicing at the normal splice acceptor site at the beginning of coding exon 2 and activates a site approximately 15 nucleotides downstream. The product of this splice variant stimulates LTR-directed gene expression. This alternative splice acceptor site is also used by a biologically active provirus with an efficiency of approximately 5% compared with the upstream site. These data suggest that a novel tat protein is encoded during the course of viral infection.

Cloning of HTLV-4 and its relation to simian and human immunodeficiency viruses.

Although much is now known of the strain variation among the type-1 human immunodeficiency virus (HIV-1), which is the cause of AIDS (acquired immune deficiency syndrome) in the United States, Europe, and Central Africa, much less is yet known about a second group of viruses that have been found in West Africans. One member of this group, named human T-cell lymphotropic virus type 4 (HTLV-4), has been isolated from healthy Senegalese. Another is the virus isolated from West Africans with AIDS-like illness and originally called LAV-2 but now renamed HIV-2. Both these viruses seem to be less closely related to HIV-1 than they are to a virus of healthy African green monkeys, known variously as simian T-cell lymphotropic virus type 3 (STLV-3) or simian immunodeficiency virus (SIV), which in turn is related to viruses isolated from healthy sooty mangabeys and captive macaques with a form of immunodeficiency (to distinguish these viruses they are referred to as STLV-3 (or SIV)agm, STLV-3mac, or STLV-3smm). To clarify the relationship between the various HIVs, STLV-3s and HTLV-4 we are determining and comparing the molecular and biological characteristics of several of them. Following our recent publication of a restriction-site map of STLV-3agm, we now report that the equivalent map of three isolates of HTLV-4 is remarkably similar to it. In addition we present comparative sequence data on the long terminal repeats (LTR) of HTLV-4, STLV-3agm, HIV-1 and HIV-2, together with evidence that cloned HTLV-4 uses the same receptor as HIV-1 and induces some, but not all, of the cytopathic effects attributed to most isolates of HIV-1 and HIV-2.

Satellite DNA-correlated nucleosomal proteins in Drosophila virilis.

Three major satellite DNAs comprise 40-45% of the genome of Drosophila virilis. Since these satellites are not substrates for most restriction enzymes, we were able to digest D. virilis nuclei with HaeIII and micrococcal nuclease and isolate chromatin fractions containing variable levels of satellite DNA. Electrophoretic analysis of these chromatin fractions revealed that the level of the acid-soluble chromosomal protein, cp17.3, was directly related to the percentage of satellite DNA in chromatin. The correlation between cp17.3 and satellite DNA abundance suggests that cp17.3 is involved in the heterochromatic condensation of satellite DNAs. cp17.3 occurs at a frequency of one molecule per 10-20 nucleosomes. It is detected in an electrophoretically distinguishable class of mononucleosomes, provisionally identified as MN1uH2A, which contains ubiquitinated histone H2A (uH2a) but lacks histone H1. It is not detected in MN1, a second class of mononucleosomes, which lacks uH2A and H1. Since cp17.3 is correlated with satellite DNAs and present in nucleosome cores, it might be a histone variant specifically associated with satellite DNAs.

Mutagenicity of dieldrin in the Salmonella-microsome test.

Dieldrin was assayed in vitro for its mutagenic activity in several strains of S. typhimurium with and without Aroclor 1254 treated mouse liver homogenate (S-9M). The mutagenicity of the insecticide was expressed maximally in all strains in the presence of liver S-9M and less so in three strains without liver microsomes.