Structural significance of the GTP-binding domain of ras p21 studied by site-directed mutagenesis.

Point mutations of p21 proteins were constructed by oligonucleotide-directed mutagenesis of the v-rasH oncogene, which substituted amino acid residues within the nucleotide-binding consensus sequence, GXG GXGK. When the glycine residue at position 10, 13, or 15 was substituted with valine, the viral rasH product p21 lost its GTP-binding and autokinase activities. Other substitutions at position 33, 51, or 59 did not impair its binding activity. G418-resistant NIH 3T3 cell lines were derived by transfection with constructs obtained by inserting the mutant proviral DNA into the pSV2neo plasmid. Clones with a valine mutation at position 13 or 15 were incapable of transforming cells, while all other mutants with GTP-binding activity were competent. A mutant with a substitution of valine for glycine at position 10 which had lost its ability to bind GTP and its autokinase activity was fully capable of transforming NIH 3T3 cells. These cells grew in soft agar and rapidly formed tumors in nude mice. The p21 of cell lines derived from tumor explants still lacked the autokinase activity. These findings suggest that the glycine-rich consensus sequence is important in controlling p21 activities and that certain mutations may confer to p21 its active conformation without participation of ligand binding.

Neutralizing monoclonal antibody against ras oncogene product p21 which impairs guanine nucleotide exchange.

The neutralizing monoclonal antibody Y13-259 severely hampers the nucleotide exchange reaction between p21-bound and exogenous guanine nucleotides but does not interfere with the association of GDP to p21. These results suggest that the nucleotide exchange reaction is critical for p21 function. Interestingly, the v-ras p21 has a much faster dissociation rate than the p21 of the c-ras proto-oncogene.

Sulfonic acid dyes: inhibition of the human immunodeficiency virus and mechanism of action.

Over 50 different commercially available sulfonic acid-containing dyes were analyzed for their ability to prevent HIV-1-induced cell killing and in inhibiting HIV-1 replication. Compounds of remarkably similar structure, but with differing patterns of sulfonic acid group substitutions, had a wide range of potency in inhibiting HIV-1. Chicago sky blue (CSB) was highly effective in the inhibition of HIV-1 with less toxicity to CEM-SS cells than most of the other sulfonated dyes tested. Synthesis of CSB was undertaken to produce a product greater than 98% pure and this compound was used to elucidate the possible mechanisms by which this class of structurally related compounds inhibits HIV-1. Addition of CSB to cells infected at high multiplicity at any time up to 24 h after infection, unlike dideoxycytidine (ddC) or oxathiin carboxanilide (OC), inhibited HIV-1-induced cell killing. Other postinfection time course studies revealed that CSB had to be present for 24 h or longer immediately after infection to be protective. Virus binding to cells occurred in the presence of CSB, but the requirement for virion envelope-cell membrane fusion was delayed. CSB was a potent inhibitor of the reverse transcriptase (RT) of both HIV-1 and HIV-2, although it was less active against HIV-2 in a cell killing-based assay. CSB also inhibited Rauscher and LP-BM5 murine leukemia viruses. CSB appears to disrupt the interaction between viral proteins and cell membranes, both in the fusion step early in the infection cycle and in the development of syncytia in the late stages of virus infection.

Design, synthesis, and biological evaluation of cosalane, a novel anti-HIV agent which inhibits multiple features of virus reproduction.

Cosalane (3), a novel anti-HIV agent having a disalicylmethane unit linked to C-3 of cholestane by a three-carbon linker, was synthesized from commercially available starting materials by a convergent route. Cosalane proved to be a potent inhibitor of HIV with a broad range of activity against a variety of laboratory, drug-resistant, and clinical HIV-1 isolates, HIV-2, and Rauscher murine leukemia virus. The cytotoxicity of cosalane is relatively low as reflected by an in vitro therapeutic index of > 100. Although cosalane inhibits HIV-1 reverse transcriptase and protease, time of addition experiments indicate that it prevents the cytopathic effect of HIV by acting earlier than reverse transcription in the viral replication cycle. The available evidence indicates that the primary mechanism of action of cosalane involves inhibition of gp120-CD4 binding as well as inhibition of a postattachment event prior to reverse transcription.

Inhibition of in vitro and in vivo HIV replication by a distamycin analogue that interferes with chemokine receptor function: a candidate for chemotherapeutic and microbicidal application.

Select chemokine receptors act as coreceptors for HIV-1 entry into human cells and represent targets for antiviral therapy. In this report we describe a distamycin analogue, 2,2'-[4, 4'-[[aminocarbonyl]amino]bis[N,4'-di[pryrrole-2-carboxamide- 1, 1'-dimethyl]]-6,8-naphthalenedisulfonic acid]hexasodium salt (NSC 651016), that selectively inhibited chemokine binding to CCR5, CCR3, CCR1, and CXCR4, but not to CXCR2 or CCR2b, and blocked chemokine-induced calcium flux. Inhibition was not due to nonspecific charge interactions at the cell surface, but was based on a specific competition for the ligand receptor interaction sites since the inhibitory effect was specific for some but not all chemoattractant receptors. NSC 651016 inhibited in vitro replication of a wide range of HIV-1 isolates, as well as HIV-2 and SIV, and exhibited in vivo anti-HIV-1 activity in a murine model. In contrast, a distamycin analogue with similar structure and charge and the monomeric form of NSC 651016 demonstrated no inhibitory effects. These data demonstrate that molecules which interfere with HIV-1 entry into cells by targeting specific chemokine coreceptors can provide a viable approach to anti-HIV-1 therapy. NSC 651016 represents an attractive candidate for the chemotherapeutic treatment of HIV-1 infection and as a microbicide to prevent the sexual transmisssion of HIV-1. Moreover, NSC 651016 can serve as a template for medicinal chemical modifications leading to more effective antivirals.

Inhibition of bovine immunodeficiency virus by anti-HIV-1 compounds in a cell culture-based assay.

The bovine immunodeficiency virus (BIV) and human immunodeficiency virus types 1 and 2 (HIV-1 and -2) are members of the lentivirus genus of retroviruses. Although DNA sequences of these viruses have diverged considerably, the BIV genome organization, function of structural and regulatory genes, and replication cycle are very similar to that of HIV-1, making BIV a potentially useful model to study compounds with anti-HIV-1 activity. A cell culture-based antiviral assay was developed to test compounds for inhibition of BIV replication. The assay uses an embryonic rabbit epithelial (EREp) cell line that is highly sensitive to BIV infection and cytopathology. The 50% effective concentrations (EC50) at which the virus was inhibited in EREp cells were determined for 13 nucleoside analog, non-nucleoside, tumor-suppressive, or membrane-surface inhibitory compounds. The nucleoside analogs (3'-azido-2',3'-dideoxythymidine, 2',3'-dideoxyinosine and 2',3'-dideoxycytosine), surface-membrane inhibitors (dextran sulfate, hypericin, Chicago Sky Blue and quinobene), the nucleoside reductase inhibitor (hydroxyurea), and a tumor-suppressive phorbol ester (prostratin) inhibited BIV with EC50 values similar to those derived in HIV-1 lymphocyte (CD4+)-based assays. BIV was markedly more resistant to inhibition with HIV-1-specific non-nucleoside reverse transcriptase inhibitors (NNRTIs) (thiazolobenzimidazole, oxathiin carboxanilide and thiocarbamate) than was HIV-1, which parallels results with NNRTIs in HIV-2 assays.

Inhibition of human immunodeficiency virus replication by the sulfonated stilbene dye resobene.

The anti-HIV sulfonated dye, resobene, was found to be a potent inhibitor of the attachment of HIV to target cells, the fusion of envelope- and CD4-expressing cells, and the cell-to-cell transmission of virus. Resobene inhibited the infection of phenotypically distinct, established human cell lines and fresh human peripheral blood lymphocytes and macrophages by laboratory-derived isolates of human immunodeficiency virus type 1 (HIV-1) and type 2 (HIV-2), and a panel of biologically diverse primary clinical isolates, including syncytium-inducing and non-syncytium-inducing viruses and strains representative of the various virus clades found worldwide. The compound was also active against all drug-resistant virus isolates tested. Cell-based and biochemical mechanism of action studies demonstrated that the compound inhibits the attachment of infectious virus and fusion of virus-infected cells to uninfected target cells by binding to the cationic V3 loop of the envelope glycoprotein. Resobene effectively inhibited the infection of cell populations which do and do not express cell surface CD4. Resobene prevented infection of the cervical epithelial cell line ME180, suggesting the compound may effectively act as a topical microbicide to prevent the sexual transmission of HIV.

Novel sulfonated and phosphonated analogs of distamycin which inhibit the replication of HIV.

A series of novel distamycin-related polyanionic compounds were compared for their anti-HIV activity. Several were highly potent inhibitors of HIV virus-induced cell killing and viral replication of a wide variety of laboratory isolates, as well as a monocytotropic virus and a clinical isolate in human peripheral blood lymphocytes. These compounds are structurally different from other sulfonic acid containing compounds reported to be potent inhibitors of the human immunodeficiency virus (HIV) in two respects: (1) they are structurally related to the non-toxic minor groove DNA binder distamycin; and (2) a number of them contain the aromatic phosphonic acid group. The compounds that were evaluated can be categorized into monomeric or dimeric ureido structural classes incorporating the bisamido-N-methylpyrrolenaphthalene-sulfonic acid group, with differences in the number and position of the sulfonic acids on the naphthalene rings. Broader structure-activity studies were made possible through the synthesis and evaluation of the compounds containing only a single N-methylpyrrole unit, those incorporating the N-methylpyrazole structure, and compounds having the isosteric phosphonic acid group substituted for the sulfonic acid group. One of the most potent of the inhibitors was 2,2'[4,4'[[aminocarbonyl]amino]bis[N,4'-di[pyrrole-2-carboxamide- 1,1'-dimethyl]]-4,6,8 naphthalenetrisulfonic acid] hexasodium salt, NSC 651015. This compound, the phosphonic acid analog NSC 662162, and the monomeric compound NSC 651018 were studied to determine the mechanism of their inhibitory activity. Mechanistic studies revealed that inhibition was due to the disruption of virus attachment to CD(4+)-susceptible cells and a further restraint on fusion of virus and cell membranes. The relative tolerance of these compounds in mice suggests that sufficient antiviral concentrations could be reached in vivo and thus may prove valuable in the treatment of AIDS patients.

Interaction of ras oncogene product p21 with guanine nucleotides.

The nucleotide exchange reaction was observed with purified ras oncogene product p21 overproduced in Escherichia coli (Hattori, S. et al. (1985) Mol. Cell Biol. 5, 1449-1455) under various conditions. (NH4)2SO4 increased the rate of dissociation of bound GDP from c-rasH and v-rasH p21. The dissociation kinetics were those of a first order reaction, and there was a linear relationship between the rate constant and the (NH4)2SO4 concentration. At any concentration of (NH4)2SO4, the exchange rate was faster with v-rasH p21 than that with c-rasH p21. EDTA and (NH4)2SO4 synergetically stimulated the dissociation reaction. Nucleotide-free p21 was prepared by gel filtration on Sephadex G-25 in the presence of 5 mM EDTA and 200 mM (NH4)2SO4 at room temperature. The free p21 was quite thermolabile, but the addition of GDP or GTP completely protected p21 from thermal inactivation. The dissociation constants for GDP and GTP were determined with free p21 to be 8.9 and 8.2 nM, respectively, for v-rasH p21, and 1.0 and 2.6 nM for c-rasH p21. In the presence of 200 mM (NH4)2SO4, these dissociation constants increased 3- to 12-fold.

Assessment of a cytoprotection assay for the discovery and evaluation of anti-human immunodeficiency virus compounds utilizing a genetically-impaired virus.

A biologically contained cytoprotection assay was developed to screen inhibitors of the human immunodeficiency virus without the need for high level containment or practices. The virus used has multiple point mutations that have destroyed its ability to produce both Rev and Tat, proteins essential for virus replication in vitro. The original cell line employed (CEM-SSTART) contains a genetic construct that allows for the continuous expression of both Rev and Tat, and a subclone (1A2) was developed that provides for maximum acute cytopathic effect. The National Cancer Institute's AIDS drug screening assay was used to test known drugs with both HIVIIIB virus in the T4 lymphocytic cell line CEM-SS and mutant virus in the 1A2 subclone. This cell-based assay uses the tetrazolium salt, XTT, as an indicator of cellular metabolism after the cells have been infected with virus. The results of extensive testing have shown that the assay using mutant virus is comparable to the current NCI AIDS drug screen. After 42 days in 1A2 or CEM-SS cell culture, the virus or the integrated genome did not revert to wild-type, and the virus produced in 1A2 cells was unable to replicate in PBMCs. Mutant viral stocks were devoid of wild-type virus as determined by a PCR assay that would have found 60-600 copies of mutant RNA. These materials, which are now available to the scientific community (NIH AIDS Research and Reference Reagent Program), should be useful tools to screen and test compounds for potential inhibition of HIV in laboratories not equipped to maintain and use wild-type infectious virus.

A sensitive assay for anti-HIV-1 drug discovery in a biological safety level-2 laboratory.

Studies involving infectious, wild type HIV-1 must be performed under strict BSL-3 practice. We have employed a defective (deltaTat/Rev)MC99 and cloned 1A2 line, ie, mutated HIV-1 and Tat/Rev transfected cells to verify anti-HIV-1 activity in a BSL-2 laboratory. A number of extracts from various parts of 11 species of plants were studied. Results were correlated with those of an anti-HIV-1 reverse transcriptase (RT) assay.

NgoII, a restriction endonuclease from Neisseria gonorrhoeae.

EndoR . NgoII, a class II restriction endonuclease isolated from Neisseria gonorrhoeae, was purified to electrophoretic homogeneity. We were able to separate it from another restriction endonuclease of N. gonorrhoeae, NgoI, by phosphocellulose chromatography. NgoII is an isoschizomer of HaeIII, a restriction endonuclease of Haemophilus aegyptius, and was found to recognize the deoxyribonucleic acid nucleotide base sequence GGCC. NgoII was able to digest phage lambda deoxyribonucleic acid over a wide pH range, with optimal activity at pH 8.5. The enzyme has an absolute requirement for Mg2+; maximal enzyme activity was observed at 1 mM Mg2+. The active enzyme has a molecular weight of 65,000 and appears to be composed of six subunits of identical molecular weight (11,000). No methylase activity could be detected in the purified enzyme preparation.

Identification of a new sequence-specific endonuclease, NgoII, from Neisseria gonorrhoeae.

A class II restriction endonuclease which recognizes the same nucleotide sequence as EndoR.HaeIII has been found in four of seven isolates of Neisseria gonorrhoeae tested.

Mutations of the ras gene product p21 that abolish guanine nucleotide binding.

We have constructed several point mutations affecting the GTP-binding site of p21, the ras-encoded protein. Both lysine (116K) and tyrosine (116Y) mutations of asparagine-116, which, by analogy with the crystal structure of elongation factor Tu (EF-Tu), has critical interactions with the guanine base, abolish GTP binding and transforming activities of p21. These activities are retained by proteins with a mutation at position 117 or 118. Both 116K and 116Y mutant p21s, when overproduced in Escherichia coli, are apparently devoid of GTP-binding and autokinase activities. Similarly, the mutant DNAs do not transform NIH 3T3 cells in a focus-forming assay. By cotransfection with pSV-neo, cell clones resistant to the neomycin analog G418 have been isolated. Cells transfected with 116K or 116Y mutant DNA are contact inhibited. In contrast to competent clones, the defective mutants have no detectable phosphorylated p21. The present results suggest that the basic structure of the GTP-binding site is conserved between p21 and EF-Tu and that this binding site is crucial for ras gene function.

Novel phosphorylation of c-ras p21 by protein kinases.

Novel p21 phosphorylation was found in cells expressing high levels of this product of c-H- and c-K-ras genes. Phorbol 12,13-dibutyrate, a protein kinase C (PKC) activator, and permeable c-AMP derivatives, which activate protein kinase A (PKA), stimulated phosphorylation of K-ras(4B) p21 in 416B cells 3 to 5 fold. By tryptic peptide mapping, it was found that both PKC and PKA phosphorylated in vitro the K-ras p21 at the same site as was found in p21 from cells labeled with [32P]orthophosphate in vivo. A common site of H-ras p21 was also phosphorylated by both PKC and PKA, although phosphopeptides of H-ras p21 were distinct from those of K-ras p21. The construction of a mutant by site-directed mutagenesis allowed the identification of serine-177 as the phosphorylation site of H-ras p21. This novel phosphorylation site lies in the hypervariable region, which links the globular catalytic domain of p21 to the membrane-anchoring site at the C-terminus, a location suggesting that this phosphorylation plays a role in modulating transmembrane signaling.

Characterization and location of myc homologous sequences in human cytomegalovirus DNA.

Specific DNA fragments from human cytomegalovirus (HCMV) strains Towne and AD169 exhibited homology to myc DNA sequences under hybridization conditions corresponding to a 22-28% base mismatch. In a specific subset of hybridizing HCMV fragments, the homology was restricted to the 5' half of viral v-myc and the 5' half of human c-myc. No hybridization was observed between HCMV fragments and the 3' v-myc and 3' human c-myc probes. In Towne DNA, myc homologous sequences mapped in four regions within the long unique segment (0.070-0.094, 0.134-0.156, 0.454-0.470, and 0.591-0.605 map unit) and one region in each of the short terminal repeats (0.832-0.847 and 0.984-1.0 map unit). In strain AD169, myc homology mapped in three regions within the long unique segment (0.123-0.147, 0.174-0.198, and 0.583-0.606 map unit) and one region in each of the short terminal repeats (0.833-0.863 and 0.976-1.0 map unit). By utilizing probes specific for the 5' and 3' portions of v-myc and human c-myc, we established that the regions of homology in a specific subset of HCMV restriction fragments corresponded to the 5' half of myc and were not due to MC29 viral helper sequences, flanking cellular sequences, or binding of probe to G.C-rich DNA.

Isolation of human cytomegalovirus DNA from infected cells.

Human cytomegalovirus (HCMV) DNA was extracted from infected human embryo fibroblast cultures using the Hirt, Triton-NaCl, and total extraction methods. The Hirt method gave a maximum yield of 60% and was 5- to 10-fold more efficient than the Triton-NaCl method for the extraction of HCMV DNA. However, both methods gave comparable yields ranging from 70 to 75% for the extraction of herpes simplex virus type 1 (HSV1) DNA from infected cells. The Hirt-extracted HCMV DNA contained from 5 to 10% contaminating mitochondrial DNA which could be removed after centrifugation in CsCl-ethidium bromide density gradients. The Hirt-extracted HCMV DNA was analyzed by velocity sedimentation in sucrose gradients and was found to sediment similar to HSV1 55S DNA. Additionally, the Hirt-extracted HCMV DNA was shown to be similar to virion-extracted HCMV DNA in plaquing efficiency and by HindIII restriction digest patterns.

Neoplastic transformation by a cloned human cytomegalovirus DNA fragment uniquely homologous to one of the transforming regions of herpes simplex virus type 2.

Specific DNA fragments of human cytomegalovirus strain Towne exhibited sequence homology to the transforming regions of herpes simplex virus type 2 (HSV-2) when examined by nitrocellulose filter hybridization under nonstringent conditions. Cloned Towne Xba I fragments B and C were homologous to both Bgl II transforming fragments N and C of HSV-2 DNA, whereas cloned Towne Xba I fragment E was uniquely homologous to HSV-2 Bgl II fragment C. Furthermore, Towne Xba I fragment E exhibited homology to a unique fragment of cytomegalovirus strain AD169 but lacked homology to the recently identified Xba I transforming (focus-forming) fragment N. Normal diploid Syrian hamster embryo cells transfected with cloned Towne Xba I fragment E displayed colonies of refractile, rapidly dividing cells which escaped senescence to form immortal cell lines. At early passages, these lines exhibited growth in 2% serum and formed small (less than 0.1 mm) colonies in 0.3% agarose. Serial passaging resulted in the appearance of large (greater than 0.25 mm) colonies in agarose, indicating the involvement of more than one step in Towne Xba I fragment E-induced transformation of the diploid hamster embryo cells. NIH 3T3 cells transfected with Towne Xba I fragment E rapidly displayed large colonies in agarose and tumors in vivo.

Tumorigenicity, metastasis and suppression of MHC class-I expression in murine fibroblasts transformed by mutant v-ras deficient in GTP binding.

We have introduced point mutations in v-rasH to study their effects on biochemical and biological properties of the ras-encoded protein p21. Several of these mutant proteins do not bind GTP and thus lack GTPase activity, while others were shown to have their GTP binding reduced. We have introduced these ras mutants into NIH 3T3 fibroblastoid cells to study major parameters of clinical importance which are associated with neoplastic transformation, particularly MHC expression in cells, metastasis and tumorigenesis in both nude mice and immune competent mice. Our data show that certain mutations in v-ras differentially affect the expression of the transformed phenotype. Mutant ras molecules deficient in GTP binding fail to generate rapidly progressing tumors in immune competent mice, and not all morphologically transformed cells were capable of experimental metastasis. Cells transformed by certain v-ras mutants form tumors in immunocompetent mice and show reduced expression of MHC class-I antigens. Other cells are morphologically transformed and tumorigenic in athymic nude mice, but fail to form tumors in normal mice and show levels of MHC class-I antigen expression similar to non-transformed 3T3 cells. The inverse relationship between MHC class-I-antigen expression and the degree of transformation in fibroblastoid cells suggests that the ras gene product could be involved in regulating MHC expression.

Inhibition of human immunodeficiency virus type 1 transcription and replication by DNA sequence-selective plant lignans.

A plant lignan, 3'-O-methyl nordihydroguaiaretic acid (3'-O-methyl NDGA, denoted Malachi 4:5-6 or Mal.4; molecular weigth 316), was isolated from Larrea tridentata and found to be able to inhibit human immunodeficiency virus (HIV) Tat-regulated transactivation in vivo, induce protection of lymphoblastoid CEM-SS cells from HIV (strain IIIB) killing, and suppress the replication of five HIV-1 strains (WM, MN, VS, JR-CSF, and IIIB) in mitogen-stimulated peripheral blood mononuclear cells, all in a dose-dependent manner. Mal.4 inhibits both basal transcription and Tat-regulated transactivation in vitro. The target of Mal.4 has been localized to nucleotides -87 to -40 of the HIV long terminal repeat. Mal.4 directly and specifically interferes with the binding of Sp1 to Sp1 sites in the HIV long terminal repeat. By inhibiting proviral expression, Mal.4 may be able to interrupt the life cycles of both wild-type and reverse transcriptase or protease mutant viruses in HIV-infected patients.