Antibody binding in proximity to the receptor/glycoprotein complex leads to a basal level of virus neutralization.

Hypothetically, antibodies may neutralize enveloped viruses by diverse mechanisms, such as disruption of receptor binding, interference with conformational changes required for virus entry, steric hindrance, or virus aggregation. Here, we demonstrate that retroviral infection mediated by the avian sarcoma-leukosis virus (ASLV-A) envelope glycoproteins can be neutralized by an antibody directed against a functionally unimportant component of a chimeric receptor protein. Thus, the binding of an antibody in proximity to the retroviral envelope glycoprotein-receptor complex, without binding to the entry machinery itself, results in neutralization. This finding provides additional support for the hypothesis that steric hindrance is sufficient for antibody-mediated neutralization of retroviruses.

Expanded tropism and altered activation of a retroviral glycoprotein resistant to an entry inhibitor peptide.

The envelope of class I viruses can be a target for potent viral inhibitors, such as the human immunodeficiency virus type 1 (HIV-1) inhibitor enfuvirtide, which are derived from the C-terminal heptad repeat (HR2) of the transmembrane (TM) subunit. Resistance to an HR2-based peptide inhibitor of a model retrovirus, subgroup A of the Avian Sarcoma and Leukosis Virus genus (ASLV-A), was studied by examining mutants derived by viral passage in the presence of inhibitor. Variants with reduced sensitivity to inhibitor were readily selected in vitro. Sensitivity determinants were identified for 13 different isolates, all of which mapped to the TM subunit. These determinants were identified in two regions: (i) the N-terminal heptad repeat (HR1) and (ii) the N-terminal segment of TM, between the subunit cleavage site and the fusion peptide. The latter class of mutants identified a region outside of the predicted HR2-binding site that can significantly alter sensitivity to inhibitor. A subset of the HR1 mutants displayed the unanticipated ability to infect nonavian cells. This expanded tropism was associated with increased efficiency of envelope triggering by soluble receptor at low temperatures, as measured by protease sensitivity of the surface subunit (SU) of envelope. In addition, expanded tropism was linked for the most readily triggered mutants with increased sensitivity to neutralization by SU-specific antiserum. These observations depict a class of HR2 peptide-selected mutations with a reduced activation threshold, thereby allowing the utilization of alternative receptors for viral entry.

Heptad repeat 2-based peptides inhibit avian sarcoma and leukosis virus subgroup a infection and identify a fusion intermediate.

Fusion proteins of enveloped viruses categorized as class I are typified by two distinct heptad repeat domains within the transmembrane subunit. These repeats are important structural elements that assemble into the six-helix bundles characteristic of the fusion-activated envelope trimer. Peptides derived from these domains can be potent and specific inhibitors of membrane fusion and virus infection. To facilitate our understanding of retroviral entry, peptides corresponding to the two heptad repeat domains of the avian sarcoma and leukosis virus subgroup A (ASLV-A) TM subunit of the envelope protein were characterized. Two peptides corresponding to the C-terminal heptad repeat (HR2), offset from one another by three residues, were effective inhibitors of infection, while two overlapping peptides derived from the N-terminal heptad repeat (HR1) were not. Analysis of envelope mutants containing substitutions within the HR1 domain revealed that a single amino acid change, L62A, significantly reduced sensitivity to peptide inhibition. Virus bound to cells at 4 degrees C became sensitive to peptide within the first 5 min of elevating the temperature to 37 degrees C and lost sensitivity to peptide after 15 to 30 min, consistent with a transient intermediate in which the peptide binding site is exposed. In cell-cell fusion experiments, peptide inhibitor sensitivity occurred prior to a fusion-enhancing low-pH pulse. Soluble receptor for ASLV-A induces a lipophilic character in the envelope which can be measured by stable liposome binding, and this activation was found to be unaffected by inhibitory HR2 peptide. Finally, receptor-triggered conformational changes in the TM subunit were also found to be unaffected by inhibitory peptide. These changes are marked by a dramatic shift in mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, from a subunit of 37 kDa to a complex of about 80 kDa. Biotinylated HR2 peptide bound specifically to the 80-kDa complex, demonstrating a surprisingly stable envelope conformation in which the HR2 binding site is exposed. These experiments support a model in which receptor interaction promotes formation of an envelope conformation in which the TM subunit is stably associated with its target membrane and is able to bind a C-terminal peptide.

Characterization of a naphthalene derivative inhibitor of retroviral integrases.

The retroviral integrase protein (IN) is essential for virus replication and, therefore, an attractive target for the development of inhibitors to treat human immunodeficiency virus (HIV) infection. Diverse classes of compounds that are active against this protein have been discovered using in vitro assays. Here we describe the synthesis of a novel compound, 3,8-dibromo-7-amino-4-hydroxy-2-naphthalenesulfonic acid (2BrNSA), which inhibits the in vitro activities of the full-length HIV-1 and avian sarcoma virus (ASV) integrases, and the isolated catalytic core fragment of the ASV protein (residues 52-207). The compound also inhibits retroviral reverse transcriptase in vitro, but the IC(50) for the HIV-1 enzyme is almost two orders of magnitude higher than for HIV-1 integrase. The inhibitor was found to be active in cell culture, preventing reporter gene transduction of HeLa cells by both ASV and HIV-1 vectors. Neither viral attachment nor uptake into cells appeared to be affected in these transfections, whereas accumulation of vector DNA and its joining to host DNA were both drastically reduced in the presence of the inhibitor. Propagation of two different strains of replication-competent HIV-1 in human peripheral blood mononuclear cells (PBMCs) was also reduced by the inhibitor, allowing survival of a substantial number of cells in the treated cultures. Based on these and other results we speculate that binding of 2BrNSA to integrase in infected cells interferes not only with its catalytic activity but also with critical interactions that are required for the formation or function of the reverse transcriptase complex. Its activity in cell culture suggests that this inhibitor may provide a valuable new lead for further development of drugs that target early steps in the HIV life cycle.

Evidence that the retroviral DNA integration process triggers an ATR-dependent DNA damage response.

Caffeine is an efficient inhibitor of cellular DNA repair, likely through its effects on ATM (ataxia telangiectasia mutated) and ATR (ATM and Rad3-related) kinases. Here, we show that caffeine treatment causes a dose-dependent reduction in the total amount of HIV-1 and avian sarcoma virus retroviral vector DNA that is joined to host DNA in the population of infected cells and also in the number of transduced cells. These changes were observed at caffeine concentrations that had little or no effect on overall cell growth, synthesis, and nuclear import of the viral DNA, or the activities of the viral integrase in vitro. Substantial reductions in the amount of host-viral-joined DNA in the infected population, and in the number of transductants, were also observed in the presence of a dominant-negative form of the ATR protein, ATRkd. After infection, a significant fraction of these cells undergoes cell death. In contrast, retroviral transduction is not impeded in ATM-deficient cells, and addition of caffeine leads to the same reduction that was observed in ATM-proficient cells. These results suggest that activity of the ATR kinase, but not the ATM kinase, is required for successful completion of the viral DNA integration process and/or survival of transduced cells. Components of the cellular DNA damage repair response may represent potential targets for antiretroviral drug development.

Functional oligomeric state of avian sarcoma virus integrase.

Retroviral integrase, one of only three enzymes encoded by the virus, catalyzes the essential step of inserting a DNA copy of the viral genome into the host during infection. Using the avian sarcoma virus integrase, we demonstrate that the enzyme functions as a tetramer. In presteady-state active site titrations, four integrase protomers were required for a single catalytic turnover. Volumetric determination of integrase-DNA complexes imaged by atomic force microscopy during the initial turnover additionally revealed substrate-induced assembly of a tetramer. These results suggest that tetramer formation may be a requisite step during catalysis with ramifications for antiviral design strategies targeting the structurally homologous human immunodeficiency virus, type 1 (HIV-1) integrase.

Nuclear entry and CRM1-dependent nuclear export of the Rous sarcoma virus Gag polyprotein.

The retroviral Gag polyprotein directs budding from the plasma membrane of infected cells. Until now, it was believed that Gag proteins of type C retroviruses, including the prototypic oncoretrovirus Rous sarcoma virus, were synthesized on cytosolic ribosomes and targeted directly to the plasma membrane. Here we reveal a previously unknown step in the subcellular trafficking of the Gag protein, that of transient nuclear localization. We have identified a targeting signal within the N-terminal matrix domain that facilitates active nuclear import of the Gag polyprotein. We also found that Gag is transported out of the nucleus through the CRM1 nuclear export pathway, based on observations that treatment of virus-expressing cells with leptomycin B resulted in the redistribution of Gag proteins from the cytoplasm to the nucleus. Internal deletion of the C-terminal portion of the Gag p10 region resulted in the nuclear sequestration of Gag and markedly diminished budding, suggesting that the nuclear export signal might reside within p10. Finally, we observed that a previously described matrix mutant, Myr1E, was insensitive to the effects of leptomycin B, apparently bypassing the nuclear compartment during virus assembly. Myr1E has a defect in genomic RNA packaging, implying that nuclear localization of Gag might be involved in viral RNA interactions. Taken together, these findings provide evidence that nuclear entry and egress of the Gag polyprotein are intrinsic components of the Rous sarcoma virus assembly pathway.

Ubiquitin is part of the retrovirus budding machinery.

Retroviruses contain relatively large amounts of ubiquitin, but the significance of this finding has been unknown. Here, we show that drugs that are known to reduce the level of free ubiquitin in the cell dramatically reduced the release of Rous sarcoma virus, an avian retrovirus. This effect was suppressed by overexpressing ubiquitin and also by directly fusing ubiquitin to the C terminus of Gag, the viral protein that directs budding and particle release. The block to budding was found to be at the plasma membrane, and electron microscopy revealed that the reduced level of ubiquitin results in a failure of mature virus particles to separate from each other and from the plasma membrane during budding. These data indicate that ubiquitin is actually part of the budding machinery.

Sparoxomycins A1 and A2, new inducers of the flat reversion of NRK cells transformed by temperature sensitive Rous sarcoma virus. I. Taxonomy of the producing organism, fermentation and biological activity.

Streptomyces sparsogenes SN-2325 isolated from a soil sample collected in Hokkaido, was found to produce sparoxomycins A1 and A2, new modulators of proliferation of mammalian cells. Sparoxomycins A1 and A2 reverse the morphology of temperature-sensitive mutant Rous sarcoma virus-infected NRK cells (src(ts)-NRK cells) from the transformed phenotype to the normal phenotype at the permissive temperature.

Isolation of virus-neutralizing RNAs from a large pool of random sequences.

RNA and ribonuclease-resistant RNA analogs that bound and neutralized Rous sarcoma virus (RSV) were isolated from a large pool of random sequences by multiple cycles of in vitro selection using infectious viral particles. The selected RNA pool of RSV-binding sequences at a concentration of 0.16 microM completely neutralized the virus. Of 19 sequences cloned from the selected pool, 5 inhibited RSV infection. The selected RNA and RNA analogs were shown to neutralize RSV by interacting with the virus, rather than by adversely affecting the host cells. The selection of the anti-RSV RNA and RNA analogs by intact virions immediately suggests the potential application of this approach to develop RNA and RNA analogs as inhibitors of other viruses such as human immunodeficiency virus.

Synthesis and antiviral activities of 9-cyclopentylpurine derivatives.

The synthesis and anti-RSV (Rouse Sarcoma Virus) activity of HMCA, (+/-)-9-(2-hydroxymethyl-cyclopenthyl)-adenine, and its derivatives are described. It has been demonstrated that trans-HMCA has greater anti-RSV activity in tissue culture than cis-HMCA.

Dietary arginine influences Rous sarcoma growth in a major histocompatibility B complex progressor genotype.

L-Arginine (L-Arg) can serve as a substrate for the production of reactive nitrogen intermediates. One of these metabolites, nitric oxide, has been shown to possess significant antitumor properties in vitro. To investigate the importance of this system in vivo, we have examined the dietary L-Arg host tumor interaction in the chicken. Since chickens are incapable of de novo L-Arg synthesis, concentration of this amino acid is readily controlled by diet. Line UNH 105 New Hampshire chickens having the major histocompatibility complex genotype, B24/B24, were used to study in vivo effects of dietary L-Arg on Rous sarcoma growth. After 5 weeks on a standard diet, 119 chicks were fed either a basal (0.92% L-Arg) diet or a high arginine (2.40% L-Arg) diet. One week later, chicks were wing-web inoculated with subgroup A Rous sarcoma virus. Tumor growth was monitored weekly for 12 weeks after inoculation. Plasma L-Arg levels and body weights from birds on each dietary treatment were analyzed. Neither body weight gains nor latent period for tumor development was affected by diet. However, plasma L-Arg levels were significantly different between dietary treatments (basal, 0.245 +/- 0.01 mumol/ml; high, 0.738 +/- 0.03 mumol/ml). In addition, mean tumor size scores were significantly (P less than 0.05) lower over time in chickens fed the high L-Arg diet. The results suggest that dietary L-Arg in excess of the amount required for growth reduces tumor load.

Ascorbate stabilizes the differentiated state and reduces the ability of Rous sarcoma virus to replicate and to uniformly transform cell cultures.

In primary avian tendon cells, Rous sarcoma virus can coexist or completely take over the cell. Infection, at high multiplicity or under conditions that promote high virus production (no ascorbate and high serum concentrations), results in almost complete oncogenic transformation of the culture. This is indicated in part by a radical change in morphology, growth at high cell density, and a dramatic drop in the production of procollagen from approximately 50% to approximately 3% of total protein synthesis. In contrast, infection at low multiplicity, infection with a replication defective virus, or the presence of ascorbate restrict the ability of the virus to transform the culture. Thus, there appears to be a balance between the normal and transformed states of the cell that can be shifted depending on the cellular environment and the level of infection. Ascorbate stabilizes the normal state by reducing virus production and promoting the synthesis of differentiated proteins.

Antiretroviral activities of anthraquinones and their inhibitory effects on reverse transcriptase.

Alizarin complexone (AC), alizarin Red S (ARS) and various other anthraquinones were evaluated for their inhibitory effects on Rous-associated virus 2 reverse transcriptase (RAV-2 RT). Some 1,2-dihydroxyanthraquinones were active against this enzyme and AC was the most potent inhibitor among these compounds [50% inhibitory concentration (IC50): 3.8 micrograms/ml]. AC slightly inhibited Rous sarcoma virus RT (RSV RT) and human immunodeficiency virus type 1 RT (HIV-1 RT) (IC50: 100 micrograms/ml and 45 micrograms/ml, respectively). However, AC efficiently inhibited focus formation by Rous sarcoma virus (RSV) and cytopathogenicity of human immunodeficiency virus type 1 (HIV-1). Simultaneous administration of AC with RSV to newborn chickens also delayed tumor induction by RSV.

Synthesis and structure-activity relationships of 2',3'-dideoxypurine nucleosides as potential antiretroviral agents.

In order to study the structure-activity relationships of 2',3'-dideoxypurine nucleosides as potential anti-retroviral agents, various purine derivatives have been synthesized and tested against rous sarcoma virus (RSV) in chick embryo fibroblast (CEF) cells, and against human immunodeficiency virus (HIV) in human CD4+ T cells (ATH8). Some of the new purine derivatives found to be significant antiretroviral activities. And the correlations of activity between anti-RSV and anti-HIV have shown fairly good values so far.

The effects of 3-deazaguanine on Rous sarcoma virus transformation.

An expression of cellular transformation in chicken embryo fibroblast infected with Rous sarcoma virus (RSV) was inhibited by 3-deazaguanine (3-DG). Re-transformation of normal rat kidney cells infected with a temperature-sensitive mutant of RSV was suppressed by 3-DG when the cells were shifted from the non-permissive temperature (38 degrees C) to the permissive temperature (33 degrees C). Removal of the inhibitor enabled the cells to revert to the transformed state. Finally, 3-DG had little or no effect on replication of RSV in the chicken embryo fibroblast cells.

Preparation of new 7-hydroxyguanine derivatives and their biological activities.

We prepared new 7-hydroxyguanine derivatives, 7-hydroxyguanosine 5'-monophosphate and N2-tetrahydropyranyl-7-hydroxyguanine, and compared biological activities of 7-hydroxyguanine derivatives including nucleosides acquired previously. 7-Hydroxyguanine and its nucleotide inhibited the focus formation of Rous sarcoma virus. Antitumor activities of these derivatives against mouse leukemia L1210 were not so different from one another. Anti-proliferative activities of the derivatives on various human cell lines were significantly different from one another.

Morphological aspects of inhibition of transformation of rat kidney cells infected with a temperature-sensitive mutant of the Rous sarcoma virus by rat fibroblast interferon.

Normal rat kidney cells (NRK) infected with a temperature-sensitive (ts) mutant of Rous Sarcoma virus (RSV) at the permissive temperature (33 degrees C) exhibit the morphological and growth characteristics of the transformed state. By the use of this system, it has been shown that the addition of rat fibroblast interferon to infected cells grown at a nonpermissive temperature (38 degrees C) prevents re-transformation when they are introduced to the permissive temperature. Merely treating cells at the permissive temperature with rat fibroblast interferon causes them to change from the transformed phenotype to a normal phenotype. These effects require the continuous presence of interferon and are quite species specific as observed with the ineffectiveness of mouse fibroblast interferon. Therefore, the results suggest that interferon might restore the growth control of transformed cells which could in part accounts for its anti-tumor effect.

Induction by herbimycin A of contact inhibition in v-src-expressed cells.

Herbimycin A, an inhibitor of pp60src tyrosin kinase, caused src oncogene-expressed cells to become sensitive to contact inhibition, but did not affect ras oncogene-expressed cells. The cell lines tested were temperature sensitive v-src- and temperature sensitive v-ras-integrated nontransformed rat kidney cell line (NRK) (srctsNRK and rastsNRK, respectively) and a wild-type v-src-integrated NIH3T3 (src3T3). srctsNRK cells in densely populated cultures (plated at 1.25 x 10(4) cells/cm2), grown at 33 degrees C in the presence of 0.45 micrograms/ml of herbimycin A, ceased the cell cycle at the G0-G1 stage within 2 days, and the cells showed normal morphology. Upon removal of herbimycin A, the quiescent cells resumed the cell cycle in concert with morphological alteration from 'normal' to 'transformed', and proceeded through the S and M stages successively in a synchronized manner. Cells in the late S stage, compared with those in other stages of the cell cycle, were more sensitive to the killing effect of 5-fluorodeoxyuridine. Such synchronism of the cell cycle was not observed with sparsely populated cultures (2.5 x 10(3) cells/cm2); the cells resumed their asynchronous growth after removal of herbimycin A, although their morphology returned to 'transformed' as in the experiment with the densely populated cultures. The induction by herbimycin A of contact inhibition in densely populated cultures was also observed with src3T3 (grown at 37 degrees C) but not with rastsNRK (grown at 33 degrees C).