FK506, a secondary metabolite produced by Streptomyces, presents a novel antiviral activity against Orthopoxvirus infection in cell culture.

AIMS: To investigate the antiviral potential of the macrolide FK506, produced by Streptomyces tsukubaensis, against Orthopoxvirus infection in cell culture, and determine the replicative stage of viral cycle affected by the treatment. METHODS AND RESULTS: Cell lines were infected with different Orthopoxviruses and treated with FK506. The macrolide inhibited the replication of the prototypic Orthopoxvirus, vaccinia virus strain WR, with an IC50 of 12.05 micromol l(-1). Progeny production of other Orthopoxviruses was also inhibited by FK506 at noncytotoxic concentrations, as evaluated by the neutral-red uptake assay and metabolic labelling of cellular proteins. By Western blot assay, we detected a severe inhibition (approximately 87.6% +/- 2.78%) of VV strain WR post-replicative protein synthesis. A similar reduction of virus DNA accumulation, as observed by slot-blot assay, probably accounts for the subsequent inhibition of virus late proteins. CONCLUSIONS: The macrolide FK506, isolated from S. tsukubaensis, presents a novel anti-poxvirus activity, probably targeting the stage of DNA replication during Orthopoxvirus infection. SIGNIFICANCE AND IMPACT OF THE STUDY: The secondary metabolite FK506, isolated from the culture filtrate of S. tsukubaensis, shows a pleiotropic range of activities, and might be a valuable tool as a lead structure in the generation of non-immunosuppressant analogues with strong anti-poxvirus activity.

Protein synthesis inhibitory activity in culture filtrates from new strains of Streptomyces isolated from Brazilian tropical soils.

AIMS: To investigate the effect of the culture supernatants from three newly isolated Streptomyces strains, 221, 235 and 606 on eukaryotic cells. METHODS AND RESULTS: Cell lines were treated with the culture filtrates and assayed for protein synthesis by metabolic labelling, followed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis analysis. RNA synthesis was investigated by [5-3H]uridine incorporation. The three culture filtrates presented a strong inhibitory activity, reducing total protein synthesis of different eukaryotic cell lines by more than 85%. No effect on cellular RNA synthesis was detected. The culture filtrates did not affect the growth of the prokaryotic cells tested. CONCLUSIONS: These new Streptomyces strains, recently isolated from Brazilian tropical soils, produce molecule(s) with inhibitory activity specific to eukaryote protein synthesis. SIGNIFICANCE AND IMPACT OF THE STUDY: Streptomyces strains 221, 235 and 606, probably representing new species, might produce new bioactive compound(s), and can be used as valuable tools to study the protein synthesis pathway in eukaryotes.

An emergent poxvirus from humans and cattle in Rio de Janeiro State: Cantagalo virus may derive from Brazilian smallpox vaccine.

The biological properties of poxvirus isolates from skin lesions on dairy cows and milkers during recent exanthem episodes in Cantagalo County, Rio de Janeiro State, Brazil, were more like vaccinia virus (VV) than cowpox virus. PCR amplification of the hemagglutinin (HA) gene substantiated the isolate classification as an Old World orthopoxvirus, and alignment of the HA sequences with those of other orthopoxviruses indicated that all the isolates represented a single strain of VV, which we have designated Cantagalo virus (CTGV). HA sequences of the Brazilian smallpox vaccine strain (VV-IOC), used over 20 years ago, and CTGV showed 98.2% identity; phylogeny inference of CTGV, VV-IOC, and 12 VV strains placed VV-IOC and CTGV together in a distinct clade. Viral DNA restriction patterns and protein profiles showed a few differences between VV-IOC and CTGV. Together, the data suggested that CTGV may have derived from VV-IOC by persisting in an indigenous animal(s), accumulating polymorphisms, and now emerging in cattle and milkers as CTGV. CTGV may represent the first case of long-term persistence of vaccinia in the New World.

Inhibition of vaccinia virus replication by cyclosporin A analogues correlates with their affinity for cellular cyclophilins.

The mechanism by which cyclosporin A (CsA) inhibits vaccinia virus (VV) replication is still unclear. The present study addresses the question of whether CsA-binding proteins named cyclophilins (Cyps) are involved in the anti-VV activity of CsA. Six CsA analogues were analysed, and their affinity for Cyps in VV-infected BSC-40 cells and their potency as inhibitors of VV replication were evaluated. It was demonstrated that analogues with strong Cyp-binding activity, such as CsC, CsG and [MeAla6]CsA, also exhibit a strong antiviral effect. In contrast, drugs with low ([MeBm2t1]CsA and CsH) or no ([MeLeu11]CsA) affinity for Cyps show poor or no antiviral activity. The data obtained suggest a correlation between the ability of CsA to block VV replication and Cyp binding activity, and indicate the involvement of Cyps in the VV replicative cycle. They also suggest that the anti-VV action of CsA may occur by a pathway distinct from that involved in the immunosuppressive effect of the drug.

Cyclosporin A inhibits vaccinia virus replication in vitro.

The effect of the immune modulator, Cyclosporin A (CsA) on vaccinia virus replication has been examined in cell cultures. In the present study we report that CsA is anti-viral towards vaccinia virus. Viral yield was inhibited by more than 97% after 24 h postinfection in the presence of 16 microM to 40 microM CsA. An analysis of the infectious cycle in greater detail revealed that CsA did not effect the total level of [35S] methionine incorporation into vaccinia infected cells. However, both early and late viral gene expression were inhibited by CsA. Late viral protein synthesis appeared to be more sensitive to the drug. At least one late viral polypeptide of approximately Mr 38,000 was virtually undetected up to 8 h postinfection in the presence of 40 microM CsA. Host protein synthesis which is normally inhibited by the virus was not turned off until very late in infection. Viral DNA replication was also inhibited by the addition of CsA at levels comparable to those observed for late protein synthesis.

Detection of RNA-protein complex in vaccinia virus core in vitro transcription system.

The incubation of vaccinia virus cores in appropriate conditions promotes the release of core proteins into a supernatant fraction. Under transcription assay conditions core mRNAs are extruded in association with viral core proteins, however the presence of these proteins within the core particle is not essential for RNA synthesis and extrusion. The RNA-protein complex is resistant to micrococcal nuclease. Five proteins of 60K, 43K, 28K, 18K and 14.5K with RNA-binding abilities have been identified by [32P]RNA overlay protein blot assays. These proteins are likely to be a component of the viral ribonucleoprotein complex since core basic proteins with similar MrS have been identified and at least one RNA-binding protein is predicted in the vaccinia virus genome.

Proteins released from vaccinia cores are probably not involved in protein synthesis inhibition in vitro.

1. It is known that vaccinia core proteins are released into the supernatant fraction when cores are incubated under appropriate conditions. When prepared in the absence of viral transcription this fraction inhibits in vitro protein synthesis. 2. We show here that after incubation, the cores loose the capability to inhibit protein synthesis. Furthermore, we show that no inhibition of translation is observed with supernatant fractions prepared from transcribing cores. 3. SDS-PAGE analysis of the supernatant fraction of cores obtained in the presence and absence of viral transcription indicated that their protein composition is similar and that approximately 17 peptides are released from the cores and that 4 are phosphorylated. 4. We conclude that the proteins associated with vaccinia cores and released in the presence of ribonucleotides are not involved in protein synthesis inhibition.

Protein synthesis in vaccinia virus-infected cells. I. Effect of hypertonic shock recovery.

Human Hep-2 cells were submitted to hypertonic shock (210 mM NaCl) to block host protein synthesis before infection with vaccinia virus. With the start of infection, the medium isotonicity (116 mM NaCl) was restored, and the effect of viral infection on the recovery of host polyribosomes and protein synthesis was studied. Although host translation blockage was released together with infection, vaccinia virus did not affect immediately host protein synthesis. During the first hour of recovery, infected cells could perfectly rebuild the polyribosome profile and recuperate the rate of protein synthesis. Also, during recovery, formation of the initiation complex for protein synthesis was not affected by viral infection. In this period, viral mRNA and proteins were detected by slot blot and SDS-polyacrylamide gel electrophoresis. The inhibitory effect of vaccinia virus on host translation was observed after the second hour of infection. These findings suggest that vaccinia virus-mediated shutoff occurs in a later period during infection, in parallel with viral mRNA accumulation in the polyribosomes and after the on-set of viral DNA replication.

Proteins released from vaccinia cores are probably not involved in protein synthesis inhibition in vitro

It is known that vaccinia core proteins are released into the supernatant fraction when cores are incubated under appropriate conditions. When prepared in the absence of viral transcription this fraction inhibits in vitro protein synthesis. We show here that after incubation, the cores loose the capability to inhibit protein synthesis. Furthermore, we show that no inhibition of translation is observed with supernatant fractions prepared from transcribing cores. SDS-PAGE analysis of the supernatantt fraction of cores obtained in the presence and absence of viral transcription indicated that their protein composiition is similar and approximately 17 peptides are released from the cores and that 4 are phosphorylated. We conclude that the proteins associated with vaccinia cores and released in the presence of ribonucleotides are not involved in protein synthesis inhibition

Vaccinia core transcription prevents in vitro depolymerization of polyribosomes in rabbit reticulocyte lysate.

When a rabbit reticulocyte lysate is incubated in the presence of vaccinia cores, protein synthesis is impaired at the level of the initiation step and the polyribosomes are depolymerized. However, when the same system is coupled with virus transcription: a) protein synthesis is restored, b) the initiation step is not inhibited, and c) the polyribosomes are not disaggregated. A viral factor activated in the absence of virus transcription and not activated when RNA synthesis occurs may be involved in the early mechanism of protein synthesis inhibition by vaccinia virus.

Vaccinia core transcription prevents in vitro depolymerization of polyribosomes in rabbit reticulocyte lysate

When a rabbit reticulocyte lysate is incubated in the presence of vaccina cores, protein synthesis is umpaired at the level of the initiation step and the polyribosomes are depolymerized. However, when the same system is coupled with virus transcription: a) protein synthesis is restored, b) the initiation step is not inhibited, and c) the polyribosomes are not disaggregated. A viral factor activated in the absence of virus transcription and not activated when RNA synthesis occurs may be involved in the early mechanism of protein synthesis inhibition by vaccinia virus