Richard K. Bestwick

Inhibition, Escape, and Attenuated Growth of Severe Acute Respiratory Syndrome Coronavirus Treated with Antisense Morpholino Oligomers

ABSTRACT The recently emerged severe acute respiratory syndrome coronavirus (SARS-CoV) is a potent pathogen of humans and is capable of rapid global spread. Peptide-conjugated antisense morpholino oligomers (P-PMO) were designed to bind by base pairing to specific sequences in the SARS-CoV (Tor2 strain) genome. The P-PMO were tested for their capacity to inhibit production of infectious virus as well as to probe the function of conserved viral RNA motifs and secondary structures. Several virus-targeted P-PMO and a random-sequence control P-PMO showed low inhibitory activity against SARS coronavirus. Certain other virus-targeted P-PMO reduced virus-induced cytopathology and cell-to-cell spread as a consequence of decreasing viral amplification. Active P-PMO were effective when administered at any time prior to peak viral synthesis and exerted sustained antiviral effects while present in culture medium. P-PMO showed low nonspecific inhibitory activity against translation of nontargeted RNA or growth of the arenavirus lymphocytic choriomeningitis virus. Two P-PMO targeting the viral transcription-regulatory sequence (TRS) region in the 5′ untranslated region were the most effective inhibitors tested. After several viral passages in the presence of a TRS-targeted P-PMO, partially drug-resistant SARS-CoV mutants arose which contained three contiguous base point mutations at the binding site of a TRS-targeted P-PMO. Those partially resistant viruses grew more slowly and formed smaller plaques than wild-type SARS-CoV. These results suggest PMO compounds have powerful therapeutic and investigative potential toward coronavirus infection.

Inhibition of Multiple Subtypes of Influenza A Virus in Cell Cultures with Morpholino Oligomers

ABSTRACT Peptide-conjugated phosphorodiamidate morpholino oligomers (P-PMO) are single-stranded nucleic acid-like antisense agents that can reduce gene expression by sterically blocking complementary RNA sequence. P-PMO are water soluble and nuclease resistant, and they readily achieve uptake into cells in culture under standard conditions. Eight P-PMO, each 20 to 22 bases in length, were evaluated for their ability to inhibit influenza A virus (FLUAV) A/PR/8/34 (H1N1) replication in cell culture. The P-PMO were designed to base pair with FLUAV RNA sequences that are highly conserved across viral subtypes and considered critical to the FLUAV biological-cycle, such as gene segment termini and mRNA translation start site regions. Several P-PMO were highly efficacious, each reducing viral titer in a dose-responsive and sequence-specific manner in A/PR/8/34-infected cells. Two P-PMO, one designed to target the AUG translation start site region of PB1 mRNA and the other the 3′-terminal region of nucleoprotein viral genome RNA, also proved to be potent against several other FLUAV strains, including A/WSN/33 (H1N1), A/Memphis/8/88 (H3N2), A/Eq/Miami/63 (H3N8), A/Eq/Prague/56 (H7N7), and the highly pathogenic A/Thailand/1(KAN-1)/04 (H5N1). The P-PMO exhibited minimal cytotoxicity in cell viability assays. High efficacy by two of the P-PMO against multiple FLUAV subtypes suggests that these oligomers represent a broad-spectrum therapeutic approach against a high percentage of known FLUAV strains.

Role of a membrane glycoprotein in friend virus-induced erythroleukemia: Studies of mutant and revertant viruses

We previously reported the isolation and characterization of spontaneous, transmissible mutants of Friend spleen focus-forming virus (SFFV) that are nonpathogenic in adult NIH/Swiss mice and that contain abnormalities in nonoverlapping regions of their envelope glycoprotein (env) genes (M. Ruta, R. Bestwick, C. Machida, and D. Kabat, 1983, Proc. Natl. Acad. Sci. USA 80, 4704-4708). In newborn NIH/Swiss mice, these mutant SFFVs form revertants that are pathogenic in mice of all ages. At least two of three studied revertants contain second site env mutations which affect the sizes and proteolytic fragmentation patterns of their encoded glycoproteins. A variety of structural and genetic evidence suggests that the xenotropic- and ecotropic-related regions of the SFFV glycoprotein fold into separate globular domains that are connected by a flexible proline-rich joint. A glutamyl peptide bond within this joint is exceptionally susceptible to cleavage with Staphylococcus aureus V8 protease. Moreover, disulfide bonds occur within the xenotropic-related domain, but not between the globular domains. These results provide strong additional evidence that the env gene is required for SFFV pathogenesis, and they provide a new system for identifying the features of glycoprotein structure and localization which are essential for its leukemogenic activity.

An enhancer sequence instability that diversifies the cell repertoire for expression of a murine leukemia virus.

Studies of recombinants between murine leukemia viruses (MuLVs) that cause thymic or erythroid leukemias have shown that enhancer sequences in the long-terminal repeats (LTRs) can determine the target tissues for pathogenesis. It has been inferred that the enhancers may specifically target viral expression into the cells that then become neoplastic. However, the neoplasms in those studies formed after latencies and contained ultimate viruses (called MCFs) that differed from the injected viruses in their enhancer sequences and envelope (env) genes. Transcriptional activities of LTRs from these proximal and ultimate viruses have not been thoroughly analyzed in different hematopoietic lineages. We present evidence that the enhancer of Friend spleen focus-forming virus (SFFV), an ultimate erythroleukemogenic retrovirus, contains an unstable 42-nucleotide direct repeat. Other ultimate erythroleukemogenic MuLVs (Friend MCFs) contain an enhancer nearly identical to that of SFFV both in its sequence and in its specific instability. The instability occurs in sequences that contain inverted repeats and we propose that it occurs by a simple reverse transcriptase hop mechanism. We constructed plasmids that contain the two forms of the SFFV LTR linked to the bacterial chloramphenicol acetyltransferase (CAT) gene, and we compared these in transient transfection assays with LTR-CAT plasmids constructed from Friend and Moloney MuLVs. The assays employed erythroleukemia cells, thymic lymphoma cells, and fibroblasts. The tropisms of expression correlated only weakly with tissue specificities of pathogenesis and each LTR was active in all cells. The SFFV 42-nucleotide duplication reduced expression in erythroid cells and increased expression in fibroblasts. We conclude that retroviral enhancers do not stringently direct gene expression into specific cell lineages, but on the contrary they are leaky and contain replicative instabilities that also may facilitate viral entrenchment throughout the host. These results have important implications for understanding murine retroviral evolution and the multi-step process of leukemogenesis.

Inhibition and escape of SARS-CoV treated with antisense morpholino oligomers.

Identification of potential SARS-CoV antiviral compounds has progressed swiftly, thanks in part to the availability of bioinformatic and virus structural data. Antivirals that target the SARS-CoV superfamily 1 helicase and the 3C-related serine proteinase with low micromolar EC50 values have been reported. The papain-related cysteine proteinase may prove to be an unsuitable target, as a coronavirus molecular clone lacking one of the two known cleavage sites for this enzyme displayed only minor growth defects in cell culture. Other confirmed and putative viral enzymes including the polymerase, poly(U)specific endo-ribonuclease homolog, S-adenosyl-methionine-dependent ribose 2’-Omethyltransferase, and cyclic phosphodiesterase represent plausible anti-SARS targets. Antivirals targeting the interaction of the viral spike protein with the ACE-2 receptor, or with the spike-mediated fusion event, and showing micromolar-scale efficiency in cell culture, have been reported. Several groups have also reported antiviral in vitro efficacy with siRNAs. The antisense agents directed against single-stranded RNA are known to act by two general mechanisms: by causing damage to an RNA strand containing the complementary “target” sequence through priming of endogenous RNase H activity, or by stably binding to and steric interference with targeted RNA function. Phosphorodiamidate morpholino oligomers (PMO) act by the latter mechanism, duplexing to specific RNA sequence by Watson-Crick base pairing and forming a steric block. The most frequently successful targeting strategies for PMO-based gene knockdown involve interfering with translation initiation or masking splice sites. We recently demonstrated antiviral effects in vitro for one peptide-conjugated PMO (P-PMO) complementary to the AUG translation start site region of a murine coronavirus replicase

Frequent hereditable shutdown of murine retrovirus gene expression in murine cell lines.

Friend spleen focus-forming virus shuts down its gene expression frequently (ca. 10(-3) per generation) in a cis-dominant hereditable fashion in various murine cells but much less frequently in rat cells (less than 10(-6) per generation). Thus, nonexpresser variants were isolated at high frequency from murine cell lines by immunoselection directed against virus-encoded cell surface glycoproteins and also simply by subcloning cells from lines which had been cultured for many generations. Studies of independently infected cell clones indicate that shutdown is a property of the cell line rather than of the specific proviral site. Nucleic acid blot analyses suggest that shutdown correlates with decreased transcription. Moreover, preliminary evidence indicates that other murine retroviruses also shut down frequently in murine but not in rat cells and that shutdown of replication-competent murine leukemia viruses with accompanying loss in interference to superinfection may be the rate-limiting reaction enabling cells to acquire multiple proviruses in their chromosomes. High-frequency shutdown in vivo would have important pathological consequences.