Search

Influenza A viruses constitute a major and ongoing global public health concern. Current antiviral strategies target viral gene products; however, the emergence of drug-resistant viruses highlights the need for novel antiviral approaches. Cleavage of the influenza virus hemagglutinin (HA) by host cell proteases is crucial for viral infectivity and therefore presents a potential drug target. Peptide-conjugated phosphorodiamidate morpholino oligomers (PPMO) are single-stranded-DNA-like antisense agents that readily enter cells and can act as antisense agents by sterically blocking cRNA. Here, we evaluated the effect of PPMO targeted to regions of the pre-mRNA or mRNA of the HA-cleaving protease TMPRSS2 on proteolytic activation and spread of influenza viruses in human Calu-3 airway epithelial cells. We found that treatment of cells with a PPMO (T-ex5) designed to interfere with TMPRSS2 pre-mRNA splicing resulted in TMPRSS2 mRNA lacking exon 5 and consequently the expression of a truncated and enzymatically inactive form of TMPRSS2. Altered splicing of TMPRSS2 mRNA by the T-ex5 PPMO prevented HA cleavage in different human seasonal and pandemic influenza A viruses and suppressed viral titers by 2 to 3 log10 units, strongly suggesting that TMPRSS2 is responsible for HA cleavage in Calu-3 airway cells. The data indicate that PPMO provide a useful reagent for investigating HA-activating proteases and may represent a promising strategy for the development of novel therapeutics to address influenza infections. [ABSTRACT FROM AUTHOR]

Abstract: A significant consequence of equine arteritis virus (EAV) infection of horses is persistence of the virus in a variable percentage of infected stallions. We recently established an in vitro model of EAV persistence in cell culture for the purpose of furthering our understanding of EAV biology in general and viral persistence in the stallion in particular. In this study we investigated whether persistently infected HeLa cells could be cured of EAV infection by treatment with an antisense peptide-conjugated phosphorodiamidate morpholino oligomer (PPMO) designed to target the 5′-terminal region of the EAV genome. We found that persistently infected HeLa cells passaged three times in the presence of 5–10μM EAV-specific PPMO produced no detectable virus. The PPMO-cured HeLa cells were free of infectious virus, viral antigen and EAV RNA as measured by plaque assay, indirect immunofluorescence assay and RT-PCR, respectively. Furthermore, when re-challenged with EAV at several passages after discontinuation of PPMO treatments, PPMO-cured HeLa cells were found to be refractory to re-infection and to the re-establishment of viral persistence. While these findings demonstrate that PPMO can be used to eliminate persistent EAV infection in cell culture, the efficacy of PPMO against EAV in vivo remains to be addressed. [Copyright &y& Elsevier]

Abstract: Measles virus (MeV) is a highly contagious human pathogen. Despite the success of measles vaccination programs, measles is still responsible for an estimated 245,000 deaths each year. There are currently no antiviral compounds available for the treatment of measles. Peptide-conjugated phosphorodiamidate morpholino oligomers (PPMO) are antisense compounds that enter cells readily and can interfere with mRNA function by steric blocking. A panel of PPMO was designed to target various sequences of MeV RNA that are known to be important for viral replication. Five PPMO, targeting MeV genomic RNA or mRNA, inhibited the replication of MeV, in a dose-responsive and sequence-specific manner in cultured cells. One of the highly active PPMO (PPMO 454), targeting a conserved sequence in the translation start site of the mRNA coding for the nucleocapsid protein, inhibited multiple genotypes of MeV. This report provides evidence that PPMO treatment represents a promising approach for developing antiviral agents against measles and other paramyxoviruses. [Copyright &y& Elsevier]

Respiratory syncytial virus (RSV) is a major cause of lower respiratory tract infection in infants, young children, and high-risk adults. Currently, there is no vaccine to prevent RSV infection, and the available therapeutic agents are of limited utility. Peptide-conjugated phosphorodiamidate morpholino oligomers (PPMOs) are a class of antisense agents that can enter cells readily and interfere with viral protein expression through steric blocking of complementary RNA. Two antisense PPMOs, designed to target sequence that includes the 5′-terminal region and translation start-site region of RSV L mRNA, were tested for anti-RSV activity in cultures of two human-airway cell lines. Both PPMOs showed minimal cytotoxicity and one of them, (AUG-2), reduced viral titers by >2.0 log10. Intranasal (i.n.) treatment of BALB/c mice with AUG-2 PPMO before the RSV inoculation produced a reduction in viral titer of 1.2 log10 in lung tissue at day 5 postinfection (p.i.), and attenuated pulmonary inflammation at day 7 postinfection. These data show that the AUG-2 PPMO possesses potent anti-RSV activity and is worthy of further investigation as a candidate for potential therapeutic application.Molecular Therapy (2008) 16 6, 1120–1128 doi:10.1038/mt.2008.81 [ABSTRACT FROM AUTHOR]

Foot-and-mouth disease (FMD) is a highly contagious viral disease of cloven-hoofed ungulates that can lead to severe losses in the livestock production and export industries. Although vaccines have been extensively used to control FMD, there is no antiviral therapy available to treat ongoing infections with FMD virus (FMDV). Six peptide-conjugated morpholino oligomers (PPMOs) with sequences complementary to various 21-nucleotide segments of the 5' and 3' untranslated regions (UTRs) of the FMDV genome (strain A24 Cruzeiro/Brazil/1955 [A24Cru]) were evaluated in cell cultures. Three of the PPMOs, targeting domain 5 of the internal ribosome entry site (5D PPMO), and the two translation start codon regions (AUG1 and AUG2 PPMOs), showed high levels of anti-FMDV activity. A dose-dependent and sequence-specific reduction in viral titers of greater than 5 log10, with a concomitant reduction of viral protein and RNA expression, was achieved at low micromolar concentrations. Under identical conditions, three other PPMOs targeting the 5'-terminal region of the genome, the cis-acting replication element in the 5' UTR, and the 3' "ab" stem-loop showed less dramatic titer reductions of 1.5 log10 to 2 log10. Treatment with 5D PPMO reduced the titers of FMDV strains representing five different serotypes by 2 log10 to 4 log10 compared to those of the controls. A24Cru-infected BHK-21 cells treated repeatedly with 5D or AUG2 PPMO generated resistant viruses for which phenotypic and genotypic properties were defined. Notably, three passages with low concentrations of the AUG1 PPMO extinguished all traces of detectable virus. The results indicate that PPMOs have potential for treating FMDV infections and that they also represent useful tools for studying picornaviral translation and evolution. [ABSTRACT FROM AUTHOR]

Background: As the causative agent of COVID-19, SARS-CoV-2 is a pathogen of immense importance to global public health. Development of innovative direct-acting antiviral agents is sorely needed to address this virus. Peptide-conjugated morpholino oligomers (PPMO) are antisense compounds composed of a phosphorodiamidate morpholino oligomer covalently conjugated to a cell-penetrating peptide. PPMO require no delivery assistance to enter cells and are able to reduce expression of targeted RNA through sequence-specific steric blocking.Methods: Five PPMO designed against sequences of genomic RNA in the SARS-CoV-2 5'-untranslated region and a negative control PPMO of random sequence were synthesized. Each PPMO was evaluated for its effect on the viability of uninfected cells and its inhibitory effect on the replication of SARS-CoV-2 in Vero-E6 cell cultures. Cell viability was evaluated with an ATP-based method using a 48 h PPMO treatment time. Viral growth was measured with quantitative RT-PCR and TCID50 infectivity assays from experiments where cells received a 5 h PPMO treatment time.Results: PPMO designed to base-pair with sequence in the 5' terminal region or the leader transcription regulatory sequence region of SARS-CoV-2 genomic RNA were highly efficacious, reducing viral titres by up to 4-6 log10 in cell cultures at 48-72 h post-infection, in a non-toxic and dose-responsive manner.Conclusions: The data indicate that PPMO have the ability to potently and specifically suppress SARS-CoV-2 growth and are promising candidates for further preclinical development. [ABSTRACT FROM AUTHOR]

Five dengue (DEN) virus-specific R5F2R4 peptide-conjugated phosphorodiamidate morpholino oligomers (P4-PMOs) were evaluated for their ability to inhibit replication of DEN virus serotype 2 (DEN-2 virus) in mammalian cell culture. Initial growth curves of DEN-2 virus 16681 were obtained in Vero cells incubated with 20 µM P4-PMO compounds. At 6 days after infection, a P4-PMO targeting the 3′-terminal nucleotides of the DEN-2 virus genome and a random-sequence P4-PMO showed relatively little suppression of DEN-2 virus titer (0.1 and 0.9 log10, respectively). P4-PMOs targeting the AUG translation start site region of the single open reading frame and the 5′ cyclization sequence region had moderate activity, generating 1.6- and 1.8-log10 reductions. Two P4-PMO compounds, 5′SL and 3′CS (targeting the 5′-terminal nucleotides and the 3′ cyclization sequence region, respectively), were highly efficacious, each reducing the viral titer by greater than 5.7 log10 compared to controls at 6 days after infection with DEN-2 virus. Further experiments showed that 5′SL and 3′CS inhibited DEN-2 virus replication in a dose-dependent and sequence-specific manner. Treatment with 10 µM 3′CS reduced the titers of all four DEN virus serotypes, i.e., DEN-1 (strain 16007), DEN-2 (16681), DEN-3 (16562), and DEN-4 (1036) viruses by over 4 log10, in most cases to below detectable limits. The extent of 3′CS efficacy was affected by the timing of compound application in relation to viral infection of the cells. The 5′SL and 3′CS P4-PMOs did not suppress the replication of West Nile virus NY99 in Vero cells. These data indicate that further evaluation of the 5′SL and 3′CS compounds as potential DEN virus therapeutics is warranted. [ABSTRACT FROM AUTHOR]

The recent emergence of novel pathogenic human and animal coronaviruses has highlighted the need for antiviral therapies that are effective against a spectrum of these viruses. We have used several strains of murine hepatitis virus (MHV) in cell culture and in vivo in mouse models to investigate the antiviral characteristics of peptide-conjugated antisense phosphorodiamidate morpholino oligomers (P-PMOs). Ten P-PMOs directed against various target sites in the viral genome were tested in cell culture, and one of these (5TERM), which was complementary to the 5′ terminus of the genomic RNA, was effective against six strains of MHV. Further studies were carried out with various arginine-rich peptides conjugated to the 5TERM PMO sequence in order to evaluate efficacy and toxicity and thereby select candidates for in vivo testing. In uninfected mice, prolonged P-PMO treatment did not result in weight loss or detectable histopathologic changes. 5TERM P-PMO treatment reduced viral titers in target organs and protected mice against virus-induced tissue damage. Prophylactic 5TERM P-PMO treatment decreased the amount of weight loss associated with infection under most experimental conditions. Treatment also prolonged survival in two lethal challenge models. In some cases of high-dose viral inoculation followed by delayed treatment, 5TERM P-PMO treatment was not protective and increased morbidity in the treated group, suggesting that P-PMO may cause toxic effects in diseased mice that were not apparent in the uninfected animals. However, the strong antiviral effect observed suggests that with further development, P-PMO may provide an effective therapeutic approach against a broad range of coronavirus infections. [ABSTRACT FROM AUTHOR]