RBM24 inhibits the translation of SARS-CoV-2 polyproteins by targeting the 5'-untranslated region.

SARS-CoV-2 is a betacoronavirus with single-stranded positive-sense RNA, which is a serious global threat to human health. Understanding the molecular mechanism of viral replication is crucial for the development of antiviral drugs. The synthesis of viral polyproteins is a crucial step in viral progression. The synthesis of viral polyproteins in coronaviruses is regulated by the 5'-untranslated region (UTR); however, the detailed regulatory mechanism needs further investigation. The present study demonstrated that the RNA binding protein, RBM24, interacts with the RNA genome of SARS-CoV-2 via its RNA recognition submotifs (RNPs). The findings revealed that RBM24 recognizes and binds to the GUGUG element at stem-loop 4 (SL4) in the 5'-UTR of SARS-CoV-2. The interaction between RBM24 and 5'-UTR prevents 80S ribosome assembly, which in turn inhibits polyproteins translation and the replication of SARS-CoV-2. Notably, other RNA viruses, including SARS-CoV, MERS-CoV, Ebolavirus, rhinovirus, West Nile virus, Zika virus, Japanese encephalitis virus, yellow fever virus, hepatitis C virus, and human immunodeficiency virus-1 also contain one or several G(U/C/A)GUG sequences in the 5'-UTR, which is also targeted by RBM24. In conclusion, the present study demonstrated that RBM24 functions by interacting with the 5'-UTR of SARS-CoV-2 RNA, and elucidated that RBM24 could be a host restriction factor for SARS-CoV-2 and other RNA viruses.

Comparison of viral propagation and drug response among SARS-CoV-2 VOCs using replicons capable of recapitulating virion assembly and release.

Several variants of concern (VOCs) have emerged since the WIV04 strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was first isolated in January 2020. Due to mutations in the spike (S) protein, these VOCs have evolved to enhance viral infectivity and immune evasion. However, whether mutations of the other viral proteins lead to altered viral propagation and drug resistance remains obscure. The replicon is a noninfectious viral surrogate capable of recapitulating certain steps of the viral life cycle. Although several SARS-CoV-2 replicons have been developed, none of them were derived from emerging VOCs and could only recapitulate viral genome replication and subgenomic RNA (sgRNA) transcription. In this study, SARS-CoV-2 replicons derived from the WIV04 strain and two VOCs (the Beta and Delta variants) were prepared by removing the S gene from their genomes, while other structural genes remained untouched. These replicons not only recapitulate viral genome replication and sgRNA transcription but also support the assembly and release of viral-like particles, as manifested by electron microscopic assays. Thus, the S-deletion replicon could recapitulate virtually all the post-entry steps of the viral life cycle and provides a versatile tool for measuring viral intracellular propagation and screening novel antiviral drugs, including inhibitors of virion assembly and release. Through the quantification of replicon RNA released into the supernatant, we demonstrate that viral intracellular propagation and drug response to remdesivir have not yet substantially changed during the evolution of SARS-CoV-2 from the WIV04 strain to the Beta and Delta VOCs.

Efficient assembly of a large fragment of monkeypox virus genome as a qPCR template using dual-selection based transformation-associated recombination.

Transformation-associated recombination (TAR) has been widely used to assemble large DNA constructs. One of the significant obstacles hindering assembly efficiency is the presence of error-prone DNA repair pathways in yeast, which results in vector backbone recircularization or illegitimate recombination products. To increase TAR assembly efficiency, we prepared a dual-selective TAR vector, pGFCS, by adding a PADH1-URA3 cassette to a previously described yeast-bacteria shuttle vector, pGF, harboring a PHIS3-HIS3 cassette as a positive selection marker. This new cassette works as a negative selection marker to ensure that yeast harboring a recircularized vector cannot propagate in the presence of 5-fluoroorotic acid. To prevent pGFCS bearing ura3 from recombining with endogenous ura3-52 in the yeast genome, a highly transformable Saccharomyces cerevisiae strain, VL6-48B, was prepared by chromosomal substitution of ura3-52 with a transgene conferring resistance to blasticidin. A 55-kb genomic fragment of monkeypox virus encompassing primary detection targets for quantitative PCR was assembled by TAR using pGFCS in VL6-48B. The pGFCS-mediated TAR assembly showed a zero rate of vector recircularization and an average correct assembly yield of 79% indicating that the dual-selection strategy provides an efficient approach to optimizing TAR assembly.

Effects of CRM1-dependent nuclear export inhibition on viral structural protein nuclear accumulation during Autographa californica multiple nucleopolyhedrovirus infection.

Autographa californica multiple nucleopolyhedrovirus (AcMNPV) assembles its nucleocapsids and occlusion-derived virions (ODVs) in the nucleus, which requires AcMNPV regulation for viral structural proteins to accumulate in the nucleus during its replication in cells. It is generally accepted that the nuclear import receptor plays a predominant role in this process. CRM1 is a nuclear export receptor that forms an export complex with its cargo protein to exit the nucleus. We previously discovered that AcMNPV inhibited CRM1-dependent nuclear export by the viral protein Ac34. This finding suggested that Ac34 could sequester CRM1-dependent proteins in the nucleus and play a novel role in the nuclear accumulation of viral structural proteins. Using the CRM1 inhibitor leptomycin B (LMB), we demonstrated that CRM1 inhibition promoted AcMNPV replication, as LMB treatment readily increased the virus titer, and even functionally surrogate Ac34 to rescue the infectivity of an ac34-knockout virus. To elucidate whether CRM1 inhibition contributes to the nuclear accumulation of viral structural proteins, we systematically analyzed the impact of CRM1 inhibition on viral protein spatial distribution patterns. We found that the nucleocapsid protein Ac102 and ODV envelope protein E26 were retained in the nucleus in response to CRM1 inhibition by Ac34. This finding indicates that AcMNPV is evolving to simultaneously exploit bidirectional nucleocytoplasmic trafficking to assist in viral replication.

[Regulation of pyocyanin biosynthesis by transcriptional factor sigma38 in Pseudomonas aeruginosa PAO1].

Pyocyanin, an important virulence factor, is synthesized and secreted by Pseudomonas aeruginosa PAO1and plays a critical role in pathogen-host interaction during infection. Sigma38 (σ38, σS) is a central regulator for many virulence production in pathogens. Objective: Our aim is to identify expression and regulation of two phenazine-producing operons mediated by the sigma38 factor in Pseudomonas aeruginosa PAO1. Methods: We first cloned the flanking fragments of rpoS from the chromosomal DNA of P. aeruginosa PAO1 and constructed the deletion mutant ΔrpoS with the insertion of gentamycin resistance cassette (aacC1). Complementation of rpoS was then carried out after construction and introduction of pME10S (containing the whole rpoS region). Finally, we created the mutant ΔrpoSphz1 and ΔrpoSphz2, and measured pyocyanin production by these mutants in GA medium, using the parental strain Δphz1 and Δphz2 as controls. Results: In GA medium, pyocyanin production by mutant ΔrpoS increased dramatically in comparison with the wild-type strain PAO1. Production of pyocyanin, however, was decreased to the level of the wild-type strain with complementation of the derivative ΔrpoS harboring pME10S. Mutant ΔrpoSphz2 produced much more pyocyanin than mutant Δphz2. Mutant ΔrpoSphz1, however, produced much less pyocyanin than mutant Δphz1. Conclusion: By positively regulating the expression of phz2 and negatively regulating the phz1, sigma38 factor exerts negative modulation on pyocyanin biosynthesis in P. aeruginosa PAO1.