A genome engineering technology is used to change a single amino acid in the viruses’ main protease enzyme to match that of circulating Omicron isolates to demonstrate antiviral efficacy of approved drugs and uncover mutants with reduced drug sensitivity.
Abstract
In addition to causing cold and flu-like symptoms, Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) can also cause chronic longer-term diseases. Antiviral drugs, especially used combinatorially, have the potential to reduce the severity of individual infections and prevent the development of chronic disease. One of the safest and most versatile reverse genetics systems for SARS-CoV-2 studies is a bacterial artificial chromosome (BAC)-based system harboring the WA1 strain full-length genome and attenuating deletions in the accessory open reading frame 3a and 7b proteins (ORF3a and ORF7b, respectively). Here, a scarless genome engineering technique called En Passant mutagenesis was used to change one amino acid in the viral main protease (Mpro P132) into the residue present in contemporary Omicron strains (H132), in order to more accurately study protease inhibitors and resistance mechanisms. This recombinant, attenuated viral system yields antiviral EC50 values for the active component of approved drugs including nirmatrelvir (Paxlovid) and ensitrelvir (Xocova) and, importantly, also enables a parallel assessment of drug efflux. For instance, the antiviral potency of nirmatrelvir improves 50-fold by inhibiting the P-Glycoprotein (P-Gp) transporter with ritonavir or tariquidar, whereas the potency of ensitrelvir is unaffected. This system also enables the safe isolation and characterization of viral variants with reduced sensitivity to drugs, as evidenced by Mpro M49L compromising the efficacy of ensitrelvir. Together, these systems combine to provide safe, reliable, and quantitative approaches for Mpro variant analysis and drug testing without the biosafety concerns of conducting these experiments using wildtype isolates. IMPORTANCE Safe genetic systems for studying coronavirus biology and developing next generation antivirals are important. One of the most versatile systems leverages a bacterial artificial chromosome to efficiently propagate and engineer a full-length SARS-CoV-2 genome. This system is also safe because it has crippling deletion mutations that limit virus replication to a small number of cell lines. Here, we use a genome engineering technology to change a single amino acid in the viruses’ main protease enzyme to match that of circulating Omicron isolates. The resulting attenuated virus was also used to demonstrate antiviral efficacy of approved drugs and uncover mutants with reduced drug sensitivity. The emergent mutants match those in a subset of circulating strains further demonstrating broad relevance.
The phylogenetic relationship of the A222V substitution in the S protein relative to various global isolates is investigated, indicating high mutation rates in the S gene, characterised by diverse point mutations.
Kiky Martha, Ariesaka, M. M. Nuryady et al.· 0 citations
The co-circulation of multiple viruses can lead to distinct pathological outcomes, yet how severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection influences other viral infections remains poorly understood, despite its documented high frequency during the pandemic. In this study, we investigated how the proteolytic activity of SARS-CoV-2 3C-like protease (3CLpro) influences the replication of influenza A virus. In silico analysis identified candidate 3CLpro cleavage sites across numerous viral proteins, and biochemical assays confirmed that 3CLpro catalyzes the degradation of influenza virus nucleoprotein (NP) and neuraminidase (NA) in a manner requiring its protease activity. This degradation of NP and PA, which are essential for viral genome packaging and transcription, disrupted the influenza replicative cycle and suppressed viral replication, both upon ectopic 3CLpro expression and during SARS-CoV-2 infection. Our data uncover a direct, enzyme-based mechanism by which SARS-CoV-2 can suppress influenza virus replication during coinfection. We provide a molecular explanation for the sharp, global decline in influenza activity observed during the COVID-19 pandemic and illustrate how enzymatic weapons of one virus can be repurposed to restrain a competing pathogen.
Liubing Du, Xiaoyu Cai, Zi-Tao Peng et al.· Virologica Sinica· 0 citations
The ability of respiratory viruses to exploit host immune responses to promote transmission is a defining feature of pandemics. SARS-CoV-2 remains a major global public health threat because of its persistent evolution and capacity to counteract evolving immune defenses. Although the immune evasion properties of the SARS-CoV-2 Spike protein are well characterized, the contributions of other viral proteins to transmission remain poorly understood. Here, we used the infant mouse model to define the role of the accessory protein ORF8 in SARS-CoV-2 spread. We demonstrate that ORF8 supports efficient upper respiratory tract (URT) infection, infectious virus shedding, and host-to-host transmission. Mice infected with a recombinant SARS-CoV-2 strain lacking ORF8 (rΔORF8) had less infectious virus recovered from URT tissues and nasal secretions and transmitted less efficiently than mice infected with the isogenic ancestral strain rWA-1, which contains an intact ORF8. Recombinant viruses encoding naturally occurring ORF8 mutations exhibited distinct transmission phenotypes, with ORF8-deficient viruses resembling rΔORF8. Infection with ORF8-sufficient viruses induced greater macrophage recruitment, inflammatory cytokine production, and type-I interferon (IFN-I) signaling programs than ORF8-deficient viruses. Intranasal IFNβ supplementation partially restored URT shedding by rΔORF8-infected mice and rescued transmission to contacts, whereas blockade of the type I interferon receptor (IFNAR) in rWA-1-infected index mice reduced contact infection and transmission. Together, these findings demonstrate that ORF8 promotes SARS-CoV-2 transmission by engaging an IFN-I-associated inflammatory and secretory program in the URT that supports virus shedding from the infected host. These data identify ORF8 as a viral determinant of host mucosal responses that promote contagiousness. Importance Efficient host-to-host transmission underlies the success of respiratory viruses. Although SARS-CoV-2 research has largely focused on the Spike protein, accessory proteins can also shape viral fitness and spread. We previously identified ORF8 as a determinant of SARS-CoV-2 transmission. Here we show that ORF8 promotes SARS-CoV-2 infectious viral shedding and transmission by engaging IFN-I-associated inflammatory and secretory responses in the URT. These findings reveal how a SARS-CoV-2 accessory protein can exploit mucosal antiviral responses to increase host contagiousness.
G. Ciabattoni, Stacey Bartlett, M. McGrath et al.· bioRxiv· 0 citations
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is still a major public health issue, even today. Among the SARS-CoV-2 nonstructural proteins, the main protease (Mpro) plays a critical role in viral polyprotein processing and is therefore indispensable for viral replication. For this reason, it represents one of the most promising therapeutic targets for the development of antiviral agents against SARS-CoV-2. Currently, only one protease antiviral agent (nirmatrelvir) has received emergency approval for COVID-19 treatment, the disease caused by SARS-CoV-2 infection. However, the emergence of viral mutations may compromise its efficacy, highlighting the urgent need to develop new, safe, and effective protease antiviral agents. In the present work, we designed and synthesized new SARS-CoV-2 Mpro small-molecule inhibitors endowed with a pyrimidine scaffold. A series of derivatives were evaluated in both biochemical and cell-based assays to assess their antiviral efficacy, with some of them being able to inhibit the SARS-CoV-2 Mpro activity and to suppress viral replication. Docking studies were confirmed by site-directed mutagenesis, and the mechanism of action of the most promising compound was elucidated.
Salvatore Nieddu, Giuseppe Ruggieri, Riccardo De Santis et al.· ACS Infectious Diseases· 0 citations
Highly pathogenic RNA viruses, such as Ebola, SARS-CoV-2, and influenza, cause severe disease in humans. High mutation rates, which enable RNA viruses to evade immunity and escape antivirals, and their ability to spread from animals to humans and cause pandemics and outbreaks, make RNA viruses significant threats to public health. Diseases caused by Ebola, SARS-CoV-2, and influenza are prevented and treated with only a limited number of approved antiviral drugs, the effectiveness of which is limited by mutations in the viral targets. It is crucial to understand the structural determinants, molecular mechanisms, and host interactions of pathogenic RNA viruses to develop effective antiviral strategies. In this review, we discuss selected RNA viruses, focusing on the structure of their RNA polymerases and interactions with host factors during the different stages of the viral lifecycle, as well as the traditional antivirals targeting these structures and pathways. Furthermore, emerging concepts such as liquid–liquid phase separation and biomolecular condensates, and novel promising antiviral strategies are discussed. Understanding shared and distinct structures, molecular mechanisms, and host interactions across highly pathogenic RNA viruses enables the discovery of new and more effective antiviral strategies, ultimately improving clinical outcomes against evolving RNA viruses.
Hana Krnjić, Adna Hrapović, Aiša Galijatović et al.· Viruses· 0 citations
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