Barreto Duran, E. | Search bioRxiv

bioRxiv2026

Scarless SARS-CoV-2 Genome Engineering and Variant Analysis

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.

microbiology↗

bioRxiv2025

Mus Musculus papillomavirus MmuPV1 resists restriction by human APOBEC3B

The single-stranded DNA deaminase APOBEC3B (A3B) is capable of potently restricting the replication of a range of viruses including retroviruses (cDNA) and herpesviruses (genomic DNA). However, these and likely other DNA virus families have evolved host species-specific counter-defenses that are equally potent and serve to protect viral DNA from restriction. Although high-risk human papillomavirus (HPV) infection triggers A3B upregulation, potentially as part of an antiviral response, the impact of this restriction factor on papillomavirus replication and pathogenesis has yet to be assessed. To study human A3B antiviral function in the absence of a species-specific counter-defense, here we ask whether human A3B is capable of restricting Mus musculus papillomavirus (MmuPV1) in cellulo and in vivo. First, we created human A3B and catalytic mutant A3B-E255A expressing FVB/N mice. Second, MmuPV1 gene expression and replication was quantified in primary keratinocytes from these animals and, surprisingly, enzymatically active human A3B caused no measurable impairment in viral transcript or DNA accumulation. Third, A3B, catalytic mutant A3B-E255A, and nontransgenic FVB/N animals were infected with MmuPV1 and similar pathologies were found regardless of A3B functionality. Thus, despite likely never being exposed to human A3B during evolution, MmuPV1 appears to be unaffected by this potent, primate-specific antiviral factor. These results suggest that MmuPV1 and perhaps papillomaviruses more broadly possess a conserved mechanism to efficiently escape restriction by human A3B and related DNA deaminases. IMPORTANCEHuman papillomaviruses (HPVs) are nearly ubiquitous, and persistent infection with high-risk types causes approximately 5% of cancers worldwide. Although HPV vaccination is effective for preventing infection, insufficient global coverage and a rising incidence of HPV-associated malignancies, such as oropharyngeal carcinoma, highlight the need to understand innate virus clearance mechanisms. APOBEC3 enzymes are a central component of the mammalian innate immune system and are hypothesized to restrict papillomavirus infection, particularly between species. Here, we establish mice that express the human antiviral enzyme APOBEC3B (A3B). Surprisingly, we find that human A3B is incapable of blocking the replication of a murine papillomavirus (Mus musculus papillomavirus 1, MmuPV1) in relevant primary cells from these animals or in infected tissues in vivo. These findings highlight the complexity of teasing apart host-pathogen interactions and suggest that papillomaviruses may have a general mechanism for escaping restriction by antiviral enzymes such as A3B.

microbiology↗