Search

Coronavirus disease 2019 is a respiratory infectious disease caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Evidence on the pathogenesis of SARS-CoV-2 is accumulating rapidly. In addition to structural proteins such as Spike and Envelope, the functional roles of non-structural and accessory proteins in regulating viral life cycle and host immune responses remain to be understood. Here, we show that open reading frame 8 (ORF8) acts as messenger for inter-cellular communication between alveolar epithelial cells and macrophages during SARS-CoV-2 infection. Mechanistically, ORF8 is a secretory protein that can be secreted by infected epithelial cells via both conventional and unconventional secretory pathways. Conventionally secreted ORF8 is glycosylated and loses the ability to recognize interleukin 17 receptor A of macrophages, possibly due to the steric hindrance imposed by N-glycosylation at Asn78. However, unconventionally secreted ORF8 does not undergo glycosylation without experiencing the ER-Golgi trafficking, thereby activating the downstream NF-κB signaling pathway and facilitating a burst of cytokine release. Furthermore, we show that ORF8 deletion in SARS-CoV-2 attenuates inflammation and yields less lung lesions in hamsters. Our data collectively highlights a role of ORF8 protein in the development of cytokine storms during SARS-CoV-2 infection., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Lin et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Modified-live herpesvirus vaccines are widely used in humans and animals, but field strains can emerge that have a higher virulence and break vaccinal protection. Since the introduction of the first vaccine in the 1970s, Marek's disease virus overcame the vaccine barrier by the acquisition of numerous genomic mutations. However, the evolutionary adaptations in the herpesvirus genome responsible for the vaccine breaks have remained elusive. Here, we demonstrate that point mutations in the multifunctional meq gene acquired during evolution can significantly alter virulence. Defined mutations found in highly virulent strains also allowed the virus to overcome innate cellular responses and vaccinal protection. Concomitantly, the adaptations in meq enhanced virus shedding into the environment, likely providing a selective advantage for the virus. Our study provides the first experimental evidence that few point mutations in a single herpesviral gene result in drastically increased virulence, enhanced shedding, and escape from vaccinal protection., Competing Interests: The authors have declared that no competing interests exist.

Codon pair bias deoptimization (CPBD) has enabled highly efficient and rapid attenuation of RNA viruses. The technique relies on recoding of viral genes by increasing the number of codon pairs that are statistically underrepresented in protein coding genes of the viral host without changing the amino acid sequence of the encoded proteins. Utilization of naturally underrepresented codon pairs reduces protein production of recoded genes and directly causes virus attenuation. As a result, the mutant virus is antigenically identical with the parental virus, but virulence is reduced or absent. Our goal was to determine if a virus with a large double-stranded DNA genome, highly oncogenic Marek's disease virus (MDV), can be attenuated by CPBD. We recoded UL30 that encodes the catalytic subunit of the viral DNA polymerase to minimize (deoptimization), maximize (optimization), or preserve (randomization) the level of overrepresented codon pairs of the MDV host, the chicken. A fully codon pair-deoptimized UL30 mutant could not be recovered in cell culture. The sequence of UL30 was divided into three segments of equal length and we generated a series of mutants with different segments of the UL30 recoded. The codon pair-deoptimized genes, in which two segments of UL30 had been recoded, showed reduced rates of protein production. In cultured cells, the corresponding viruses formed smaller plaques and grew to lower titers compared with parental virus. In contrast, codon pair-optimized and -randomized viruses replicated in vitro with kinetics that were similar to those of the parental virus. Animals that were infected with the partially codon pair-deoptimized virus showed delayed progression of disease and lower mortality rates than codon pair-optimized and parental viruses. These results demonstrate that CPBD of a herpesvirus gene causes attenuation of the recoded virus and that CPBD may be an applicable strategy for attenuation of other large DNA viruses.