Establishment of a reverse genetics system for SARS-CoV-2 using circular polymerase extension reaction

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been identified as the causative agent of coronavirus disease 2019 (COVID-19). Although multiple mutations have been observed in SARS-CoV-2, functional analysis of each mutation of SARS-CoV-2 has been limited by the lack of convenient mutagenesis methods. In this study, we establish a PCR-based, bacterium-free method to generate SARS-CoV-2 infectious clones. Recombinant SARS-CoV-2 could be rescued at high titer with high accuracy after assembling 10 SARS-CoV-2 cDNA fragments by circular polymerase extension reaction (CPER) and transfection of the resulting circular genome into susceptible cells. The construction of infectious clones for reporter viruses and mutant viruses could be completed in two simple steps: introduction of reporter genes or mutations into the desirable DNA fragments (similar to 5,000 base pairs) by PCR and assembly of the DNA fragments by CPER. This reverse genetics system may potentially advance further understanding of SARS-CoV-2

Various miRNAs compensate the role of miR-122 on HCV replication.

One of the determinants for tissue tropism of hepatitis C virus (HCV) is miR-122, a liver-specific microRNA. Recently, it has been reported that interaction of miR-122 to HCV RNA induces a conformational change of the 5'UTR internal ribosome entry site (IRES) structure to form stem-loop II structure (SLII) and hijack of translating 80S ribosome through the binding of SLIII to 40S subunit, which leads to efficient translation. On the other hand, low levels of HCV-RNA replication have also been detected in some non-hepatic cells; however, the details of extrahepatic replication remain unknown. These observations suggest the possibility that miRNAs other than miR-122 can support efficient replication of HCV-RNA in non-hepatic cells. Here, we identified a number of such miRNAs and show that they could be divided into two groups: those that bind HCV-RNA at two locations (miR-122 binding sites I and II), in a manner similar to miR-122 (miR-122-like), and those that target a single site that bridges sites I and II and masking both G28 and C29 in the 5'UTR (non-miR-122-like). Although the enhancing activity of these non-hepatic miRNAs were lower than those of miR-122, substantial expression was detected in various normal tissues. Furthermore, structural modeling indicated that both miR-122-like and non-miR-122-like miRNAs not only can facilitate the formation of an HCV IRES SLII but also can stabilize IRES 3D structure in order to facilitate binding of SLIII to the ribosome. Together, these results suggest that HCV facilitates miR-122-independent replication in non-hepatic cells through recruitment of miRNAs other than miR-122. And our findings can provide a more detailed mechanism of miR-122-dependent enhancement of HCV-RNA translation by focusing on IRES tertiary structure

Secretory glycoprotein NS1 plays a crucial role in the particle formation of flaviviruses

Flaviviruses, which are globally distributed and cause a spectrum of potentially severe illnesses, pose a major threat to public health. Although Flaviviridae viruses, including flaviviruses, possess similar genome structures, only the flaviviruses encode the non-structural protein NS1, which resides in the endoplasmic reticulum (ER) and is secreted from cells after oligomerization. The ER-resident NS1 is known to be involved in viral genome replication, but the essential roles of secretory NS1 in the virus life cycle are not fully understood. Here we characterized the roles of secretory NS1 in the particle formation of flaviviruses. We first identified an amino acid residue essential for the NS1 secretion but not for viral genome replication by using protein-protein interaction network analyses and mutagenesis scanning. By using the recombinant flaviviruses carrying the identified NS1 mutation, we clarified that the mutant flaviviruses employed viral genome replication. We then constructed a recombinant NS1 with the identified mutation and demonstrated by physicochemical assays that the mutant NS1 was unable to form a proper oligomer or associate with liposomes. Finally, we showed that the functions of NS1 that were lost by the identified mutation could be compensated for by the in trans-expression of E-rns of pestiviruses and host exchangeable apolipoproteins, which participate in the infectious particle formation of pestiviruses and hepaciviruses in the family Flaviviridae, respectively. Collectively, our study suggests that secretory NS1 plays a role in the particle formation of flaviviruses through its interaction with the lipid membrane. Author summaryIt is difficult to characterize the function of NS1 in the post-genome replication stages in the virus life cycle of flaviviruses. Here, by means of protein-protein interaction network analyses and mutagenesis scanning, we identified a unique mutation in NS1 by which the protein loses its secretory capacity while retaining its genome replication activity. Physicochemical assays using the mutant NS1 revealed that oligomerization of NS1 is responsible for the lipid association and secretion of NS1. In addition, we established a complementation assay that can evaluate the particle formation of Flaviviridae viruses. By using recombinant flaviviruses possessing the identified mutation in NS1, we clarified that NS1 is involved in particle formation. Our findings reveal that the flavivirus NS1 has at least two roles in the virus life cycles-namely, a role in infectious particle formation and a role in viral genome replication

Characterization of recombinant Flaviviridae viruses possessing a small reporter-tag

The family Flaviviridae consists of four genera, Flavivirus, Pestivirus, Pegivirus, and Hepacivirus, and comprises important pathogens of human and animals. Although the construction of recombinant viruses carrying reporter genes including fluorescent and bioluminescent proteins has been reported, the stable insertion of foreign genes into viral genomes retaining infectivity remains difficult. Here, we applied the 11-amino-acid subunit derived from NanoLuc luciferase to the engineering of the Flaviviridae viruses, and then examined the biological characteristics of the viruses. We successfully generated recombinant viruses carrying the split-luciferase gene including dengue virus, Japanese encephalitis virus, hepatitis C virus (HCV), and bovine viral diarrhea virus. The stability of the viruses was confirmed by five rounds of serial passages in respective susceptible cell lines. The propagation of the recombinant luciferase viruses in each cell line was comparable to that of the parental viruses. By using a purified counterpart luciferase protein, this split-luciferase assay can be applicable in various cell lines, even when it is difficult to transduce the counterpart gene. The efficacy of antiviral reagents against the recombinant viruses could be monitored by the reduction of luciferase expression, which was correlated with that of viral RNA, and the recombinant HCV was also useful to examine viral dynamics in vivo Taken together, our findings indicate that the recombinant Flaviviridae viruses possessing the split NanoLuc luciferase gene generated herein provide powerful tools to understand viral life cycle and pathogenesis, and a robust platform to develop novel antivirals against Flaviviridae viruses.IMPORTANCE The construction of reporter viruses possessing a stable transgene capable of expressing specific signals is crucial to investigations of viral life cycle and pathogenesis and the development of antivirals. However, it is difficult to maintain the stability of a large foreign gene such as those for fluorescent and bioluminescent proteins after insertion into a viral genome. Here, we successfully generated recombinant Flaviviridae viruses carrying the 11-amino-acid subunit derived from NanoLuc luciferase, and demonstrated that these viruses are applicable to in vitro and in vivo experiments, suggesting that these recombinant Flaviviridae viruses are powerful tools for increasing our understanding of viral life cycle and pathogenesis, and that these recombinant viruses will provide a robust platform to develop antivirals against Flaviviridae viruses

Propagation of Con1C3/JFH_122KO in 751-122KO cells.

<p>(A) Infectious titer in the culture medium on serial passage of 751-122KO#1 or Huh7.5.1 cells. Red circles indicate the passage in 751-122KO cells, and the other circles indicate the passage in Huh7.5.1 cells. Three independent passages (#4–6, #4–8, #7–8) are shown. (B) Nuclear translocation of IPS-GFP (arrows) in Huh7.5.1 and 751-122KO cells upon infection with Con1C3/JFH and Con1C3/JFH_122KO. (C) Con1C3/JFH and Con1C3/JFH_122KO were inoculated into 751-122KO#1 and Huh7.5.1 cells, and the levels of intracellular HCV-RNA replication were determined. Error bars indicate the standard deviation of the mean and asterisks indicate significant differences (**P < 0.01) versus the results for the control. (D) 293T-CLDN cells infected with either Con1C3/JFH or Con1C3/JFH_122KO were treated with IFNα and BILN and then the intracellular HCV-RNA level was determined at 12, 24 and 48 hpi. Error bars indicate the standard deviation of the mean and asterisks indicate significant differences (**P < 0.01) versus the results for the control.</p

Characterization of miR-122-independent propagation of HCV

<div><p>miR-122, a liver-specific microRNA, is one of the determinants for liver tropism of hepatitis C virus (HCV) infection. Although miR-122 is required for efficient propagation of HCV, we have previously shown that HCV replicates at a low rate in miR-122-deficient cells, suggesting that HCV-RNA is capable of propagating in an miR-122-independent manner. We herein investigated the roles of miR-122 in both the replication of HCV-RNA and the production of infectious particles by using miR-122-knockout Huh7 (Huh7-122KO) cells. A slight increase of intracellular HCV-RNA levels and infectious titers in the culture supernatants was observed in Huh7-122KO cells upon infection with HCV. Moreover, after serial passages of HCV in miR-122-knockout Huh7.5.1 cells, we obtained an adaptive mutant, HCV_122KO, possessing G28A substitution in the 5’UTR of the HCV genotype 2a JFH1 genome, and this mutant may help to enhance replication complex formation, a possibility supported by polysome analysis. We also found the introduction of adaptive mutation around miR-122 binding site in the genotype 1b/2a chimeric virus, which originally had an adenine at the nucleotide position 29. HCV_122KO exhibited efficient RNA replication in miR-122-knockout cells and non-hepatic cells without exogenous expression of miR-122. Competition assay revealed that the G28A mutant was dominant in the absence of miR-122, but its effects were equivalent to those of the wild type in the presence of miR-122, suggesting that the G28A mutation does not confer an advantage for propagation in miR-122-rich hepatocytes. These observations may explain the clinical finding that the positive rate of G28A mutation was higher in miR-122-deficient PBMCs than in the patient serum, which mainly included the hepatocyte-derived virus from HCV-genotype-2a patients. These results suggest that the emergence of HCV mutants that can propagate in non-hepatic cells in an miR-122-independent manner may participate in the induction of extrahepatic manifestations in chronic hepatitis C patients.</p></div

G28A mutants can replicate efficiently in an Ago2-independent manner.

<p>(A) Intracellular HCV-RNA levels (left panel) and infectious titers in the culture supernatants (right panel) of Huh7-122KO and Huh7-122KOR cells infected with either HCV or HCV_122KO in the presence of either control-LNA or LNA-miR-122 were determined at 72 hpi. (B) Ago2 complexes in 751-122KO and Huh7.5.1 cells infected with HCV were immunoprecipitated by either anti-IgG or anti-Ago2 mouse antibody at 12 dpi. Levels of Ago2 and HCV-RNA in the precipitates were determined by immunoblotting and qRT-PCR, respectively. Error bars indicate the standard deviation of the mean and asterisks indicate significant differences (*P < 0.05; **P < 0.01) versus the results for the control.</p