Publications

The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), the causative agent of COVID-19 illness, has caused millions of infections worldwide. In SARS coronaviruses, the non-structural protein 16 (nsp16), in conjunction with nsp10, methylates the 5'-end of virally encoded mRNAs to mimic cellular mRNAs, thus protecting the virus from host innate immune restriction. We report here the high-resolution structure of a ternary complex of SARS-CoV-2 nsp16 and nsp10 in the presence of cognate RNA substrate analogue and methyl donor, S-adenosyl methionine (SAM). The nsp16/nsp10 heterodimer is captured in the act of 2'-O methylation of the ribose sugar of the first nucleotide of SARS-CoV-2 mRNA. We observe large conformational changes associated with substrate binding as the enzyme transitions from a binary to a ternary state. This induced fit model provides mechanistic insights into the 2'-O methylation of the viral mRNA cap. We also discover a distant (25 Å) ligand-binding site unique to SARS-CoV-2, which can alternatively be targeted, in addition to RNA cap and SAM pockets, for antiviral development.

An infectious coronavirus disease 2019 (COVID-19) emerged in the city of Wuhan (China) in December 2019, causing a pandemic that has dramatically impacted public health and socioeconomic activities worldwide. A previously unknown coronavirus, Severe Acute Respiratory Syndrome CoV-2 (SARS-CoV-2), has been identified as the causative agent of COVID-19. To date, there are no United States (US) Food and Drug Administration (FDA)-approved vaccines or therapeutics available for the prevention or treatment of SARS-CoV-2 infection and/or associated COVID-19 disease, which has triggered a large influx of scientific efforts to develop countermeasures to control SARS-CoV-2 spread. To contribute to these efforts, we have developed an infectious cDNA clone of the SARS-CoV-2 USA-WA1/2020 strain based on the use of a bacterial artificial chromosome (BAC). Recombinant (r)SARS-CoV-2 was readily rescued by transfection of the BAC into Vero E6 cells. Importantly, the BAC-derived rSARS-CoV-2 exhibited growth properties and plaque sizes in cultured cells comparable to those of the SARS-CoV-2 natural isolate. Likewise, rSARS-CoV-2 showed similar levels of replication to that of the natural isolate in nasal turbinates and lungs of infected golden Syrian hamsters. This is, to our knowledge, the first BAC based reverse genetics system for the generation of infectious rSARS-CoV-2 that displays similar features in vivo to that of a natural viral isolate. This SARS-CoV-2 BAC-based reverse genetics will facilitate studies addressing several important questions in the biology of SARS-CoV-2, as well as the identification of antivirals and development of vaccines for the treatment of SARS-CoV-2 infection and associated COVID-19 disease.

The receptor-binding domain (RBD) of the SARS-CoV-2 spike protein is a candidate vaccine antigen that binds angiotensin-converting enzyme 2 (ACE2), leading to virus entry. Here, it is shown that rapid conversion of recombinant RBD into particulate form via admixing with liposomes containing cobalt-porphyrin-phospholipid (CoPoP) potently enhances the functional antibody response. Antigen binding via His-tag insertion into the CoPoP bilayer results in a serum-stable and conformationally intact display of the RBD on the liposome surface. Compared to other vaccine formulations, immunization using CoPoP liposomes admixed with recombinant RBD induces multiple orders of magnitude higher levels of antibody titers in mice that neutralize pseudovirus cell entry, block RBD interaction with ACE2, and inhibit live virus replication. Enhanced immunogenicity can be accounted for by greater RBD uptake into antigen-presenting cells in particulate form and improved immune cell infiltration in draining lymph nodes. QS-21 inclusion in the liposomes results in an enhanced antigen-specific polyfunctional T cell response. In mice, high dose immunization results in minimal local reactogenicity, is well-tolerated, and does not elevate serum cobalt levels. Taken together, these results confirm that particulate presentation strategies for the RBD immunogen should be considered for inducing strongly neutralizing antibody responses against SARS-CoV-2.

Zika virus (ZIKV) was identified in 1947 in the Zika forest of Uganda and it has emerged recently as a global health threat, with recurring outbreaks and its associations with congenital microcephaly through maternal fetal transmission and Guillain-Barré syndrome. Currently, there are no United States (US) Food and Drug Administration (FDA)-approved vaccines or antivirals to treat ZIKV infections, which underscores an urgent medical need for the development of disease intervention strategies to treat ZIKV infection and associated disease. Drug repurposing offers various advantages over developing an entirely new drug by significantly reducing the timeline and resources required to advance a candidate antiviral into the clinic. Screening the ReFRAME library, we identified ten compounds with antiviral activity against the prototypic mammarenavirus lymphocytic choriomeningitis virus (LCMV). Moreover, we showed the ability of these ten compounds to inhibit influenza A and B virus infections, supporting their broad-spectrum antiviral activity. In this study, we further evaluated the broad-spectrum antiviral activity of the ten identified compounds by testing their activity against ZIKV. Among the ten compounds, Azaribine (SI-MTT = 146.29), AVN-944 (SI-MTT = 278.16), and Brequinar (SI-MTT = 157.42) showed potent anti-ZIKV activity in post-treatment therapeutic conditions. We also observed potent anti-ZIKV activity for Mycophenolate mofetil (SI-MTT = 20.51), Mycophenolic acid (SI-MTT = 36.33), and AVN-944 (SI-MTT = 24.51) in pre-treatment prophylactic conditions and potent co-treatment inhibitory activity for Obatoclax (SI-MTT = 60.58), Azaribine (SI-MTT = 91.51), and Mycophenolate mofetil (SI-MTT = 73.26) in co-treatment conditions. Importantly, the inhibitory effect of these compounds was strain independent, as they similarly inhibited ZIKV strains from both African and Asian/American lineages. Our results support the broad-spectrum antiviral activity of these ten compounds and suggest their use for the development of antiviral treatment options of ZIKV infection.

SARS-CoV-2, the causative agent of COVID-19, has been responsible for a million deaths worldwide as of September 2020. At the time of this writing, there are no available US FDA-approved therapeutics for the treatment of SARS-CoV-2 infection. Here, we describe a detailed protocol to generate recombinant (r)SARS-CoV-2 using reverse-genetics approaches based on the use of a bacterial artificial chromosome (BAC). This method will allow the production of mutant rSARS-CoV-2-which is necessary for understanding the function of viral proteins, viral pathogenesis and/or transmission, and interactions at the virus-host interface-and attenuated SARS-CoV-2 to facilitate the discovery of effective countermeasures to control the ongoing SARS-CoV-2 pandemic. © 2020 Wiley Periodicals LLC. Basic Protocol: Generation of recombinant SARS-CoV-2 using a bacterial artificial chromosome Support Protocol: Validation and characterization of rSARS-CoV-2.

Vaccine and antiviral development against SARS-CoV-2 infection or COVID-19 disease would benefit from validated small animal models. Here, we show that transgenic mice expressing human angiotensin-converting enzyme 2 (hACE2) by the human cytokeratin 18 promoter (K18 hACE2) represent a susceptible rodent model. K18 hACE2 transgenic mice succumbed to SARS-CoV-2 infection by day 6, with virus detected in lung airway epithelium and brain. K18 ACE2 transgenic mice produced a modest TH1/2/17 cytokine storm in the lung and spleen that peaked by day 2, and an extended chemokine storm that was detected in both lungs and brain. This chemokine storm was also detected in the brain at day 6. K18 hACE2 transgenic mice are, therefore, highly susceptible to SARS-CoV-2 infection and represent a suitable animal model for the study of viral pathogenesis, and for identification and characterization of vaccines (prophylactic) and antivirals (therapeutics) for SARS-CoV-2 infection and associated severe COVID-19 disease.

Keratinocytes express many pattern recognition receptors that enhance the skin's adaptive immune response to epicutaneous antigens. We have shown that these pattern recognition receptors are expressed below tight junctions (TJ), strongly implicating TJ disruption as a critical step in antigen responsiveness. To disrupt TJs, we designed peptides inspired by the first extracellular loop of the TJ transmembrane protein CLDN1. These peptides transiently disrupted TJs in the human lung epithelial cell line 16HBE and delayed TJ formation in primary human keratinocytes. Building on these observations, we tested whether vaccinating mice with an epicutaneous influenza patch containing TJ-disrupting peptides was an effective strategy to elicit an immunogenic response. Application of a TJ-disrupting peptide patch resulted in barrier disruption as measured by increased transepithelial water loss. We observed a significant increase in antigen-specific antibodies when we applied patches with TJ-disrupting peptide plus antigen (influenza hemagglutinin) in either a patch-prime or a patch-boost model. Collectively, these observations demonstrate that our designed peptides perturb TJs in human lung as well as human and murine skin epithelium, enabling epicutaneous vaccine delivery. We anticipate that this approach could obviate currently used needle-based vaccination methods that require administration by health care workers and biohazard waste removal.

Infectious bursal disease virus (IBDV) of the Birnaviridae family leads to immunosuppression of young chickens by destroying B cells in the bursa of Fabricius (BFs). Given the increasing number of variant IBDV strains, we urgently require a method to produce attenuated virus for vaccine development. To accomplish this goal, the dual-promoter plasmids in which the RNA polymerase II and RNA polymerase I (Pol I) promoters were placed upstream of the IBDV genomic sequence, which was followed by mouse Pol I terminator and a synthetic polyadenylation signal, were developed for rapid generation of IBDV. This approach did not require trans-supplementation of plasmids for the expression of VP1 and VP3, the main components of IBDV ribonucleoprotein (RNP). Based on the finding in this study that the IBDV RNP activity was partially retained by VP1-FLAG, we successfully rescued the replication-competent IBDV/1FLAG expressing VP1-FLAG. Compared with its parental counterpart, IBDV/1FLAG formed smaller size plaques in cultured cells and induced the same 100% immune protection in vivo However, neither retarded development nor severe BFs lesion was observed in the IBDV/1FLAG-inoculated chickens. Collectively, this is the first report that viral RNP activity was affected by the addition of an epitope tag on the componential viral proteins. Furthermore, this work demonstrates the rapid generation of attenuated IBDV from dual-promoter plasmids via reducing viral RNP activity by a fused FLAG tag on the C terminus of VP1. This would be a convenient strategy to attenuate epidemic variant IBDV strains for rapid and efficient vaccine development.IMPORTANCE Immunosuppression in chickens as a result of infectious bursal disease virus (IBDV) infection leads to significant economic losses in the poultry industry worldwide every year. Currently, vaccination is still the best way to prevent the prevalence of IBDV. However, with the occurrence of increasing numbers of variant IBDV strains, it is challenging to develop antigen-matched live attenuated vaccine. Here, we first developed a dual-promoter reverse-genetic system for the rapid generation of IBDV. Using this system, the attenuated IBDV/1FLAG expressing VP1-FLAG, which displays the decreased viral RNP activity, was rescued. Moreover, IBDV/1FLAG inoculation induced a similar level of neutralizing antibodies to that of its parental counterpart, protecting chickens against lethal challenge. Our study, for the first time, describes a dual-promoter reverse-genetic approach for the rapid generation of attenuated IBDV while maintaining entire parental antigenicity, suggesting a potential new method to attenuate epidemic variant IBDV strains for vaccine development.

Satellite viruses, most commonly found in plants, rely on helper viruses to complete their replication cycle. The only known example of a human satellite virus is the hepatitis D virus (HDV), and it is generally thought to require hepatitis B virus (HBV) to form infectious particles. Until 2018, HDV was the sole representative of the genus Deltavirus and was thought to have evolved in humans, the only known HDV host. The subsequent identification of HDV-like agents in birds, snakes, fish, amphibians, and invertebrates indicated that the evolutionary history of deltaviruses is likely much longer than previously hypothesized. Interestingly, none of the HDV-like agents were found in coinfection with an HBV-like agent, suggesting that these viruses use different helper virus(es). Here we show, using snake deltavirus (SDeV), that HBV and hepadnaviruses represent only one example of helper viruses for deltaviruses. We cloned the SDeV genome into a mammalian expression plasmid, and by transfection could initiate SDeV replication in cultured snake and mammalian cell lines. By superinfecting persistently SDeV-infected cells with reptarenaviruses and hartmaniviruses, or by transfecting their surface proteins, we could induce production of infectious SDeV particles. Our findings indicate that deltaviruses can likely use a multitude of helper viruses or even viral glycoproteins to form infectious particles. This suggests that persistent infections, such as those caused by arenaviruses and orthohantaviruses used in this study, and recurrent infections would be beneficial for the spread of deltaviruses. It seems plausible that further human or animal disease associations with deltavirus infections will be identified in the future.IMPORTANCE Deltaviruses need a coinfecting enveloped virus to produce infectious particles necessary for transmission to a new host. Hepatitis D virus (HDV), the only known deltavirus until 2018, has been found only in humans, and its coinfection with hepatitis B virus (HBV) is linked with fulminant hepatitis. The recent discovery of deltaviruses without a coinfecting HBV-like agent in several different taxa suggested that deltaviruses could employ coinfection by other enveloped viruses to complete their life cycle. In this report, we show that snake deltavirus (SDeV) efficiently utilizes coinfecting reptarena- and hartmaniviruses to form infectious particles. Furthermore, we demonstrate that cells expressing the envelope proteins of arenaviruses and orthohantaviruses produce infectious SDeV particles. As the envelope proteins are responsible for binding and infecting new host cells, our findings indicate that deltaviruses are likely not restricted in their tissue tropism, implying that they could be linked to animal or human diseases other than hepatitis.

Reptarenaviruses cause Boid Inclusion Body Disease (BIBD), and co-infections by several reptarenaviruses are common in affected snakes. Reptarenaviruses have only been found in captive snakes, and their reservoir hosts remain unknown. In affected animals, reptarenaviruses appear to replicate in most cell types, but their complete host range, as well as tissue and cell tropism are unknown. As with other enveloped viruses, the glycoproteins (GPs) present on the virion's surface mediate reptarenavirus cell entry, and therefore, the GPs play a critical role in the virus cell and tissue tropism. Herein, we employed single cycle replication, GP deficient, recombinant vesicular stomatitis virus (VSV) expressing the enhanced green fluorescent protein (scrVSV∆G-eGFP) pseudotyped with different reptarenavirus GPs to study the virus cell tropism. We found that scrVSV∆G-eGFPs pseudotyped with reptarenavirus GPs readily entered mammalian cell lines, and some mammalian cell lines exhibited higher, compared to snake cell lines, susceptibility to reptarenavirus GP-mediated infection. Mammarenavirus GPs used as controls also mediated efficient entry into several snake cell lines. Our results confirm an important role of the virus surface GP in reptarenavirus cell tropism and that mamma-and reptarenaviruses exhibit high cross-species transmission potential.