Synthesis and biological evaluation of novel peptidomimetic inhibitors of the coronavirus 3C-like protease.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and related variants, are responsible for the devastating coronavirus disease 2019 (COVID-19) pandemic. The SARS-CoV-2 main protease (Mpro) plays a central role in the replication of the virus and represents an attractive drug target. Herein, we report the discovery of novel SARS-CoV-2 Mpro covalent inhibitors, including highly effective compound NIP-22c which displays high potency against several key variants and clinically relevant nirmatrelvir Mpro E166V mutants.

Discovery of Nirmatrelvir Resistance Mutations in SARS-CoV-2 3CLpro: A Computational-Experimental Approach.

The COVID-19 pandemic has emphasized the urgency for effective antiviral therapies against SARS-CoV-2. Targeting the main protease (3CLpro) of the virus has emerged as a promising approach, and nirmatrelvir (PF-07321332), the active component of Pfizer's oral drug Paxlovid, has demonstrated remarkable clinical efficacy. However, the emergence of resistance mutations poses a challenge to its continued success. In this study, we employed alchemical free energy perturbation (FEP) alanine scanning to identify nirmatrelvir-resistance mutations within SARS-CoV-2 3CLpro. FEP identified several mutations, which were validated through in vitro IC50 experiments and found to result in 8- and 72-fold increases in nirmatrelvir IC50 values. Additionally, we constructed SARS-CoV-2 omicron replicons containing these mutations, and one of the mutants (S144A/E166A) displayed a 20-fold increase in EC50, confirming the role of FEP in identifying drug-resistance mutations. Our findings suggest that FEP can be a valuable tool in proactively monitoring the emergence of resistant strains and guiding the design of future inhibitors with reduced susceptibility to drug resistance. As nirmatrelvir is currently widely used for treating COVID-19, this research has important implications for surveillance efforts and antiviral development.

Nirmatrelvir Resistance in SARS-CoV-2 Omicron_BA.1 and WA1 Replicons and Escape Strategies.

The antiviral component of Paxlovid, nirmatrelvir (NIR), forms a covalent bond with Cys145 of SARS-CoV-2 nsp5. To explore NIR resistance we designed mutations to impair binding of NIR over substrate. Using 12 Omicron (BA.1) and WA.1 SARS-CoV-2 replicons, cell-based complementation and enzymatic assays, we showed that in both strains, E166V imparted high NIR resistance (∼55-fold), with major decrease in WA1 replicon fitness (∼20-fold), but not BA.1 (∼2-fold). WA1 replicon fitness was restored by L50F. These differences may contribute to a potentially lower barrier to resistance in Omicron than WA1. E166V is rare in untreated patients, albeit more prevalent in paxlovid-treated EPIC-HR clinical trial patients. Importantly, NIR-resistant replicons with E166V or E166V/L50F remained susceptible to a) the flexible GC376, and b) PF-00835231, which forms additional interactions. Molecular dynamics simulations show steric clashes between the rigid and bulky NIR t-butyl and ß-branched V166 distancing the NIR warhead from its Cys145 target. In contrast, GC376, through "wiggling and jiggling" accommodates V166 and still covalently binds Cys145. PF-00835231 uses its strategically positioned methoxy-indole to form a ß-sheet and overcome E166V. Drug design based on strategic flexibility and main chain-targeting may help develop second-generation nsp5-targeting antivirals efficient against NIR-resistant viruses.

Baicalein and Baicalin Inhibit SARS-CoV-2 RNA-Dependent-RNA Polymerase.

Coronavirus Disease 2019 (COVID-19) is a deadly emerging infectious disease caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). Because SARS-CoV-2 is easily transmitted through the air and has a relatively long incubation time, COVID-19 has rapidly developed into a global pandemic. As there are no antiviral agents for the prevention and treatment of this severe pathogen except for remdesivir, development of antiviral therapies to treat infected individuals remains highly urgent. Here, we showed that baicalein and baicalin exhibited significant antiviral activity against SARS-CoV-2, the causative agent of COVID-19 through in vitro studies. Our data through cell-based and biochemical studies showed that both compounds act as SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) inhibitors directly and inhibit the activity of the SARS-CoV-2 RdRp, but baicalein was more potent. We also showed specific binding of baicalein to the SARS-CoV-2 RdRp, making it a potential candidate for further studies towards therapeutic development for COVID-19 as a selective non-nucleoside polymerase inhibitor.

Single-cell Multiplexed Fluorescence Imaging to Visualize Viral Nucleic Acids and Proteins and Monitor HIV, HTLV, HBV, HCV, Zika Virus, and Influenza Infection.

Capturing the dynamic replication and assembly processes of viruses has been hindered by the lack of robust in situ hybridization (ISH) technologies that enable sensitive and simultaneous labeling of viral nucleic acid and protein. Conventional DNA fluorescence in situ hybridization (FISH) methods are often not compatible with immunostaining. We have therefore developed an imaging approach, MICDDRP (multiplex immunofluorescent cell-based detection of DNA, RNA and protein), which enables simultaneous single-cell visualization of DNA, RNA, and protein. Compared to conventional DNA FISH, MICDDRP utilizes branched DNA (bDNA) ISH technology, which dramatically improves oligonucleotide probe sensitivity and detection. Small modifications of MICDDRP enable imaging of viral proteins concomitantly with nucleic acids (RNA or DNA) of different strandedness. We have applied these protocols to study the life cycles of multiple viral pathogens, including human immunodeficiency virus (HIV)-1, human T-lymphotropic virus (HTLV)-1, hepatitis B virus (HBV), hepatitis C virus (HCV), Zika virus (ZKV), and influenza A virus (IAV). We demonstrated that we can efficiently label viral nucleic acids and proteins across a diverse range of viruses. These studies can provide us with improved mechanistic understanding of multiple viral systems, and in addition, serve as a template for application of multiplexed fluorescence imaging of DNA, RNA, and protein across a broad spectrum of cellular systems.

Publisher Correction: HIV-1 replication complexes accumulate in nuclear speckles and integrate into speckle-associated genomic domains.

A Correction to this paper has been published: https://doi.org/10.1038/s41467-020-20152-w.

Virulent infection of outbred Hartley guinea pigs with recombinant Pichinde virus as a surrogate small animal model for human Lassa fever.

Arenaviruses, such as Lassa virus (LASV), can cause severe and fatal hemorrhagic fevers (e.g., Lassa fever, LF) in humans with no vaccines or therapeutics. Research on arenavirus-induced hemorrhagic fevers (AHFs) has been hampered by the highly virulent nature of these viral pathogens, which require high biocontainment laboratory, and the lack of an immune-competent small animal model that can recapitulate AHF disease and pathological features. Guinea pig infected with Pichinde virus (PICV), an arenavirus that does not cause disease in humans, has been established as a convenient surrogate animal model for AHFs as it can be handled in a conventional laboratory. The PICV strain P18, derived from sequential passaging of the virus 18 times in strain 13 inbred guinea pigs, causes severe febrile illness in guinea pigs that is reminiscent of lethal LF in humans. As inbred guinea pigs are not readily available and are difficult to maintain, outbred Hartley guinea pigs have been used but they show a high degree of disease heterogeneity upon virulent P18 PICV infection. Here, we describe an improved outbred guinea-pig infection model using recombinant rP18 PICV generated by reverse genetics technique followed by plaque purification, which consistently shows >90% mortality and virulent infection. Comprehensive virological, histopathological, and immunohistochemical analyses of the rP18-virus infected animals show similar features of human LASV infection. Our data demonstrate that this improved animal model can serve as a safe, affordable, and convenient surrogate small animal model for studying human LF pathogenesis and for evaluating efficacy of preventative or therapeutic approaches.

HIV-1 replication complexes accumulate in nuclear speckles and integrate into speckle-associated genomic domains.

The early steps of HIV-1 infection, such as uncoating, reverse transcription, nuclear import, and transport to integration sites are incompletely understood. Here, we imaged nuclear entry and transport of HIV-1 replication complexes in cell lines, primary monocyte-derived macrophages (MDMs) and CD4+ T cells. We show that viral replication complexes traffic to and accumulate within nuclear speckles and that these steps precede the completion of viral DNA synthesis. HIV-1 transport to nuclear speckles is dependent on the interaction of the capsid proteins with host cleavage and polyadenylation specificity factor 6 (CPSF6), which is also required to stabilize the association of the viral replication complexes with nuclear speckles. Importantly, integration site analyses reveal a strong preference for HIV-1 to integrate into speckle-associated genomic domains. Collectively, our results demonstrate that nuclear speckles provide an architectural basis for nuclear homing of HIV-1 replication complexes and subsequent integration into associated genomic loci.

Pichinde Virus Infection of Outbred Hartley Guinea Pigs as a Surrogate Animal Model for Human Lassa Fever: Histopathological and Immunohistochemical Analyses.

Lassa virus (LASV) is a mammarenavirus (arenavirus) that causes zoonotic infection in humans that can lead to fatal hemorrhagic Lassa fever (LF) disease. Currently, there are no FDA-approved vaccines or therapeutics against LASV. Development of treatments against LF and other related arenavirus-induced hemorrhagic fevers (AHFs) requires relevant animal models that can recapitulate clinical and pathological features of AHF diseases in humans. Laboratory mice are generally resistant to LASV infection, and non-human primates, while being a good animal model for LF, are limited by their high cost. Here, we describe a small, affordable, and convenient animal model that is based on outbred Hartley guinea pigs infected with Pichinde virus (PICV), a mammarenavirus that is non-pathogenic in humans, for use as a surrogate model of human LF. We conducted a detailed analysis of tissue histopathology and immunohistochemical analysis of different organs of outbred Hartley guinea pigs infected with different PICV strains that show differential disease phenotypes and pathologies. Comparing to infection with the avirulent PICV strain (P2 or rP2), animals infected with the virulent strain (P18 or rP18) show extensive pathological changes in different organs that sustain high levels of virus replication. The similarity of tissue pathology and viral antigen distribution between the virulent PICV-guinea pig model and lethal human LASV infection supports a role of this small animal model as a surrogate model of studying human LF in order to understand its pathogenesis and for evaluating potential preventative and therapeutic options against AHFs.

Visualization of Positive and Negative Sense Viral RNA for Probing the Mechanism of Direct-Acting Antivirals against Hepatitis C Virus.

RNA viruses are highly successful pathogens and are the causative agents for many important diseases. To fully understand the replication of these viruses it is necessary to address the roles of both positive-strand RNA ((+)RNA) and negative-strand RNA ((-)RNA), and their interplay with viral and host proteins. Here we used branched DNA (bDNA) fluorescence in situ hybridization (FISH) to stain both the abundant (+)RNA and the far less abundant (-)RNA in both hepatitis C virus (HCV)- and Zika virus-infected cells, and combined these analyses with visualization of viral proteins through confocal imaging. We were able to phenotypically examine HCV-infected cells in the presence of uninfected cells and revealed the effect of direct-acting antivirals on HCV (+)RNA, (-)RNA, and protein, within hours of commencing treatment. Herein, we demonstrate that bDNA FISH is a powerful tool for the study of RNA viruses that can provide insights into drug efficacy and mechanism of action.

Establishment of Bisegmented and Trisegmented Reverse Genetics Systems to Generate Recombinant Pichindé Viruses.

Pichindé virus (PICV), isolated from rice rats in Colombia, South America, is an enveloped arenavirus with a bisegmented RNA genome. The large (L) genomic segment encodes the Z matrix protein and the L RNA-dependent RNA polymerase, whereas the small (S) genomic segment encodes the nucleoprotein (NP) and the glycoprotein (GPC). This article describes the successful development of reverse genetics systems to generate recombinant PICV with either a bisegmented or trisegmented genome. We have successfully demonstrated that these systems can generate high-titered and genetically stable replication-competent viruses from plasmid transfection into appropriate cell lines. These systems demonstrate the power and versatility of reverse genetic technology to generate recombinant arenaviruses for use in pathogenesis studies and as new viral vaccine vectors.

In vitro and in vivo characterizations of pichinde viral nucleoprotein exoribonuclease functions.

UNLABELLED: Arenaviruses cause severe hemorrhagic fever diseases in humans, and there are limited preventative and therapeutic measures against these diseases. Previous structural and functional analyses of arenavirus nucleoproteins (NPs) revealed a conserved DEDDH exoribonuclease (RNase) domain that is important for type I interferon (IFN) suppression, but the biological roles of the NP RNase in viral replication and host immune suppression have not been well characterized. Infection of guinea pigs with Pichinde virus (PICV), a prototype arenavirus, can serve as a surrogate small animal model for arenavirus hemorrhagic fevers. In this report, we show that mutation of each of the five RNase catalytic residues of PICV NP diminishes the IFN suppression activity and slightly reduces the viral RNA replication activity. Recombinant PICVs with RNase catalytic mutations can induce high levels of IFNs and barely grow in IFN-competent A549 cells, in sharp contrast to the wild-type (WT) virus, while in IFN-deficient Vero cells, both WT and mutant viruses can replicate at relatively high levels. Upon infection of guinea pigs, the RNase mutant viruses stimulate strong IFN responses, fail to replicate productively, and can become WT revertants. Serial passages of the RNase mutants in vitro can also generate WT revertants. Thus, the NP RNase function is essential for the innate immune suppression that allows the establishment of a productive early viral infection, and it may be partly involved in the process of viral RNA replication. IMPORTANCE: Arenaviruses, such as Lassa, Lujo, and Machupo viruses, can cause severe and deadly hemorrhagic fever diseases in humans, and there are limited preventative and treatment options against these diseases. Development of broad-spectrum antiviral drugs depends on a better mechanistic understanding of the conserved arenavirus proteins in viral infection. The nucleoprotein (NPs) of all arenaviruses carry a unique exoribonuclease (RNase) domain that has been shown to be critical for the suppression of type I interferons. However, the functional roles of the NP RNase in arenavirus replication and host immune suppression have not been characterized systematically. Using a prototype arenavirus, Pichinde virus (PICV), we characterized the viral growth and innate immune suppression of recombinant RNase-defective mutants in both cell culture and guinea pig models. Our study suggests that the NP RNase plays an essential role in the suppression of host innate immunity, and possibly in viral RNA replication, and that it can serve as a novel target for developing antiviral drugs against arenavirus pathogens.

Identification of virulence determinants within the L genomic segment of the pichinde arenavirus.

Several arenaviruses are responsible for causing viral hemorrhagic fevers (VHF) in humans. Lassa virus (LASV), the causative agent of Lassa fever, is a biosafety level 4 (BSL4) pathogen that requires handling in BSL4 facilities. In contrast, the Pichinde arenavirus (PICV) is a BSL2 pathogen that can cause hemorrhagic fever-like symptoms in guinea pigs that resemble those observed in human Lassa fever. Comparative sequence analysis of the avirulent P2 strain of PICV and the virulent P18 strain shows a high degree of sequence homology in the bisegmented genome between the two strains despite the polarized clinical outcomes noted for the infected animals. Using reverse genetics systems that we have recently developed, we have mapped the sequence changes in the large (L) segment of the PICV genome that are responsible for the heightened virulence phenotype of the P18 strain. By monitoring the degree of disease severity and lethality caused by the different mutant viruses, we have identified specific residues located within the viral L polymerase gene encoded on the L segment essential for mediating disease pathogenesis. Through quantitative reverse transcription-PCR (RT-PCR) analysis, we have confirmed that the same set of residues is responsible for the increased viral replicative potential of the P18 strain and its heightened disease severity in vivo. Our laboratory findings serve to reinforce field observations that a high level of viremia often correlates with severe disease outcomes in LASV-infected patients.

Characterization of virulence-associated determinants in the envelope glycoprotein of Pichinde virus.

We use a small animal model, based on guinea pigs infected with a non-pathogenic Pichinde virus (PICV), to understand the virulence mechanisms of arenavirus infections in the hosts. PICV P2 strain causes a mild febrile reaction in guinea pigs, while P18 causes severe disease with clinical and pathological features reminiscent of Lassa hemorrhagic fever in humans. The envelope glycoproteins (GPC) of P2 and P18 viruses differ at positions 119, 140, and 164, all localized to the receptor-binding G1 subunit. We found that lentiviral pseudotyped virions (VLPs) bearing P18 GPC show more efficient cell entry than those with P2 GPC, and that the E140 residue plays a critical role in this process. Infection of guinea pigs with the recombinant viruses containing the E140K change demonstrated that E140 of GPC is a necessary virulence determinant of P18 infections, possibly by enhancing the ability of virus to enter target cells.

Genotype and subtype profiling of PSI-7977 as a nucleotide inhibitor of hepatitis C virus.

PSI-7977, a prodrug of 2'-F-2'-C-methyluridine monophosphate, is the purified diastereoisomer of PSI-7851 and is currently being investigated in phase 3 clinical trials for the treatment of hepatitis C. In this study, we profiled the activity of PSI-7977 and its ability to select for resistance using a number of different replicon cells. Results showed that PSI-7977 was active against genotype (GT) 1a, 1b, and 2a (strain JFH-1) replicons and chimeric replicons containing GT 2a (strain J6), 2b, and 3a NS5B polymerase. Cross-resistance studies using GT 1b replicons confirmed that the S282T change conferred resistance to PSI-7977. Subsequently, we evaluated the ability of PSI-7977 to select for resistance using GT 1a, 1b, and 2a (JFH-1) replicon cells. S282T was the common mutation selected among all three genotypes, but while it conferred resistance to PSI-7977 in GT 1a and 1b, JFH-1 GT 2a S282T showed only a very modest shift in 50% effective concentration (EC(50)) for PSI-7977. Sequence analysis of the JFH-1 NS5B region indicated that additional amino acid changes were selected both prior to and after the emergence of S282T. These include T179A, M289L, I293L, M434T, and H479P. Residues 179, 289, and 293 are located within the finger and palm domains, while 434 and 479 are located on the surface of the thumb domain. Data from the JFH-1 replicon variants showed that amino acid changes within the finger and palm domains together with S282T were required to confer resistance to PSI-7977, while the mutations on the thumb domain serve to enhance the replication capacity of the S282T replicons.

Cap binding and immune evasion revealed by Lassa nucleoprotein structure.

Lassa virus, the causative agent of Lassa fever, causes thousands of deaths annually and is a biological threat agent, for which there is no vaccine and limited therapy. The nucleoprotein (NP) of Lassa virus has essential roles in viral RNA synthesis and immune suppression, the molecular mechanisms of which are poorly understood. Here we report the crystal structure of Lassa virus NP at 1.80 Å resolution, which reveals amino (N)- and carboxy (C)-terminal domains with structures unlike any of the reported viral NPs. The N domain folds into a novel structure with a deep cavity for binding the m7GpppN cap structure that is required for viral RNA transcription, whereas the C domain contains 3'-5' exoribonuclease activity involved in suppressing interferon induction. To our knowledge this is the first X-ray crystal structure solved for an arenaviral NP, which reveals its unexpected functions and indicates unique mechanisms in cap binding and immune evasion. These findings provide great potential for vaccine and drug development.

Molecular determinants of Pichinde virus infection of guinea pigs--a small animal model system for arenaviral hemorrhagic fevers.

Arenaviruses are enveloped single-strand RNA viruses that mostly have natural hosts in rodents. Upon infection of humans, several arenaviruses can cause severe hemorrhagic fever diseases, including Lassa fever that is endemic in West Africa. The virulence mechanism of these deadly arenaviruses can be studied in a safe and economical small animal model-guinea pigs infected by a nonpathogenic arenavirus Pichinde virus (PICV), a virulent strain of which can cause similar disease syndromes in guinea pigs as arenaviral hemorrhagic fevers in humans. We have recently developed molecular clones for both the virulent and avirulent strains of PICV. Using the available reverse genetics tools, we are characterizing the molecular determinants of virulent arenavirus infections in vivo.

Development of infectious clones for virulent and avirulent pichinde viruses: a model virus to study arenavirus-induced hemorrhagic fevers.

Several arenaviruses can cause hemorrhagic fever diseases (VHFs) in humans, the pathogenic mechanism of which is poorly understood due to their virulent nature and the lack of molecular clones. A safe, convenient, and economical small animal model of arenavirus hemorrhagic fever is based on guinea pigs infected by the arenavirus Pichinde (PICV). PICV does not cause disease in humans, but an adapted strain of PICV (P18) causes a disease in guinea pigs that mimics arenavirus hemorrhagic fever in humans in many aspects, while a low-passaged strain (P2) remains avirulent in infected animals. In order to identify the virulence determinants within the PICV genome, we developed the molecular clones for both the avirulent P2 and virulent P18 viruses. Recombinant viruses were generated by transfecting plasmids that contain the antigenomic L and S RNA segments of PICV under the control of the T7 promoter into BSRT7-5 cells, which constitutively express T7 RNA polymerase. By analyzing viral growth kinetics in vitro and virulence in vivo, we show that the recombinant viruses accurately recapitulate the replication and virulence natures of their respective parental viruses. Both parental and recombinant virulent viruses led to high levels of viremia and titers in different organs of the infected animals, whereas the avirulent viruses were effectively controlled and cleared by the hosts. These novel infectious clones for the PICV provide essential tools to identify the virulence factors that are responsible for the severe VHF-like disease in infected animals.

Genomic and biological characterization of aggressive and docile strains of lymphocytic choriomeningitis virus rescued from a plasmid-based reverse-genetics system.

Arenaviruses include several causative agents of haemorrhagic fever disease in humans. In addition, the prototypic arenavirus lymphocytic choriomeningitis virus (LCMV) is a superb model for the study of virus-host interactions, including the basis of viral persistence and associated diseases. There is little understanding about the molecular mechanisms concerning the regulation and specific role of viral proteins in modulating arenavirus-host cell interactions either associated with an acute or persistent infection, and associated disease. Here, we report the genomic and biological characterization of LCMV strains 'Docile' (persistent) and 'Aggressive' (not persistent) recovered from cloned cDNA via reverse genetics. Our results confirmed that the cloned viruses accurately recreated the in vivo phenotypes associated with the corresponding natural Docile and Aggressive viral isolates. In addition, we provide evidence that the ability of the Docile strain to persist is determined by the nature of both S and L RNA segments. Thus, our findings provide the foundation for studies aimed at gaining a detailed understanding of viral determinants of LCMV persistence in its natural host, which may aid in the development of vaccines to prevent or treat the diseases caused by arenaviruses in humans.

Genome comparison of virulent and avirulent strains of the Pichinde arenavirus.

A virulent (P18) strain of the Pichinde arenavirus produces a disease in guinea pigs that somewhat mimics human Lassa fever, whereas an avirulent (P2) strain of this virus is attenuated in infected animals. It has been speculated that the composition of viral genomes may confer the degree of virulence in an infected host; the complete sequence of the viral genomes, however, is not known. Here, we provide for the first time genomic sequences of the S and L segments for both the P2 and P18 strains. Sequence comparisons identify three mutations in the GP1 subunit of the viral glycoprotein, one in the nucleoprotein NP, and five in the viral RNA polymerase L protein. These mutations, alone or in combination, may contribute to the acquired virulence of Pichinde virus infection in animals. The three amino acid changes in the variable region of the GP1 glycoprotein subunit may affect viral entry by altering its receptor-binding activity. While NP has previously been shown to modulate host immune responses to viral infection, we found that the R374 K change in this protein does not affect the NP function of suppressing interferon-beta expression. Four out of the five amino acid changes in the L protein occur in a small region of the protein that may contribute to viral virulence by enhancing its function in viral genomic RNA synthesis.