Characterization of bi-segmented and tri-segmented recombinant Pichinde virus particles.

Mammarenaviruses include several highly virulent pathogens (e.g., Lassa virus) capable of causing severe hemorrhagic fever diseases for which there are no approved vaccines and limited treatment options. Mammarenaviruses are enveloped, bi-segmented ambisense RNA viruses. There is limited knowledge about cellular proteins incorporated into progeny virion particles and their potential biological roles in viral infection. Pichinde virus (PICV) is a prototypic arenavirus used to characterize mammarenavirus replication and pathogenesis. We have developed a recombinant PICV with a tri-segmented RNA genome as a viral vector platform. Whether the tri-segmented virion differs from the wild-type bi-segmented one in viral particle morphology and protein composition has not been addressed. In this study, recombinant PICV (rPICV) virions with a bi-segmented (rP18bi) and a tri-segmented (rP18tri) genome were purified by density-gradient ultracentrifugation and analyzed by cryo-electron microscopy and mass spectrometry. Both virion types are pleomorphic with spherical morphology and have no significant difference in size despite rP18tri having denser particles. Both virion types also contain similar sets of cellular proteins. Among the highly enriched virion-associated cellular proteins are components of the endosomal sorting complex required for transport pathway and vesicle trafficking, such as ALIX, Tsg101, VPS, CHMP, and Ras-associated binding proteins, which have known functions in virus assembly and budding. Other enriched cellular proteins include peripheral and transmembrane proteins, chaperone proteins, and ribosomal proteins; their biological roles in viral infection warrant further analysis. Our study provides important insights into mammarenavirus particle formation and aids in the future development of viral vectors and antiviral discovery.IMPORTANCEMammarenaviruses, such as Lassa virus, are enveloped RNA viruses that can cause severe hemorrhagic fever diseases (Lassa fever) with no approved vaccine and limited therapeutic options. Cellular proteins incorporated into progeny virion particles and their biological roles in mammarenavirus infection have not been well characterized. Pichinde virus (PICV) is a prototypic mammarenavirus used as a surrogate model for Lassa fever. We used cryo-electron microscopy and proteomic analysis to characterize the morphology and protein contents of the purified PICV particles that package either two (bi-segmented) or three (tri-segmented) genomic RNA segments. Our results demonstrate a similar virion morphology but different particle density for the bi- and tri-segmented viral particles and reveal major virion-associated cellular proteins. This study provides important insights into the virus-host interactions that can be used for antiviral development and optimizing arenavirus-based vaccine vectors.

Influenza A virus activates cellular Tropomyosin receptor kinase A (TrkA) signaling to promote viral replication and lung inflammation.

Influenza A virus (IAV) infection causes acute respiratory disease with potential severe and deadly complications. Viral pathogenesis is not only due to the direct cytopathic effect of viral infections but also to the exacerbated host inflammatory responses. Influenza viral infection can activate various host signaling pathways that function to activate or inhibit viral replication. Our previous studies have shown that a receptor tyrosine kinase TrkA plays an important role in the replication of influenza viruses in vitro, but its biological roles and functional mechanisms in influenza viral infection have not been characterized. Here we show that IAV infection strongly activates TrkA in vitro and in vivo. Using a chemical-genetic approach to specifically control TrkA kinase activity through a small molecule compound 1NMPP1 in a TrkA knock-in (TrkA KI) mouse model, we show that 1NMPP1-mediated TrkA inhibition completely protected mice from a lethal IAV infection by significantly reducing viral loads and lung inflammation. Using primary lung cells isolated from the TrkA KI mice, we show that specific TrkA inhibition reduced IAV viral RNA synthesis in airway epithelial cells (AECs) but not in alveolar macrophages (AMs). Transcriptomic analysis confirmed the cell-type-specific role of TrkA in viral RNA synthesis, and identified distinct gene expression patterns under the TrkA regulation in IAV-infected AECs and AMs. Among the TrkA-activated targets are various proinflammatory cytokines and chemokines such as IL6, IL-1ß, IFNs, CCL-5, and CXCL9, supporting the role of TrkA in mediating lung inflammation. Indeed, while TrkA inhibitor 1NMPP1 administered after the peak of IAV replication had no effect on viral load, it was able to decrease lung inflammation and provided partial protection in mice. Taken together, our results have demonstrated for the first time an important biological role of TrkA signaling in IAV infection, identified its cell-type-specific contribution to viral replication, and revealed its functional mechanism in virus-induced lung inflammation. This study suggests TrkA as a novel host target for therapeutic development against influenza viral disease.

Evaluating the Biological Role of Lassa Viral Z Protein-Mediated RIG-I Inhibition Using a Replication-Competent Trisegmented Pichinde Virus System in an Inducible RIG-IN Expression Cell Line.

Lassa virus (LASV) is a mammarenavirus that can cause lethal Lassa fever disease with no FDA-approved vaccine and limited treatment options. Fatal LASV infections are associated with innate immune suppression. We have previously shown that the small matrix Z protein of LASV, but not of a nonpathogenic arenavirus Pichinde virus (PICV), can inhibit the cellular RIG-I-like receptors (RLRs), but its biological significance has not been evaluated in an infectious virus due to the multiple essential functions of the Z protein required for the viral life cycle. In this study, we developed a stable HeLa cell line (HeLa-iRIGN) that could be rapidly and robustly induced by doxycycline (Dox) treatment to express RIG-I N-terminal effector, with concomitant production of type I interferons (IFN-Is). We also generated recombinant tri-segmented PICVs, rP18tri-LZ, and rP18tri-PZ, which encode LASV Z and PICV Z, respectively, as an extra mScarlet fusion protein that is nonessential for the viral life cycle. Upon infection, rP18tri-LZ consistently expressed viral genes at a higher level than rP18tri-PZ. rP18tri-LZ also showed a higher level of a viral infection than rP18tri-PZ did in HeLa-iRIGN cells, especially upon Dox induction. The heterologous Z gene did not alter viral growth in Vero and A549 cells by growth curve analysis, while LASV Z strongly increased and prolonged viral gene expression, especially in IFN-competent A549 cells. Our study provides important insights into the biological role of LASV Z-mediated RIG-I inhibition and implicates LASV Z as a potential virulence factor. IMPORTANCE Lassa virus (LASV) can cause lethal hemorrhagic fever disease in humans but other arenaviruses, such as Pichinde virus (PICV), do not cause obvious disease. We have previously shown that the Z protein of LASV but not of PICV can inhibit RIG-I, a cytosolic innate immune receptor. In this study, we developed a stable HeLa cell line that can be induced to express the RIG-I N-terminal effector domain, which allows for timely control of RIG-I activation. We also generated recombinant PICVs encoding LASV Z or PICV Z as an extra gene that is nonessential for the viral life cycle. Compared to PICV Z, LASV Z could increase viral gene expression and viral infection in an infectious arenavirus system, especially when RIG-I signaling is activated. Our study presented a convenient cell system to characterize RIG-I signaling and its antagonists and revealed LASV Z as a possible virulence factor and a potential antiviral target.

SARS-CoV-2 infection of kidney tissues in some severe and fatal cases of COVID-19.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection can lead to diverse clinical manifestations and pathologies that involve multiple organs. Even though the disease severity is manifested mainly in the respiratory tract, which is the primary target of SARS-CoV-2 infection, acute kidney injury in the form of acute tubular necrosis has also been noted in some COVID-19 cases. It is not entirely clear whether renal cells can be infected by the virus that might be involved in acute kidney disorder. In a recent publication by Radovic and colleagues, that has been selected as the editor's choice paper published in the Journal of Medical Virology, the authors provided strong histopathological and immunofluorescence evidence of SARS-CoV-2 infection and tissue injury of renal parenchymal and tubular epithelial cells, which strongly suggest an active viral replication in the kidney of some severe and fatal COVID-19 cases, and to a lesser extent, a potential role for innate immune cells in viral infection and renal disease pathogenesis.

Host-Directed Antiviral Therapy.

Antiviral drugs have traditionally been developed by directly targeting essential viral components. However, this strategy often fails due to the rapid generation of drug-resistant viruses. Recent genome-wide approaches, such as those employing small interfering RNA (siRNA) or clustered regularly interspaced short palindromic repeats (CRISPR) or those using small molecule chemical inhibitors targeting the cellular "kinome," have been used successfully to identify cellular factors that can support virus replication. Since some of these cellular factors are critical for virus replication, but are dispensable for the host, they can serve as novel targets for antiviral drug development. In addition, potentiation of immune responses, regulation of cytokine storms, and modulation of epigenetic changes upon virus infections are also feasible approaches to control infections. Because it is less likely that viruses will mutate to replace missing cellular functions, the chance of generating drug-resistant mutants with host-targeted inhibitor approaches is minimized. However, drug resistance against some host-directed agents can, in fact, occur under certain circumstances, such as long-term selection pressure of a host-directed antiviral agent that can allow the virus the opportunity to adapt to use an alternate host factor or to alter its affinity toward the target that confers resistance. This review describes novel approaches for antiviral drug development with a focus on host-directed therapies and the potential mechanisms that may account for the acquisition of antiviral drug resistance against host-directed agents.

Biological Characterization of Conserved Residues within the Cytoplasmic Tail of the Pichinde Arenaviral Glycoprotein Subunit 2 (GP2).

Several mammarenaviruses can cause deadly hemorrhagic fever infections in humans, with limited preventative and therapeutic measures available. Arenavirus cell entry is mediated by the viral glycoprotein (GP) complex, which consists of the stable signal peptide (SSP), the receptor-binding subunit GP1, and the transmembrane subunit GP2. The GP2 cytoplasmic tail (CT) is relatively conserved among arenaviruses and is known to interact with the SSP to regulate GP processing and membrane fusion, but its biological role in the context of an infectious virus has not been fully characterized. Using a Pichinde virus (PICV) GP expression vector and a PICV reverse genetics system, we systematically characterized the functional roles of 12 conserved residues within the GP2 CT in GP processing, trafficking, assembly, and fusion, as well as in viral replication. Except for P478A and K505A R508A, alanine substitutions at conserved residues abolished GP processing and membrane fusion in plasmid-transfected cells. Six invariant H and C residues and W503 are essential for viral replication, as evidenced by the fact that their mutant viruses could not be rescued. Both P480A and R482A mutant viruses were rescued, grew similarly to wild-type (WT) virus, and produced evidently processed GP1 and GP2 subunits in virus-infected cells, despite the fact that the same mutations abolished GP processing and membrane fusion in a plasmid-based protein expression system, illustrating the importance of using an infectious-virus system for analyzing viral glycoprotein function. In summary, our results demonstrate an essential biological role of the GP2 CT in arenavirus replication and suggest it as a potential novel target for developing antivirals and/or attenuated viral vaccine candidates.IMPORTANCE Several arenaviruses, such as Lassa virus (LASV), can cause severe and lethal hemorrhagic fever diseases with high mortality and morbidity, for which no FDA-approved vaccines or therapeutics are available. Viral entry is mediated by the arenavirus GP complex, which consists of the stable signal peptide (SSP), the receptor-binding subunit GP1, and the transmembrane subunit GP2. The cytoplasmic tail (CT) of GP2 is highly conserved among arenaviruses, but its functional role in viral replication is not completely understood. Using a reverse genetics system of a prototypic arenavirus, Pichinde virus (PICV), we show that the GP2 CT contains certain conserved residues that are essential for virus replication, implicating it as a potentially good target for developing antivirals and live-attenuated viral vaccines against deadly arenavirus pathogens.

Arenaviral Nucleoproteins Suppress PACT-Induced Augmentation of RIG-I Function To Inhibit Type I Interferon Production.

RIG-I is a major cytoplasmic sensor of viral pathogen-associated molecular pattern (PAMP) RNA and induces type I interferon (IFN) production upon viral infection. A double-stranded RNA (dsRNA)-binding protein, PACT, plays an important role in potentiating RIG-I function. We have shown previously that arenaviral nucleoproteins (NPs) suppress type I IFN production via their RNase activity to degrade PAMP RNA. We report here that NPs of arenaviruses block the PACT-induced enhancement of RIG-I function to mediate type I IFN production and that this inhibition is dependent on the RNase function of NPs, which is different from that of a known mechanism of other viral proteins to abolish the interaction between PACT and RIG-I. To understand the biological roles of PACT and RIG-I in authentic arenavirus infection, we analyze growth kinetics of recombinant Pichinde virus (PICV), a prototypical arenavirus, in RIG-I knockout (KO) and PACT KO mouse embryonic fibroblast (MEF) cells. Wild-type (WT) PICV grew at higher titers in both KO MEF lines than in normal MEFs, suggesting the important roles of these cellular proteins in restricting virus replication. PICV carrying the NP RNase catalytically inactive mutation could not grow in normal MEFs but could replicate to some extent in both KO MEF lines. The level of virus growth was inversely correlated with the amount of type I IFNs produced. These results suggest that PACT plays an important role in potentiating RIG-I function to produce type I IFNs in order to restrict arenavirus replication and that viral NP RNase activity is essential for optimal viral replication by suppressing PACT-induced RIG-I activation.IMPORTANCE We report here a new role of the nucleoproteins of arenaviruses that can block type I IFN production via their specific inhibition of the cellular protein sensors of virus infection (RIG-I and PACT). Our results suggest that PACT plays an important role in potentiating RIG-I function to produce type I IFNs in order to restrict arenavirus replication. This new knowledge can be exploited for the development of novel antiviral treatments and/or vaccines against some arenaviruses that can cause severe and lethal hemorrhagic fever diseases in humans.