Chaperones Hsc70 and Hsp70 play distinct roles in the replication of bocaparvovirus minute virus of canines.

Minute virus of canines (MVC) belongs to the genus Bocaparvovirus (formerly Bocavirus) within the Parvoviridae family and causes serious respiratory and gastrointestinal symptoms in neonatal canines worldwide. A productive viral infection relies on the successful recruitment of host factors for various stages of the viral life cycle. However, little is known about the MVC-host cell interactions. In this study, we identified that two cellular proteins (Hsc70 and Hsp70) interacted with NS1 and VP2 proteins of MVC, and both two domains of Hsc70/Hsp70 were mediated for their interactions. Functional studies revealed that Hsp70 was induced by MVC infection, knockdown of Hsc70 considerably suppressed MVC replication, whereas the replication was dramatically promoted by Hsp70 knockdown. It is interesting that low amounts of overexpressed Hsp70 enhanced viral protein expression and virus production, but high amounts of Hsp70 overexpression weakened them. Upon Hsp70 overexpressing, we observed that the ubiquitination of viral proteins changed with Hsp70 overexpression, and proteasome inhibitor (MG132) restored an accumulation of viral proteins. In addition, we verified that Hsp70 family inhibitors remarkably decreased MVC replication. Overall, we identified Hsc70 and Hsp70 as interactors of MVC NS1 and VP2 proteins and were involved in MVC replication, which may provide novel targets for anti-MVC approach.

AMPK restricts HHV-6A replication by inhibiting glycolysis and mTOR signaling.

AMP-activated protein kinase (AMPK) is a cellular energy sensor regulating metabolic homeostasis. In this study, we investigated the role of AMPK in response to human herpesvirus 6A (HHV-6A) infection. We show that HHV-6A infection significantly downregulates the active phosphorylated state of AMPK in infected T cells. Pharmacological activation of AMPK highly attenuated HHV-6A propagation. Mechanistically, we found that the activation of AMPK by AICAR blocked HHV-6-induced glycolysis by inhibiting glucose metabolism and lactate secretion, as well as decreasing expressions of key glucose transporters and glycolytic enzymes. In addition, mTOR signaling has been inactivated in HHV-6A infected T cells by AICAR treatment. We also showed that HHV-6A infection of human umbilical cord blood mononuclear cells (CBMCs) reduced AMPK activity whereas the activation of AMPK by metformin drastically reduced HHV-6A DNA replication and virions production. Taken together, this study demonstrates that AMPK is a promising antiviral therapeutic target against HHV-6A infection.

Multiple functions of the nonstructural protein 3D in picornavirus infection.

3D polymerase, also known as RNA-dependent RNA polymerase, is encoded by all known picornaviruses, and their structures are highly conserved. In the process of picornavirus replication, 3D polymerase facilitates the assembly of replication complexes and directly catalyzes the synthesis of viral RNA. The nuclear localization signal carried by picornavirus 3D polymerase, combined with its ability to interact with other viral proteins, viral RNA and cellular proteins, indicate that its noncatalytic role is equally important in viral infections. Recent studies have shown that 3D polymerase has multiple effects on host cell biological functions, including inducing cell cycle arrest, regulating host cell translation, inducing autophagy, evading immune responses, and triggering inflammasome formation. Thus, 3D polymerase would be a very valuable target for the development of antiviral therapies. This review summarizes current studies on the structure of 3D polymerase and its regulation of host cell responses, thereby improving the understanding of picornavirus-mediated pathogenesis caused by 3D polymerase.

Integrated Metabolomic and transcriptomic analyses reveal deoxycholic acid promotes transmissible gastroenteritis virus infection by inhibiting phosphorylation of NF-κB and STAT3.

BACKGROUND: Acute diarrhea, dehydration and death in piglets are all symptoms of transmissible gastroenteritis virus (TGEV), which results in significant financial losses in the pig industry. It is important to understand the pathogenesis and identify new antiviral targets by revealing the metabolic interactions between TGEV and host cells. RESULTS: We performed metabolomic and transcriptomic analyses of swine testicular cells infected with TGEV. A total of 1339 differential metabolites and 206 differentially expressed genes were detected post TEGV infection. The differentially expressed genes were significantly enriched in the HIF-1 signaling pathway and PI3K-Akt signaling. Integrated analysis of differentially expressed genes and differential metabolites indicated that they were significantly enriched in the metabolic processes such as nucleotide metabolism, biosynthesis of cofactors and purine metabolism. In addition, the results showed that most of the detected metabolites involved in the bile secretion was downregulated during TGEV infection. Furthermore, exogenous addition of key metabolite deoxycholic acid (DCA) significantly enhanced TGEV replication by NF-κB and STAT3 signal pathways. CONCLUSIONS: We identified a significant metabolite, DCA, related to TGEV replication. It added TGEV replication in host cells by inhibiting phosphorylation of NF-κB and STAT3. This study provided novel insights into the metabolomic and transcriptomic alterations related to TGEV infection and revealed potential molecular and metabolic targets for the regulation of TGEV infection.

Effect of miR-17 on Polygonum Cillinerve polysaccharide against transmissible gastroenteritis virus.

Transmissible gastroenteritis virus (TGEV) could cause diarrhea, vomiting, dehydration and even death in piglets, miRNA played an important role in the interaction between virus and cell. The study aimed to investigate the impact of miR-17 on the polysaccharide of Polygonum Cillinerve (PCP) in combating TGEV. miR-17 was screened and transfection validation was performed by Real-time PCR. The function of miR-17 on PK15 cells infected with TGEV and treated with PCP was investigated by DCFH-DA loading probe, JC-1 staining and Hoechst fluorescence staining. Furthermore, the effect of miR-17 on PCP inhibiting TGEV replication and apoptosis signaling pathways during PCP against TGEV infection was measured through Real-time PCR and Western blot. The results showed that miR-17 mimic and inhibitor could be transferred into PK15 cells and the expression of miR-17 significantly increased and decreased respectively compared with miR-17 mimic and inhibitor (P < 0.05). A total 250 µg/mL of PCP could inhibit cells apoptosis after transfection with miR-17. PCP (250 µg/mL and 125 µg/mL) significantly inhibited the decrease in mitochondrial membrane potential induced by TGEV after transfection with miR-17 (P < 0.05). After transfection of miR-17 mimic, PCP at concentrations of 250 µg/mL and 125 µg/mL significantly promoted the mRNA expression of P53, cyt C and caspase 9 (P < 0.05). Compared with the control group, the replication of TGEV gRNA and gene N was significantly inhibited by PCP at concentrations of 250 µg/mL and 125 µg/mL after transfection of both miR-17 mimic and inhibitor (P < 0.05). PCP at 62.5 µg/mL significantly inhibited the replication of gene S following transfection with miR-17 inhibitor (P < 0.05). These results suggested that PCP could inhibit the replication of TGEV and apoptosis induced by TGEV by regulating miR-17.

Defense signaling pathways in resistance to plant viruses: Crosstalk and finger pointing.

Resistance to infection by plant viruses involves proteins encoded by plant resistance (R) genes, viz., nucleotide-binding leucine-rich repeats (NLRs), immune receptors. These sensor NLRs are activated either directly or indirectly by viral protein effectors, in effector-triggered immunity, leading to induction of defense signaling pathways, resulting in the synthesis of numerous downstream plant effector molecules that inhibit different stages of the infection cycle, as well as the induction of cell death responses mediated by helper NLRs. Early events in this process involve recognition of the activation of the R gene response by various chaperones and the transport of these complexes to the sites of subsequent events. These events include activation of several kinase cascade pathways, and the syntheses of two master transcriptional regulators, EDS1 and NPR1, as well as the phytohormones salicylic acid, jasmonic acid, and ethylene. The phytohormones, which transit from a primed, resting states to active states, regulate the remainder of the defense signaling pathways, both directly and by crosstalk with each other. This regulation results in the turnover of various suppressors of downstream events and the synthesis of various transcription factors that cooperate and/or compete to induce or suppress transcription of either other regulatory proteins, or plant effector molecules. This network of interactions results in the production of defense effectors acting alone or together with cell death in the infected region, with or without the further activation of non-specific, long-distance resistance. Here, we review the current state of knowledge regarding these processes and the components of the local responses, their interactions, regulation, and crosstalk.

The relationship between autophagy and respiratory viruses.

Respiratory viruses have caused severe global health problems and posed essential challenges to the medical community. In recent years, the role of autophagy as a critical process in cells in viral respiratory diseases has been noticed. One of the vital catabolic biological processes in the body is autophagy. Autophagy contributes to energy recovery by targeting and selectively directing foreign microorganisms, organelles, and senescent intracellular proteins to the lysosome for degradation and phagocytosis. Activation or suppression of autophagy is often initiated when foreign pathogenic organisms such as viruses infect cells. Because of its antiviral properties, several viruses may escape or resist this process by encoding viral proteins. Viruses can also use autophagy to enhance their replication or prolong the persistence of latent infections. Here, we provide an overview of autophagy and respiratory viruses such as coronavirus, rhinovirus, parainfluenza, influenza, adenovirus, and respiratory syncytial virus, and examine the interactions between them and the role of autophagy in the virus-host interaction process and the resulting virus replication strategy.

Co-opted cytosolic proteins form condensate substructures within membranous replication organelles of a positive-strand RNA virus.

Positive-strand RNA viruses co-opt organellar membranes for biogenesis of viral replication organelles (VROs). Tombusviruses also co-opt pro-viral cytosolic proteins to VROs. It is currently not known what type of molecular organization keeps co-opted proteins sequestered within membranous VROs. In this study, we employed tomato bushy stunt virus (TBSV) and carnation Italian ringspot virus (CIRV) - Nicotiana benthamiana pathosystems to identify biomolecular condensate formation in VROs. We show that TBSV p33 and the CIRV p36 replication proteins sequester glycolytic and fermentation enzymes in unique condensate substructures associated with membranous VROs. We find that p33 and p36 form droplets in vitro driven by intrinsically disordered region. The replication protein organizes partitioning of co-opted host proteins into droplets. VRO-associated condensates are critical for local adenosine triphosphate production to support energy for virus replication. We find that co-opted endoplasmic reticulum membranes and actin filaments form meshworks within and around VRO condensates, contributing to unique composition and structure. We propose that p33/p36 organize liquid-liquid phase separation of co-opted concentrated host proteins in condensate substructures within membranous VROs. Overall, we demonstrate that subverted membranes and condensate substructures co-exist and are critical for VRO functions. The replication proteins induce and connect the two substructures within VROs.

Absence of CD80 reduces HSV-1 replication in the eye and delays reactivation but not latency levels.

Herpes simplex virus-1 (HSV-1) infections are among the most frequent serious viral eye infections in the U.S. and are a major cause of viral-induced blindness. HSV-1 infection is known to induce T cell activation, proliferation, and differentiation that play crucial roles in the development of virus-induced inflammatory lesions, leading to eye disease and causing chronic corneal damage. CD80 is a co-stimulatory molecule and plays a leading role in T cell differentiation. Previous efforts to limit lesion severity by controlling inflammation at the cellular level led us to ask whether mice knocked out for CD80 would show attenuated virus replication following reactivation. By evaluating the effects of CD80 activity on primary and latent infection, we found that in the absence of CD80, virus replication in the eyes and virus reactivation in latent trigeminal ganglia were both significantly reduced. However, latency in latently infected CD80-/- mice did not differ significantly from that in wild-type (WT) control mice. Reduced virus replication in the eyes of CD80-/- mice correlated with significantly expanded CD11c gene expression as compared to WT mice. Taken together, our results indicate that suppression of CD80 could offer significant beneficial therapeutic effects in the treatment of Herpes Stromal Keratitis (HSK).IMPORTANCEOf the many problems associated with recurrent ocular infection, reducing virus reactivation should be a major goal of controlling ocular herpes simplex virus-1 (HSV-1) infection. In this study, we have shown that the absence of CD80 reduces HSV-1 reactivation, which marks the establishment of a previously undescribed mechanism underlying viral immune evasion that could be exploited to better manage HSV infection.

Inhibitory effects of kaempferol, quercetin and luteolin on the replication of human parainfluenza virus type 2 in vitro.

The eight flavonoids, apigenin, chrysin, hesperidin, kaempferol, myricetin, quercetin, rutin and luteolin were tested for the inhibition of human parainfluenza virus type 2 (hPIV-2) replication. Three flavonoids out of the eight, kaempferol, quercetin and luteolin inhibited hPIV-2 replication. Kaempferol reduced the virus release (below 1/10,000), partly inhibited genome and mRNA syntheses, but protein synthesis was observed. It partly inhibited virus entry into the cells and virus spreading, and also partly disrupted microtubules and actin microfilaments, indicating that the virus release inhibition was partly caused by the disruption of cytoskeleton. Quercetine reduced the virus release (below 1/10,000), partly inhibited genome, mRNA and protein syntheses. It partly inhibited virus entry and spreading, and also partly destroyed microtubules and microfilaments. Luteolin reduced the virus release (below 1/100,000), largely inhibited genome, mRNA and protein syntheses. It inhibited virus entry and spreading. It disrupted microtubules and microfilaments. These results indicated that luteolin has the most inhibitory effect on hPIV-2 relication. In conclusion, the three flavonoids inhibited virus replication by the inhibition of genome, mRNA and protein syntheses, and in addition to those, by the disruption of cytoskeleton in vitro.

Macrophages: Key Cellular Players in HIV Infection and Pathogenesis.

Although cells of the myeloid lineages, including tissue macrophages and conventional dendritic cells, were rapidly recognized, in addition to CD4+ T lymphocytes, as target cells of HIV-1, their specific roles in the pathophysiology of infection were initially largely neglected. However, numerous studies performed over the past decade, both in vitro in cell culture systems and in vivo in monkey and humanized mouse animal models, led to growing evidence that macrophages play important direct and indirect roles as HIV-1 target cells and in pathogenesis. It has been recently proposed that macrophages are likely involved in all stages of HIV-1 pathogenesis, including virus transmission and dissemination, but above all, in viral persistence through the establishment, together with latently infected CD4+ T cells, of virus reservoirs in many host tissues, the major obstacle to virus eradication in people living with HIV. Infected macrophages are indeed found, very often as multinucleated giant cells expressing viral antigens, in almost all lymphoid and non-lymphoid tissues of HIV-1-infected patients, where they can probably persist for long period of time. In addition, macrophages also likely participate, directly as HIV-1 targets or indirectly as key regulators of innate immunity and inflammation, in the chronic inflammation and associated clinical disorders observed in people living with HIV, even in patients receiving effective antiretroviral therapy. The main objective of this review is therefore to summarize the recent findings, and also to revisit older data, regarding the critical functions of tissue macrophages in the pathophysiology of HIV-1 infection, both as major HIV-1-infected target cells likely found in almost all tissues, as well as regulators of innate immunity and inflammation during the different stages of HIV-1 pathogenesis.

In silico identification of chikungunya virus replication inhibitor validated using biochemical and cell-based approaches.

Discovering an alternative therapy with a long-lasting effect on symptoms caused by chikungunya virus (CHIKV) infection is prompted by the lack of a vaccine and the absence of safe, effective and non-toxic medications. One potential strategy is synthesizing or identifying small compounds that can specifically target the active site of an essential enzyme and prevent virus replication. Previous site-directed mutagenesis studies have demonstrated the crucial role of the macrodomain, which is a part of non-structural protein 3 (nsP3), in virus replication. Exploiting this fact, the macrodomain can be targeted to discover a natural substance that can inhibit its function and thereby impede virus replication. With this aim, the present study focused on potential CHIKV nsP3 macrodomain (nsP3MD ) inhibitors through in silico, in vitro and cell-based methods. Through virtual screening of the natural compound library, nine nsP3MD inhibitors were initially identified. Molecular dynamics (MD) simulations were employed to evaluate these nine compounds based on the stability of their ligand-receptor complexes and energy parameters. Target analysis and ADMET (i.e. absorption, distribution, metabolism, excretion and toxicity) prediction of the selected compounds revealed their drug-like characteristics. Subsequent in vitro investigation allowed us to narrow the selection down to one compound, N-[2-(5-methoxy-1H-indol-3-yl) ethyl]-2-oxo-1,2-dihydroquinoline-4-carboxamide, which exhibited potent inhibition of CHIKV growth. This molecule effectively inhibited CHIKV replication in the stable embryonal rhabdomyosarcoma cell line capable of producing CHIKV. Our findings demonstrate that the selected compound possesses substantial anti-CHIKV nsP3MD activity both in vitro and in vivo. This work provides a promising molecule for further preclinical studies to develop a potential drug against the CHIKV.

DEAD-box RNA helicase 21 interacts with porcine circovirus type 2 Cap protein and facilitates viral replication.

Porcine circovirus type 2 (PCV2) is the etiological agent of PCV2-associated diseases that pose a serious threat to the swine industry. PCV2 capsid (Cap) protein has been shown to interact with DEAD-box RNA helicase 21 (DDX21), an important protein that regulates RNA virus replication. However, whether the interaction between DDX21 and the PCV2 Cap regulates PCV2 replication remains unclear. Herein, by using western blotting, interaction assays, and knockdown analysis, we found that PCV2 infection induced the cytoplasmic relocation of DDX21 from the nucleolus in cultured PK-15 cells. Moreover, the nuclear localization signal (NLS) of PCV2 Cap interacted directly with DDX21. The NLS of PCV2 Cap and 763GSRSNRFQNK772 residues at the C-terminal domain (CTD) of DDX21 were essential for the dual interaction. Upon shRNA-mediated DDX21 depletion in PK-15 cells, we observed impaired PCV2 replication via a lentivirus-delivered system, as evidenced by decreased levels of viral protein expression and virus production. In contrast, the replication of PCV2 increased in transiently DDX21-overexpressing cells. Our results indicate that DDX21 interacts with PCV2 Cap and plays a crucial role in virus replication. These results provide a reference for developing novel potential targets for prevention and control of PCV2 infection.

The adenovirus DNA-binding protein DBP.

Adenoviruses are a group of double-stranded DNA viruses that can mainly cause respiratory, gastrointestinal, and eye infections in humans. In addition, adenoviruses are employed as vector vaccines for combatting viral infections, including SARS-CoV-2, and serve as excellent gene therapy vectors. These viruses have the ability to modulate the host cell machinery to their advantage and trigger significant restructuring of the nuclei of infected cells through the activity of viral proteins. One of those, the adenovirus DNA-binding protein (DBP), is a multifunctional non-structural protein that is integral to the reorganization processes. DBP is encoded in the E2A transcriptional unit and is highly abundant in infected cells. Its activity is unequivocally linked to the formation, structure, and integrity of virus-induced replication compartments, molecular hubs for the regulation of viral processes, and control of the infected cell. DBP also plays key roles in viral DNA replication, transcription, viral gene expression, and even host range specificity. Notably, post-translational modifications of DBP, such as SUMOylation and extensive phosphorylation, regulate its biological functions. DBP was first investigated in the 1970s, pioneering research on viral DNA-binding proteins. In this literature review, we provide an overview of DBP and specifically summarize key findings related to its complex structure, diverse functions, and significant role in the context of viral replication. Finally, we address novel insights and perspectives for future research.

SARS-CoV-2 Viral Replication Persists in the Human Lung for Several Weeks after Symptom Onset.

Rationale: In the upper respiratory tract, replicating (culturable) severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is recoverable for ∼4-8 days after symptom onset, but there is a paucity of data about the frequency and duration of replicating virus in the lower respiratory tract (i.e., the human lung).Objectives: We undertook lung tissue sampling (needle biopsy) shortly after death in 42 mechanically ventilated decedents during the Beta and Delta waves. An independent group of 18 ambulatory patients served as a control group.Methods: Lung biopsy cores from decedents underwent viral culture, histopathological analysis, electron microscopy, transcriptomic profiling, and immunohistochemistry.Measurements and Main Results: Thirty-eight percent (16 of 42) of mechanically ventilated decedents had culturable virus in the lung for a median of 15 days (persisting for up to 4 wk) after symptom onset. Lung viral culture positivity was not associated with comorbidities or steroid use. Delta but not Beta variant lung culture positivity was associated with accelerated death and secondary bacterial infection (P < 0.05). Nasopharyngeal culture was negative in 23.1% (6 of 26) of decedents despite lung culture positivity. This hitherto undescribed biophenotype of lung-specific persisting viral replication was associated with an enhanced transcriptomic pulmonary proinflammatory response but with concurrent viral culture positivity.Conclusions: Concurrent rather than sequential active viral replication continues to drive a heightened proinflammatory response in the human lung beyond the second week of illness and was associated with variant-specific increased mortality and morbidity. These findings have potential implications for the design of interventional strategies and clinical management of patients with severe coronavirus disease (COVID-19).

Chicken speckle-type POZ protein (SPOP) negatively regulates MyD88/NF-κB signaling pathway mediated proinflammatory cytokine production to promote the replication of Newcastle disease virus.

The speckle-type POZ protein (SPOP) is demonstrated to be a specific adaptor of the cullin-RING-based E3 ubiquitin ligase complex that participates in multiple cellular processes. Up to now, SPOP involved in inflammatory response has attracted more attention, but the association of SPOP with animal virus infection is scarcely reported. In this study, chicken MyD88 (chMyD88), an innate immunity-associated protein, was screened to be an interacting partner of chSPOP using co-immunoprecipitation (Co-IP) combined with liquid chromatography-tandem mass spectrometry methods. This interaction was further confirmed by fluorescence co-localization, Co-IP, and pull-down assays. It was interesting that exogenous recombinant protein HA-chSPOP or endogenous chSPOP alone was mainly located in the nucleus but was translocated to the cytoplasm upon co-expression with chMyD88 or lipopolysaccharide stimulation. In addition, chSPOP reduced chMyD88 expression by ubiquitination in a dose-dependent manner, and the regulation of NF-κB activity by chSPOP was dependent solely on chMyD88. Importantly, chSPOP played a negative regulatory role in the MyD88/NF-κB signaling pathway and the production of proinflammatory cytokines. Moreover, we found that velogenic Newcastle disease virus (NDV) infection changed the subcellular localization of chSPOP and the expression patterns of chSPOP and chMyD88, and overexpression of chSPOP decreased the production of proinflammatory cytokines to enhance velogenic and lentogenic NDV replication, while siRNA-mediated chSPOP knockdown obtained the opposite results, thereby indicating that chSPOP negatively regulated MyD88/NF-κB signaling pathway mediated proinflammatory cytokine production to promote NDV replication. These findings highlight the important role of the SPOP/MyD88/NF-κB signaling pathway in NDV replication and may provide insightful information about NDV pathogenesis.

Effects of G and SH Truncation on the Replication, Virulence, and Immunogenicity of Avian Metapneumovirus.

Four mutants varying the length of the G and SH genes, including a G-truncated mutant (ΔG) and three G/SH-truncated mutants (ΔSH/G-1, ΔSH/G-2, and ΔSH/G-3), were generated via serially passaging the avian metapneumovirus strain SNU21004 into the cell lines Vero E6 and DF-1 and into embryonated chicken eggs. The mutant ΔG particles resembled parental virus particles except for the variance in the density of their surface projections. G and G/SH truncation significantly affected the viral replication in chickens' tracheal ring culture and in infected chickens but not in the Vero E6 cells. In experimentally infected chickens, mutant ΔG resulted in the restriction of viral replication and the attenuation of the virulence. The mutants ΔG and ΔSH/G-1 upregulated three interleukins (IL-6, IL-12, and IL-18) and three interferons (IFNα, IFNß, and IFNγ) in infected chickens. In addition, the expression levels of innate immunity-related genes such as Mda5, Rig-I, and Lgp2, in BALB/c mice were also upregulated when compared to the parental virus. Immunologically, the mutant ΔG induced a strong, delayed humoral immune response, while the mutant ΔSH/G-1 induced no humoral immune response. Our findings indicate the potential of the mutant ΔG but not the mutant ΔSH/G-1 as a live attenuated vaccine candidate.

Molecular determinants within the C-termini of L-HDAg that regulate hepatitis D virus replication and assembly.

Background & Aims: Hepatitis D virus (HDV) is the causative agent of chronic hepatitis delta, the most severe form of viral hepatitis. HDV encodes one protein, hepatitis delta antigen (HDAg), in two isoforms: S- and L-HDAg. They are identical in sequence except that L-HDAg contains an additional 19-20 amino acids at its C-terminus, which confer regulatory roles that are distinct from those of S-HDAg. Notably, these residues are divergent between different genotypes. We aimed to elucidate the molecular determinants within the C-termini that are essential for the regulatory role of L-HDAg in HDV replication and assembly. Methods: Northern blot, reverse-transcription quantitative PCR, and a newly established HDV trans-complementary system were used in this study. Results: C-termini of L-HDAg, albeit with high sequence variation among different genotypes, are interchangeable with respect to the trans-inhibitory function of L-HDAg and HDV assembly. The C-terminus of L-HDAg features a conserved prenylation CXXQ motif and is enriched with proline and hydrophobic residues. Abolishment of the CXXQ motif attenuated the inhibitory effect of L-HDAg on HDV replication. In contrast, the enrichment of proline and hydrophobic residues per se does not modify the trans-inhibitory function of L-HDAg. Nevertheless, these residues are essential for HDV assembly. Mechanistically, prolines and hydrophobic residues contribute to HDV assembly via a mode of action independent of the prenylated CXXQ motif. Conclusions: Within the C-terminus of L-HDAg, the CXXQ motif and the enrichment of proline and hydrophobic residues are all essential determinants of L-HDAg's regulatory roles in HDV replication and assembly. This intrinsic viral regulatory mechanism we elucidated deepens our understanding of the unique life cycle of HDV. Impact and implications: Hepatitis D virus (HDV) encodes one protein, hepatitis delta antigen (HDAg), in two isoforms: S- and L-HDAg. They are identical in sequence except that L-HDAg contains an additional 19-20 amino acids at its C-terminus. This C-terminal extension in L-HDAg confers regulatory roles in the HDV life cycle that are distinct from those of S-HDAg. Herein, we found that C-termini of L-HDAg, although with high sequence variation, are interchangeable among different HDV genotypes. Within the C-terminus of L-HDAg, the prenylation motif, and the enrichment of proline and hydrophobic residues are all essential determinants of L-HDAg's regulatory roles in HDV replication and assembly.

Expression of RNautophagy/DNautophagy-related genes is regulated under control of an innate immune receptor.

Double-stranded RNA (dsRNA) is a molecular pattern uniquely produced in cells infected with various viruses as a product or byproduct of replication. Cells detect such molecules, which indicate non-self invasion, and induce diverse immune responses to eliminate them. The degradation of virus-derived molecules can also play a role in the removal of pathogens and suppression of their replication. RNautophagy and DNautophagy are cellular degradative pathways in which RNA and DNA are directly imported into a hydrolytic organelle, the lysosome. Two lysosomal membrane proteins, SIDT2 and LAMP2C, mediate nucleic acid uptake via this pathway. Here, we showed that the expression of both SIDT2 and LAMP2C is selectively upregulated during the intracellular detection of poly(I:C), a synthetic analog of dsRNA that mimics viral infection. The upregulation of these two gene products upon poly(I:C) introduction was transient and synchronized. We also observed that the induction of SIDT2 and LAMP2C expression by poly(I:C) was dependent on MDA5, a cytoplasmic innate immune receptor that directly recognizes poly(I:C) and induces various antiviral responses. Finally, we showed that lysosomes can target viral RNA for degradation via RNautophagy and may suppress viral replication. Our results revealed a novel degradative pathway in cells as a downstream component of the innate immune response and provided evidence suggesting that the degradation of viral nucleic acids via RNautophagy/DNautophagy contributes to the suppression of viral replication.