Viral Replication Research Articles

Metformin in Antiviral Therapy: Evidence and Perspectives

Metformin, a widely used antidiabetic medication, has emerged as a promising broad-spectrum antiviral agent due to its ability to modulate cellular pathways essential for viral replication. By activating AMPK, metformin depletes cellular energy reserves that viruses rely on, effectively limiting the replication of pathogens such as influenza, HIV, SARS-CoV-2, HBV, and HCV. Its role in inhibiting the mTOR pathway, crucial for viral protein synthesis and reactivation, is particularly significant in managing infections caused by HIV, CMV, and EBV. Furthermore, metformin reduces oxidative stress and reactive oxygen species (ROS), which are critical for replicating arboviruses such as Zika and dengue. The drug also regulates immune responses, cellular differentiation, and inflammation, disrupting the life cycle of HPV and potentially other viruses. These diverse mechanisms suppress viral replication, enhance immune system functionality, and contribute to better clinical outcomes. This multifaceted approach highlights metformin’s potential as an adjunctive therapy in treating a wide range of viral infections.

Virological and Immunological Characteristics of HBeAg-Positive Chronic Hepatitis B Patients With Low HBsAg Levels.

HBeAg-positive chronic hepatitis B (CHB) with low HBsAg levels represents a relatively rare serological pattern and is closely associated with the severity of liver disease. However, the underlying mechanisms in such cases remain largely unclear. Treatment-naïve HBeAg-positive CHB patients with low HBsAg levels in China were enrolled and analysed. Invitro cell experiments and immunoassays were conducted to investigate the effects of the preS2 deletion mutation on virus reproduction and host immune response. Treatment-naïve HBeAg-positive CHB patients with low HBsAg levels (low HBsAg group) exhibited higher fibrosis scores and a greater prevalence of quasispecies mutations introduced by preS2 deletion compared to patients with positive HBeAg and high HBsAg levels (high HBsAg group). Further analysis revealed that fibrosis scores in CHB patients with the preS2 deletion mutations were significantly higher compared to both in wild-type patients and the high HBsAg group. Invitro assays indicated that while this mutation may not impact HBV replication, it significantly reduced viral infectivity. The number of viral-specific IFN-γ-secreting cells induced by the mutant was significantly lower than that induced by the wild-type strain. Additionally, the levels of HBs-specific B cells and cytokine secretion from lymphocytes triggered by the mutant strain were significantly reduced. HBeAg-positive CHB patients with low HBsAg and genotype C exhibited higher noninvasive fibrosis indexes compared with typical patients, accompanied by a significant increase in quasispecies variants associated with preS2 deletion. The emergence of the preS2 deletion mutants in patients could be due to its enhanced ability to evade the host immunity.

MARCH8 Restricts RSV Replication by Promoting Cellular Apoptosis Through Ubiquitin-Mediated Proteolysis of Viral SH Protein

Numerous host factors function as intrinsic antiviral effectors to attenuate viral replication. MARCH8 is an E3 ubiquitin ligase that has been identified as a host restriction factor that inhibits the replication of various viruses. This study elucidated the mechanism by which MARCH8 restricts respiratory syncytial virus (RSV) replication through selective degradation of the viral small hydrophobic (SH) protein. We demonstrated that MARCH8 directly interacts with RSV-SH and catalyzes its ubiquitination at lysine 13, leading to SH degradation via the ubiquitin-lysosomal pathway. Functionally, MARCH8 expression enhances RSV-induced apoptosis through SH degradation, ultimately reducing viral titers. Conversely, an RSV strain harboring the SH-K13R mutation exhibited prolonged SH protein stability and attenuated apoptosis in infected cells, even in the presence of MARCH8. Targeted depletion of MARCH8 enhances cellular survival and potentially increases viral persistence. These findings demonstrate that MARCH8 promotes the early elimination of virus-infected cells by abrogating the anti-apoptotic function of SH, thereby reducing viral transmission. Our study provides novel insights into the interplay between host restriction factors and viral evasion strategies, potentially providing new therapeutic approaches for RSV infections.

Establishment of minigenomes for infectious bursal disease virus

Minigenomes (MGs) have greatly advanced research on the viral life cycle, including viral replication and transcription, virus‒host interactions, and the discovery of antivirals against RNA viruses. However, an MG for infectious bursal disease virus (IBDV) has not been well established. Here, we describe the development of IBDV MG, in which the entire coding sequences of viral genomic segments A and B are replaced with Renilla luciferase (Rluc) or enhanced green fluorescent protein (EGFP) reporter genes. Under the control of the RNA polymerase I promoter, the translation of IBDV MG is controlled by the viral proteins VP1 and VP3. Interestingly, IBDV B MG shows greater activity than does IBDV A MG. Moreover, the sense IBDV B MG was expressed at a higher level than the antisense IBDV B MG. In agreement with our previous findings, the translation of IBDV B MG controlled by VP1 and VP3 is independent of the cellular translation machinery components eukaryotic initiation factor (eIF)4E and eIF4G, but intact VP1 polymerase activity, VP3 dsRNA-binding activity, and the interaction between VP1 and VP3 are indispensable for both sense and antisense IBDV B MG activity. In addition, ribavirin, which inhibits IBDV replication, inhibits IBDV B MG activity in a dose-dependent manner. Collectively, the IBDV MG established in this study provides a powerful tool to investigate IBDV intracellular replication and transcription and virus‒host interactions and facilitates high-throughput screening for the identification of IBDV antivirals.

The N6-methyladenosine RNA epigenetic modification modulates the amplification of coxsackievirus B1 in human pancreatic beta cells

Type 1 diabetes (T1D) is characterized by a prolonged autoimmune attack resulting in the massive loss of insulin-producing beta cells. The initiation and progression of T1D depends on a complex interaction between genetic, immunological and environmental factors. Epidemiological, experimental and clinical evidence suggest a link between viral infections, particularly Coxsackievirus type B (CVB), and T1D development. Specifically, infections by the CVB serotype 1 (CVB1) contribute to the triggering of autoimmunity against beta cells in genetically predisposed individuals, and prolonged and probably non-lytic infections by CVB are associated with the development of T1D. However, the molecular mechanisms underlying CVB1 replication and establishing persistent infections in human pancreatic beta cells remain poorly understood. Here we show that the N6-methyladenosine (m6A) RNA epigenetic modification machinery regulates CVB1 amplification in the human beta cells. Using small interfering RNA (siRNA) targeting m6A writers and erasers, we observed that downregulation of m6A writers increases CVB1 amplification, while the downregulation of m6A erasers decreases it. Notably, the inhibition of Fat Mass and Obesity-associated protein (FTO), a key m6A eraser, reduced by 95% the production of infectious CVB1 in both human insulin-producing EndoC-βH1 cells and in induced pluripotent stem cell (iPSC)-derived islets. The FTO inhibitor reduced CVB1 expression within 6 h post-infection, suggesting a direct regulation of the CVB1 genome by m6A modification. Furthermore, in the absence of viral replication, FTO inhibition also decreased the translation of the incoming CVB1 genome, indicating that m6A plays a critical role in the initial stages of viral RNA translation. In addition, modulation of the m6A machinery affected the type I interferon response after poly-IC transfection, a mimic of RNA virus replication, but did not affect the cellular antiviral response in CVB1-infected cells. Altogether, these observations suggest that m6A directly affects CVB1 production. Our study provides the first evidence that the m6A epigenetic modification machinery controls CVB amplification in human pancreatic beta cells. This suggests that the m6A machinery is a potential target to control CVB infection in T1D and raises the possibility of an epigenetic control in the establishment of persistent CVB infections observed in the pancreas in individuals with type 1 diabetes.

Photodynamic Inactivation of Human Herpes Virus In Vitro with Ga(III) and Zn(II) Phthalocyanines

Photodynamic inactivation (PDI) has been revealed as a valuable approach against viral infections because of the fast therapeutic effect and low possibility of resistance development. The photodynamic inhibition of the infectivity of human herpes simplex virus type 1 (HSV-1) strain Victoria at different stages of its reproduction was studied. PDI activity was determined on extracellular virions, on the stage of their adsorption to the Madin-Darby bovine kidney (MDBK) cell line and inhibition of the viral replication stage by application of two tetra-methylpyridiloxy substituted gallium and zinc phthalocyanines (ZnPcMe and GaPcMe) upon 660 nm light exposure with a light-emitting diode (LED 660 nm). The PDI effect was evaluated on extracellular virions and virus adsorption by the terminal dilution method and the change in viral infectivity, which was compared to the untreated control group. The decrease in viral titer (Δlgs) was determined. The effect on the replicative cycle of the virus was determined using the cytopathic effect inhibition (CPE) assay. The direct influence on the virions showed a remarkable effect with a decrease in the viral titer more than 4 (Δlg > 4). The influence of the virus to the cell on the stage of adsorption was also significantly affected by the exposure time and the concentration of applied photosensitizers. A distinct inhibition was evaluated for ZnPcMe at the viral replication stage, which demonstrated a high photoinactivation index (PII = 33.0). This study suggested the high efficacy of PDI with phthalocyanines on HSV-1 virus, with full inhibition caused by the mechanism of singlet oxygen generation. These promising data are a good basis for further investigations on the PDI application against pathogenic viruses.

Fructose-1,6-diphosphate inhibits viral replication by promoting the lysosomal degradation of HMGB1 and blocking the binding of HMGB1 to the viral genome.

Fructose-1,6-diphosphate (FBP), a key glycolytic metabolite, is recognized for its cytoprotective effects during stress. However, the role of FBP in viral infections is unknown. Here, we demonstrate that virus-infected cells exhibit elevated FBP levels. Exogenous FBP inhibits both RNA and DNA virus infections in vitro and in vivo. Modulating intracellular FBP levels by regulating the expression of the metabolic enzymes FBP1 and PFK1 significantly impacts viral infections. Mechanistically, the inhibitory effects of FBP are not a result of altered viral adhesion or entry and are largely independent of type I interferon-mediated immune responses; rather, they occur through modulation of HMGB1. During viral infections, FBP predominantly reduces the protein levels of HMGB1 by facilitating its lysosomal degradation. Furthermore, FBP interacts with HMGB1 and disrupts the binding of HMGB1 to viral genomes, thereby further inhibiting viral replication. Our findings underscore the potential of FBP as a therapeutic target for controlling viral infections.

Heat shock protein 70 enhances viral replication by stabilizing Senecavirus A nonstructural proteins L and 3D

Senecavirus A (SVA) is an emerging pathogen that causes idiopathic vesicular infections in pig herds, posing a potential threat to their production performance. Heat shock protein 70 (Hsp70) is a molecular chaperone that plays an important role in host homeostasis under both physiological and stress conditions. However, the effects of Hsp70 on SVA infection and its underlying regulatory mechanisms remain unclear. Here, we confirmed that Hsp70 expression promotes SVA infection, as evidenced by the expression of viral proteins, viral titers, and the number of rSVA-eGFP-infected cells. This positive regulatory role of Hsp70 is mainly involved in post-entry stages of SVA. Viral proteins that interacted with Hsp70 were screened, and co-immunoprecipitation (co-IP) shows an interaction between Hsp70 and SVA L and 3D proteins. Subsequently, we determined that the expression of Hsp70 is beneficial for the stability of the SVA L and 3D proteins. Additionally, the substrate-binding domain (SBD) of Hsp70 plays an important role in the interaction between Hsp70 and SVA L or 3D proteins; and the deletion of this domain results in the loss of the stabilizing effect of Hsp70 on SVA L and 3D proteins and the positive regulatory effect of Hsp70 on SVA replication. These results reveal that Hsp70 promotes SVA infection by stabilizing viral L and 3D proteins and provides a strategy for preventing and controlling SVA infection.

Development Using Bioluminescence Imaging of a Recombinant Anguillid Herpesvirus 1 Vaccine Candidate Associated with Normal Replication In Vitro but Abortive Infection In Vivo

Background/Objectives: Anguillid herpesvirus 1 (AngHV-1) (recently renamed Cyvirus anguillidallo 1) is the etiologic agent of a lethal disease that affects several eel species. It is thought to be one of the main infectious agents causing a population decline in wild eels and economic loss within the eel aquaculture sector. To date, no vaccines are available against AngHV-1. Recently, we developed a safe and efficacious live attenuated recombinant vaccine against Cyprinid herpesvirus 3 (CyHV-3). This CyHV-3 recombinant vaccine encodes a deletion of ORF57. Orthologues of CyHV-3 ORF57 exist in Cyprinid herpesvirus 2 (CyHV-2, ORF57) and AngHV-1 (ORF35). Methods: In the present study, using recombinant strains and bioluminescent in vivo imaging, we investigated the effect of AngHV-1 ORF35 deletion on virus replication in vitro, virulence in vivo, and the potential of an AngHV-1 ORF35-deleted recombinant as a vaccine candidate for the mass vaccination of eels by immersion. With this goal in mind, we produced ORF35-deleted recombinants using two parental strains: a UK strain and a recombinant derived from the former strain by insertion of a Luciferase–GFP reporter cassette into a non-coding intergenic region. Results: Analyses of ORF35-deleted recombinants led to the following observations: (i) AngHV-1 ORF35 is not essential for viral growth in cell culture, and its deletion does not affect the production of extracellular virions despite reducing the size of viral plaque. (ii) In contrast to what has been observed for CyHV-3 ORF57 and CyHV-2 ORF57, in vivo bioluminescent analyses revealed that AngHV-1 ORF35 is an essential virulence factor and that its deletion led to abortive infection in vivo. (iii) Inoculation of the AngHV-1 ORF35-deleted recombinant by immersion induced a protective immune response against a wild-type challenge. This protection was shown to be dose-dependent and to rely on the infectivity of AngHV-1 ORF35-deleted virions. Conclusions: This study suggests that the AngHV-1 ORF35 protein has singular properties compared to its orthologues encoded by CyHV-2 and CyHV-3. It also supports the potential of AngHV-1 ORF35-deleted recombinants for the mass vaccination of eels by immersion.

Structural aspects of HIV-1 integrase inhibitors: SAR studies and synthetic strategies.

Acquired immunodeficiency syndrome (AIDS) poses a significant threat to life. Antiretroviral therapy is employed to diminish the replication of the human immunodeficiency virus (HIV), extending life expectancy and improving the quality of patients' lives. These HIV-1 integrase inhibitors form robust covalent interactions with Mg2+ ions, contributing to their tight binding, thereby inhibiting the integration of viral DNA into the CD4 cell DNA. The second-generation INSTIs, the most recently approved, exhibit a higher genetic barrier compared to first-generation drugs. Hence, there is a need to develop novel and safe compounds as inhibitors of HIV-1 integrase. This article presents an overview of the current landscape of anti-HIV-1 integrase inhibitors, emphasizing the structure-activity relationship (SAR) of small molecules. The molecules discussed include monocyclic rings consisting of triazoles moiety, and pyrimidine analog along with bicyclic rings with nitrogen-containing moieties. Researchers are exploring anti-HIV-1 integrase inhibitors from natural sources like marine environments, plant extracts, and microbial products, emphasizing the importance of diverse bioactive compounds in combating the virus, which have also been included in the manuscript. The current manuscript will be helpful to the scientific community engaged in the manipulation of small molecules as anti-HIV integrase inhibitors for designing newer leads.

Strategies Used by SARS-CoV-2 to Evade the Innate Immune System in an Evolutionary Perspective

By the end of 2019, the COVID-19 pandemic, resulting from the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), had diffused widely across the globe, with 770 million infected individuals and over 7 million deaths reported. In addition to its high infectivity and pathogenicity and its rapid mutation rate, the unique capacity of SARS-CoV-2 to circumvent the immune system has also contributed to the widespread nature of this pandemic. SARS-CoV-2 elicits the onset of innate immune system activation and initiates antiviral responses once it has infected the host. While battling the host’s immune responses, SARS-CoV-2 has established many countermeasures to evade attack and clearance. As the exploration of SARS-CoV-2 continues, substantial evidence has revealed that the 29 proteins synthesized by the SARS-CoV-2 genome are integral to the viral infection process. They not only facilitate viral replication and transmission, but also assist SARS-CoV-2 in escaping the host’s immune defenses, positioning them as promising therapeutic targets that have attracted considerable attention in recent studies. This review summarizes the manner in which SARS-CoV-2 interfaces with the innate immune system, with a particular focus on the continuous evolution of SARS-CoV-2 and the implications of mutations.

Challenges and global trends in combating enteric hepatitis.

Enteric hepatitis, represented by the hepatitis A virus (HAV) and hepatitis E virus (HEV), remains a significant global public health concern. While much progress has been made, many aspects of the biology and pathophysiology of HAV and HEV are still not fully understood. One of the major challenges is the absence of a reliable system for virus replication. Additionally, the lack of standardized and widely accessible diagnostic tests contributes to the underestimation of the true prevalence of these viruses. Factors such as climate change, environmental shifts, globalization and increased population mobility further complicate the spread of these infections by affecting pathogen transmission, water quality and the distribution of vectors. This review approaches the emergent research challenges and trends of enteric hepatitis and focuses on developing more efficient diagnostic tools, exploring the role of zoonotic transmission and addressing the impact of environmental and climate changes on disease dynamics, underscoring the need for collaborative, interdisciplinary efforts to effectively combat enteric hepatitis in a rapidly changing world.

A previously unidentified circRNA inhibits virus replication by regulating the miR-24-3p/KEAP1 axis.

Circular RNAs (circRNAs) exert diverse biological functions in different processes. However, the role of circRNAs during virus infection is mostly unknown. Herein, we explored the characteristics of host circRNAs using alphaherpesvirus pseudorabies virus (PRV) as a model. PRV infection upregulated the expression of circRNA circ29164, which does not encode a protein. RNA pulldown assays identified that circ29164 interacts with the microRNA ssc-miRNA-24-3p. Further analysis indicated that ssc-miR-24-3p targets the mRNA encoding kelch-like ECH-associated protein 1 (KEAP1), and circ29164 competitively binds to ssc-miR-24-3p to prevent it binding to Keap1. Apoptosis detection demonstrated that circ29164 or Keap1 overexpression, but not knockdown, induced caspase 3 activity and the release of cytochrome C from mitochondria, and inhibited PRV replication. Taken together, these data identified a previously undiscovered circRNA, circ29164, which inhibits PRV replication by competitively binding to ssc-24-3p to maintain KEAP1 levels.

Vaccination with a Replication-Dead Murine Gammaherpesvirus Lacking Viral Pathogenesis Genes Inhibits WT Virus Infection

Gammaherpesviruses are oncogenic pathogens that establish lifelong infections. There are no FDA-approved vaccines against Epstein–Barr virus or Kaposi sarcoma herpesvirus. Murine gammaherpesvirus-68 (MHV68) infection of mice provides a system for investigating gammaherpesvirus pathogenesis and testing vaccine strategies. Prime-boost vaccination with a replication-dead virus (RDV) that does not express the essential replication and transactivator protein (RTA) encoded by ORF50 (RDV-50.stop) protected against WT virus replication and reduced latency in C57BL/6 mice, and prevented lethal disease in Ifnar1−/− mice. To further improve the RDV vaccine and more closely model KSHV vaccine design, we generated an RDV lacking the unique M1-M4 genes and the non-coding tRNA-miRNA-encoded RNAs (TMERs) 6, 7, and 8 that collectively promote latency of MHV68 in vivo. Prime-boost vaccination of mice with RDV-50.stop∆M1-M4 elicited neutralizing antibodies and virus-specific CD8 T-cell responses in the lungs and spleens, the respective sites of acute replication and latency, that were comparable to RDV-50.stop vaccination. When challenged with WT MHV68, vaccinated mice exhibited a near-complete block of lytic replication and a reduction in latency and reactivation. We conclude that the unique M1-M4 genes and TMERs 6, 7, and 8, which are major determinants of WT MHV68 pathogenesis, are not required for eliciting protective immunity.

Influenza B viruses are more susceptible to high temperatures than influenza A viruses

Seasonal influenza is caused by two subtypes of influenza A virus (A/H1N1 and A/H3N2) and two lineages of influenza B viruses (B/Victoria-lineage and B/Yamagata-lineage). Seasonal influenza viruses replicate efficiently in the human upper respiratory tract, where the temperature is 33 °C. In this study, we investigated the susceptibility of seasonal influenza A and B viruses to different temperatures. We examined the differences in viral replication efficiency inside cultured cells and in infectious titre outside cultured cells at different temperatures (i.e., 33 °C, 37 °C, and 39 °C). We found that there were differences in temperature sensitivity between influenza A and B viruses, with influenza B viruses being more temperature sensitive. In addition, we found that cells cultured at 39 °C and infected with influenza B virus showed decreased expression of HA protein with receptor-binding activity on the cell surface. Our findings contribute to our understanding of the properties of seasonal influenza viruses.

Influenza a virus antiparallel helical nucleocapsid-like pseudo-atomic structure.

Influenza A viruses are responsible for human seasonal epidemics and severe animal pandemics with a risk of zoonotic transmission to humans. The viral segmented RNA genome is encapsidated by nucleoproteins (NP) and attached to the heterotrimeric polymerase, forming the viral ribonucleoproteins (vRNPs). Flexible helical vRNPs are central for viral transcription and replication. In this study, we present an advanced biological tool, the antiparallel helical RNP-like complex, assembled from recombinant N-terminally truncated NP and short synthetic RNA. The 3.0 Å cryo-electron microscopy structure details for the first time the whole RNA pathway across NP as well as NP-NP interactions that drive the antiparallel helical assembly accommodating major and minor grooves. Our findings show that the surface of the protein can harbour several conformations of the RNA, confirming that the number of nucleobases that binds to NP is not fixed, but ranges probably between 20 and 24. Taking all together, our data provide details to further understand the genome encapsidation and explain the inherent flexibility of influenza A virus vRNPs.

Optimization of antiviral dosing in Herpesviridae encephalitis: a promising approach to improve outcome?

Despite established antiviral therapy for herpes simplex (HSV), varicella zoster (VZV) and cytomegalovirus (CMV) encephalitis, outcome remains poor. To assess pharmacokinetic (PK) and -dynamic (PD) data of antiviral drugs in the central nervous system (CNS) to optimize treatment of Herpesviridae encephalitis. PUBMED search 1950 to September 2024, terms 1. "encephalitis" and ("HSV" or "VZV" or "CMV") or 2. cerebrospinal and ["(val)acyclovir" or "(val)ganciclovir" or "foscarnet" or "cidofovir"]. Antivirals against herpes viruses apparently act in a time-dependent manner. To suppress viral replication, drug concentration in the extracellular space at the site of the infection should be kept above the concentrations active in cell cultures for 24h per day. Most data reflect delayed drug entry into lumbar cerebrospinal fluid (CSF). Ratios of the areas of the concentration/time curves (AUC) in CSF and serum (AUCCSF/AUCS) of acyclovir, ganciclovir and foscarnet are 0.15-0.3 in the absence of meningeal inflammation and increase in severe meningoencephalitis. Elimination half-lives (t1/2β) are longer in CSF than in plasma. CSF concentrations are rough approximations of drug concentrations in the cerebral extracellular fluid (ECF). Lumbar CSF concentrations usually are higher than ventricular or cisternal CSF concentrations tending to overestimate cerebral ECF concentrations. Provided the availability of adequate measurements in individual CNS compartments - antiviral concentrations and efficacy may be predicted by future PK/PD modeling. Probenecid holds potential to reduce efflux of antiviral drugs from the CNS. The long t1/2β of antiviral drugs in CSF suggest relatively uniform steady state CSF levels at dosing intervals ≤12h and accumulation after repeated dosing. Probenecid is of unproven utility. To rapidly attain effective concentrations in the infected tissue, physiologically based pharmacokinetic (PBPK) and PK/PD modeling may be helpful. Until reliable PK/PD data are available, doubling the first dose should be considered.

Enteroviral 3C protease cleaves N4BP1 to impair the host inflammatory response.

Targeting cellular proteins for cleavage by enteroviral 3Cpro is a conserved strategy used by enteroviruses to promote viral replication. While the cleavage of certain host proteins by 3Cpro may not affect viral replication, it is strongly associated with the pathogenesis of viral infection. In this study, we identified and characterized N4BP1, which plays such a role, using a combination of bioinformatic, biochemical, and cell biological approaches. Our data show that multiple 3Cpros cleave N4BP1 at residue Q816 and that cleavage of endogenous N4BP1 can occur during viral infection. N4BP1 has no effect on coxsackievirus B3 replication, but 3Cpro-induced N4BP1 cleavage abolishes its regulatory function in NF-κB signaling. We also show that mouse N4bp1 resists human enteroviral 3Cpro cleavage. In contrast, rodent enteroviral EMCV 3Cpro can target human and mouse N4BP1 for cleavage at different residues, which indicates that future investigations are needed to elucidate the potential mechanisms involved.

Deubiquitinase USP37 enhances the anti-HIV-2/SIV ability of the host restriction factor SAMHD1.

The Vpx protein encoded by HIV-2/simian immunodeficiency virus (SIV) can antagonize the restriction of the host intrinsic restriction factor, SAMHD1, in nondividing cells by promoting its polyubiquitination and subsequent degradation, thereby facilitating viral replication and immune evasion. However, the role of deubiquitinating enzymes (DUBs) in the dynamics of virus and host remains poorly understood. Here, we demonstrate that DUB USP37 significantly reverses the Vpx-mediated degradation of SAMHD1 in various HIV-2/SIV subtypes by interacting with SAMHD1 and removing its ubiquitin chains. Notably, USP37 deubiquitinates SAMHD1 by directly recognizing SAMHD1 rather than by targeting the E3 ubiquitin ligase. The deubiquitinase activity of USP37 and its ubiquitin interacting motifs are essential for the deubiquitination of SAMHD1, whereas the phosphorylation state of USP37 does not influence its activity. Additionally, USP37 enhances the suppression of the retrotransposition of LINE-1 elements by SAMHD1 via stabilizing SAMHD1. Our findings provide important evidence that enhancing the deubiquitinating activity of some DUBs results in the stability of the host restriction factor and might be a viable strategy against HIV/SIV infections.IMPORTANCESAMHD1 is a multifunctional protein, including restricting virus replication, maintaining genomic integrity through DNA repair, modulating the immune response by influencing the production of type I interferons and other cytokines, and affecting cancer cell proliferation and sensitivity to chemotherapy. However, HIV-2/simian immunodeficiency virus (SIV)-encoded Vpx and the host E3 ligase TRIM21 can induce the degradation of SAMHD1 via the ubiquitin-proteasome pathway. Therefore, it is necessary to find the strategy to stabilize SAMHD1. Our study demonstrates that the deubiquitinase USP37 reverses Vpx- and TRIM21-mediated degradation of SAMHD1, thereby inhibiting SIV replication and LINE-1 activity by stabilizing SAMHD1. Thus, we report a novel role of USP37, which represents a potentially useful target for the development of new drugs.

The effective multiplicity of infection for HCMV depends on the activity of the cellular 20S proteasome.

Human cytomegalovirus (HCMV) is a betaherpesvirus capable of infecting numerous cell types and persisting throughout an infected individual's life. Disease usually occurs in individuals with compromised or underdeveloped immune systems. Several antivirals exist but have limitations relating to toxicity and resistance. HCMV replication involves upregulation of host proteasomal activities, which play important roles in the temporal stages of replication. Here, we defined the impact on replication kinetics of the proteasome inhibitor, bortezomib. We demonstrate that bortezomib significantly reduces levels of viral genomes and infectious virions produced from a population of cells. Inhibition reduced expression of viral proteins that are influenced by genome synthesis. When added prior to 24 hpi, we observe decreases in PCNA and Cdk1 while increases in p21 whose regulations contribute to efficient replication. This response synergized with an antiviral, maribavir. Since some replication occurred, we tested the hypothesis that a subset of infected cells might break through inhibition. Initially, we simulated bortezomib activities using a mechanistic computational model of late-lytic replication. Upon reducing multiplicity of infection (MOI) in silico, we observed near-identical simulated results compared to experimental data. Next, we analyzed replication using live-cell imaging. This revealed treated cultures do contain a population of cells with fully developed late-stage cytoplasmic assembly compartments but at significantly lower numbers. We refer to this as the effective MOI. Overall, our studies support a hypothesis in which 20S proteasome inhibition disrupts HCMV replication by reducing the MOI to an effective MOI, defined by a fraction of infected cells capable of progressing to fulminant infection.IMPORTANCEHuman cytomegalovirus (HCMV) infection and reactivation continues to contribute to morbidity and mortality around the world. Antiviral compounds are available but have limitations. Here, we have defined the impact of the proteasome inhibitor bortezomib on HCMV replication. Proteasomal activities play a critical role in temporal changes required for replication. We demonstrate that disrupting these activities inhibits viral replication while likely supporting increased antiviral activity of the anti-HCMV agent, maribavir. Using a combination of live-cell imaging and computational tools, we discover that a subset of infected cells progresses to fulminant infection, which we define as the effective multiplicity of infection, and this subset would otherwise be missed when analyzing the average of the population.