Viral Life Cycle Research Articles

Microhomology-mediated repair machinery and its relationship with HPV-mediated oncogenesis.

Human Papillomaviruses (HPV) are a diverse family of non-enveloped dsDNA viruses that infect the skin and mucosal epithelia. Persistent HPV infections can lead to cancer frequently involving integration of the virus into the host genome, leading to sustained oncogene expression and loss of capsid and genome maintenance proteins. Microhomology-mediated double-strand break repair, a DNA double-stranded breaks repair pathway present in many organisms, was initially thought to be a backup but it's now seen as vital, especially in homologous recombination-deficient contexts. Increasing evidence has identified microhomology (MH) near HPV integration junctions, suggesting MH-mediated repair pathways drive integration. In this comprehensive review, we present a detailed summary of both the mechanisms underlying MH-mediated repair and the evidence for its involvement in HPV integration in cancer. Lastly, we highlight the involvement of these processes in the integration of other DNA viruses and the broader implications on virus lifecycles and host innate immune response.

Scorpion Venom Antimicrobial Peptide Derivative BmKn2-T5 Inhibits Enterovirus 71 in the Early Stages of the Viral Life Cycle In Vitro.

Enterovirus 71 (EV71), a typical representative of unenveloped RNA viruses, is the main pathogenic factor responsible for hand, foot, and mouth disease (HFMD) in infants. This disease seriously threatens the health and lives of humans worldwide, especially in the Asia-Pacific region. Numerous animal antimicrobial peptides have been found with protective functions against viruses, bacteria, fungi, parasites, and other pathogens, but there are few studies on the use of scorpion-derived antimicrobial peptides against unenveloped viruses. Here, we investigated the antiviral activities of scorpion venom antimicrobial peptide BmKn2 and five derivatives, finding that BmKn2 and its derivative BmKn2-T5 exhibit a significant inhibitory effect on EV71. Although both peptides exhibit characteristics typical of amphiphilic α-helices in terms of their secondary structure, BmKn2-T5 displayed lower cellular cytotoxicity than BmKn2. BmKn2-T5 was further found to inhibit EV71 in a dose-dependent manner in vitro. Moreover, time-of-drug-addition experiments showed that BmKn2-T5 mainly restricts EV71, but not its virion or replication, at the early stages of the viral cycle. Interestingly, BmKn2-T5 was also found to suppress the replication of the enveloped viruses DENV, ZIKV, and HSV-1 in the early stages of the viral cycle, which suggests they may share a common early infection step with EV71. Together, the results of our study identified that the scorpion-derived antimicrobial peptide BmKn2-T5 showed valuable antiviral properties against EV71 in vitro, but also against other enveloped viruses, making it a potential new candidate therapeutic molecule.

Incorporating Intracellular Processes in Virus Dynamics Models.

In-host models have been essential for understanding the dynamics of virus infection inside an infected individual. When used together with biological data, they provide insight into viral life cycle, intracellular and cellular virus-host interactions, and the role, efficacy, and mode of action of therapeutics. In this review, we present the standard model of virus dynamics and highlight situations where added model complexity accounting for intracellular processes is needed. We present several examples from acute and chronic viral infections where such inclusion in explicit and implicit manner has led to improvement in parameter estimates, unification of conclusions, guidance for targeted therapeutics, and crossover among model systems. We also discuss trade-offs between model realism and predictive power and highlight the need of increased data collection at finer scale of resolution to better validate complex models.

The Global Impact of HIV: A Comprehensive Review

The Human Immunodeficiency Virus (HIV) has been a significant global health challenge since its discovery in the 1980s, leading to the devastating Acquired Immunodeficiency Syndrome (AIDS). Despite advancements in medical research and antiretroviral therapies, HIV/AIDS remains a pressing public health concern, particularly in resource-limited regions. This comprehensive review provides updated data and scientific insights on the global impact of HIV, shedding light on the current state of the epidemic and the challenges faced in combating it. The review highlights the resurgence of HIV cases in Eastern European countries, such as Ukraine, Estonia, and Latvia, with surprising increases in AIDS cases observed in East Asian countries. However, it is in sub-Saharan Africa that the burden of HIV remains most acute, with high prevalence rates and a significant number of affected individuals. The region requires continued attention and resources to address the complex challenges associated with HIV prevention, treatment, and care. The article delves into the classification and structure of HIV, emphasizing its belonging to the Lentivirinae subfamily, specifically HIV-1 and HIV-2. HIV-1, the predominant viral species globally, exhibits a distinct clinical latency phase, contributing to its persistence and infectivity. The high replication rate and genetic variability of HIV, driven by the error-prone reverse transcriptase enzyme, result in the emergence of diverse subtypes and strains across different regions. Furthermore, the article explores the structural components of HIV-1, including the envelope glycoproteins (gp120 and gp41), matrix protein, and various enzymes critical to the viral life cycle. Despite progress in medical research and access to antiretroviral therapies, HIV/AIDS continues to present challenges such as limited resources, drug resistance, and regional disparities.

Human T-cell leukemia virus type 1 uses a specific tRNAPro isodecoder to prime reverse transcription.

Human T-cell leukemia virus type 1 (HTLV-1) is the only oncogenic human retrovirus discovered to date. All retroviruses are believed to use a host cell tRNA to prime reverse transcription (RT). In HTLV-1, the primer-binding site (PBS) in the genomic RNA is complementary to the 3' 18 nucleotides (nt) of human tRNAPro The human genome encodes 20 cytoplasmic tRNAPro genes representing seven isodecoders, all of which share the same 3' 18 nt sequence but vary elsewhere. Whether all tRNAPro isodecoders are used to prime RT in cells is unknown. A previous study showed that a 3' 18 nt tRNAPro-derived fragment (tRFPro) is packaged into HTLV-1 particles and can serve as an RT primer in vitro. The role of this tRNA fragment in the viral life cycle is unclear. In retroviruses, N1-methylation of the tRNA primer at position A58 (m1A) is essential for successful plus-strand transfer. Using primer-extension assays performed in chronically HTLV-1-infected cells, we found that A58 of tRNAPro is m1A-modified, implying that full-length tRNAPro is capable of facilitating successful plus-strand transfer. Analysis of HTLV-1 RT primer extension products indicated that full-length tRNAPro is likely to be the primer. To determine which tRNAPro isodecoder is used as the RT primer, we sequenced the minus-strand strong-stop RT product containing the intact tRNA primer and established that HTLV-1 primes RT using a specific tRNAPro UGG isodecoder. Further studies are required to understand how this primer is annealed to the highly structured HTLV-1 PBS and to investigate the role of tRFPro in the viral life cycle.

Spectroscopic analysis of the bacterially expressed head domain of rotavirus VP6.

The rotavirus capsid protein VP6 forms the middle of three protein layers and is responsible for many critical steps in the viral life cycle. VP6 as a structural protein can be used in various applications including as a subunit vaccine component. The head domain of VP6 (VP6H) contains key sequences that allow the protein to trimerize and that represent epitopes that are recognized by human antibodies in the viral particle. The domain is rich in β-sheet secondary structures. Here, VP6H was solubilised from bacterial inclusion bodies and purified using a single affinity chromatography step. Spectral (far-UV circular dichroism and intrinsic tryptophan fluorescence) analysis revealed that the purified domain had native-like secondary and tertiary structures. The domain could maintain structure up to 44°C during thermal denaturation following which structural changes result in an intermediate forming and finally irreversible aggregation and denaturation. The chemical denaturation with urea and guanidinium hydrochloride produces intermediates that represent a loss in the cooperativity. The VP6H domain is stable and can fold to produce its native structure in the absence of the VP6 base domain but cannot be defined as an independent folding unit.

Epstein-Barr virus-driven metabolic alterations contribute to the viral lytic reactivation and tumor progression in nasopharyngeal carcinoma.

Metabolic reprogramming induced by Epstein-Barr virus (EBV) often mirrors metabolic changes observed in cancer cells. Accumulating evidence suggests that lytic reactivation is crucial in EBV-associated oncogenesis. The aim of this study was to explore the role of metabolite changes in EBV-associated malignancies and viral life cycle control. We first revealed that EBV (LMP1) accelerates the secretion of the oncometabolite D-2HG, and serum D-2HG level is a potential diagnostic biomarker for NPC. EBV (LMP1)-driven metabolite changes disrupts the homeostasis of global DNA methylation and demethylation, which have a significantly inhibitory effect on active DNA demethylation and 5hmC content. We found that loss of 5hmC indicates a poor prognosis for NPC patients, and that 5hmC modification is a restriction factor of EBV reactivation. We confirmed a novel EBV reactivation inhibitor, α-KG, which inhibits the expression of EBV lytic genes with CpG-containing ZREs and the latent-lytic switch by enhancing 5hmC modification. Our results demonstrate a novel mechanism of which metabolite abnormality driven by EBV controls the viral lytic reactivation through epigenetic modification. This study presents a potential strategy for blocking EBV reactivation, and provides potential targets for the diagnosis and therapy of NPC.

Stimulus-responsive assembly of nonviral nucleocapsids

Controlled assembly of a protein shell around a viral genome is a key step in the life cycle of many viruses. Here we report a strategy for regulating the co-assembly of nonviral proteins and nucleic acids into highly ordered nucleocapsids in vitro. By fusing maltose binding protein to the subunits of NC-4, an engineered protein cage that encapsulates its own encoding mRNA, we successfully blocked spontaneous capsid assembly, allowing isolation of the individual monomers in soluble form. To initiate RNA-templated nucleocapsid formation, the steric block can be simply removed by selective proteolysis. Analyses by transmission and cryo-electron microscopy confirmed that the resulting assemblies are structurally identical to their RNA-containing counterparts produced in vivo. Enzymatically triggered cage formation broadens the range of RNA molecules that can be encapsulated by NC-4, provides unique opportunities to study the co-assembly of capsid and cargo, and could be useful for studying other nonviral and viral assemblies.

The PACS-2 protein and trafficking motifs in CCHFV Gn and Gc cytoplasmic domains govern CCHFV assembly

ABSTRACT The Crimean-Congo hemorrhagic fever virus (CCHFV) is a tick-borne bunyavirus that causes high mortality in humans. This enveloped virus harbors two surface glycoproteins (GP), Gn and Gc, that are released by processing of a glycoprotein precursor complex whose maturation takes place in the ER and is completed through the secretion pathway. Here, we characterized the trafficking network exploited by CCHFV GPs during viral assembly, envelopment, and/or egress. We identified membrane trafficking motifs in the cytoplasmic domains (CD) of CCHFV GPs and addressed how they impact these late stages of the viral life cycle using infection and biochemical assays, and confocal microscopy in virus-producing cells. We found that several of the identified CD motifs modulate GP transport through the retrograde trafficking network, impacting envelopment and secretion of infectious particles. Finally, we identified PACS-2 as a crucial host factor contributing to CCHFV GPs trafficking required for assembly and release of viral particles.

Genetic background of adaptation of Crimean-Congo haemorrhagic fever virus to the different tick hosts.

Crimean-Congo haemorrhagic fever orthonairovirus (CCHFV) is a negative-sense, single-stranded RNA virus with a segmented genome and the causative agent of a severe Crimean-Congo haemorrhagic fever (CCHF) disease. The virus is transmitted mainly by tick species in Hyalomma genus but other ticks such as representatives of genera Dermacentor and Rhipicephalus may also be involved in virus life cycle. To improve our understanding of CCHFV adaptation to its tick species, we compared nucleotide composition and codon usage patterns among the all CCHFV strains i) which sequences and other metadata as locality of collection and date of isolation are available in GenBank and ii) which were isolated from in-field collected tick species. These criteria fulfilled 70 sequences (24 coding for S, 23 for M, and 23 for L segment) of virus isolates originating from different representatives of Hyalomma and Rhipicephalus genera. Phylogenetic analyses confirmed that Hyalomma- and Rhipicephalus-originating CCHFV isolates belong to phylogenetically distinct CCHFV clades. Analyses of nucleotide composition among the Hyalomma- and Rhipicephalus-originating CCHFV isolates also showed significant differences, mainly in nucleotides located at the 3rd codon positions indicating changes in codon usage among these lineages. Analyses of codon adaptation index (CAI), effective number of codons (ENC), and other codon usage statistics revealed significant differences between Hyalomma- and Rhipicephalus-isolated CCHFV strains. Despite both sets of strains displayed a higher adaptation to use codons that are preferred by Hyalomma ticks than Rhipicephalus ticks, there were distinct codon usage preferences observed between the two tick species. These findings suggest that over the course of its long co-evolution with tick vectors, CCHFV has optimized its codon usage to efficiently utilize translational resources of Hyalomma species.

Rutin prevents EqHV-8 induced infection and oxidative stress via Nrf2/HO-1 signaling pathway.

The Nuclear factor erythroid 2-related factor 2 (Nrf2)/heme oxygenase-1 (HO-1) signaling pathway has been extensively studied for its role in regulating antioxidant and antiviral responses. The Equid herpesvirus type 8 (EqHV-8) poses a significant threat to the equine industry, primarily manifesting as respiratory disease, abortions, and neurological disorders in horses and donkeys. Oxidative stress is considered a key factor associated with pathogenesis of EqHV-8 infection. Unfortunately, there is currently a dearth of therapeutic interventions available for the effective control of EqHV-8. Rutin has been well documented for its antioxidant and antiviral potential. In current study we focused on the evaluation of Rutin as a potential therapeutic agent against EqHV-8 infection. For this purpose, we encompassed both in-vitro and in-vivo investigations to assess the effectiveness of Rutin in combatting EqHV-8 infection. The results obtained from in vitro experiments demonstrated that Rutin exerted a pronounced inhibitory effect on EqHV-8 at multiple stages of the viral life cycle. Through meticulous experimentation, we elucidated that Rutin's antiviral action against EqHV-8 is intricately linked to the Nrf2/HO-1 signaling pathway-mediated antioxidant response. Activation of this pathway by Rutin was found to significantly impede EqHV-8 replication, thereby diminishing the viral load. This mechanistic insight not only enhances our understanding of the antiviral potential of Rutin but also highlights the significance of antioxidant stress responses in combating EqHV-8 infection. To complement our in vitro findings, we conducted in vivo studies employing a mouse model. These experiments revealed that Rutin administration resulted in a substantial reduction in EqHV-8 infection within the lungs of the mice, underscoring the compound's therapeutic promise in vivo. In summation, our finding showed that Rutin holds promise as a novel and effective therapeutic agent for the prevention and control of EqHV-8 infections.

Triggering Degradation of Host Cellular Proteins for Robust Propagation of Influenza Viruses.

Following infection, influenza viruses strive to establish a new host cellular environment optimized for efficient viral replication and propagation. Influenza viruses use or hijack numerous host factors and machinery not only to fulfill their own replication process but also to constantly evade the host's antiviral and immune response. For this purpose, influenza viruses appear to have formulated diverse strategies to manipulate the host proteins or signaling pathways. One of the most effective tactics is to specifically induce the degradation of the cellular proteins that are detrimental to the virus life cycle. Here, we summarize the cellular factors that are deemed to have been purposefully degraded by influenza virus infection. The focus is laid on the mechanisms for the protein ubiquitination and degradation in association with facilitated viral amplification. The fate of influenza viral infection of hosts is heavily reliant on the outcomes of the interplay between the virus and the host antiviral immunity. Understanding the processes of how influenza viruses instigate the protein destruction pathways could provide a foundation for the development of advanced therapeutics to target host proteins and conquer influenza.

Identification critical host factors for Japanese encephalitis virus replication via CRISPR screening of human sgRNA library

Japanese encephalitis virus (JEV) is a pathogen with a substantial impact on both livestock and human health. However, the critical host factors in the virus life cycle remain poorly understood. Using a library comprising 123411 small guide RNAs (sgRNAs) targeting 19050 human genes, we conducted a genome-wide clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-based screen to identify essential genes for JEV replication. By employing knockout or knockdown techniques on genes, we identified eleven human genes crucial for JEV replication, such as prolactin releasing hormone receptor (PRLHR), activating signal cointegrator 1 complex subunit 3 (ASCC3), acyl-CoA synthetase long chain family member 3 (ACSL3), and others. Notably, we found that PRLHR knockdown blocked the autophagic flux, thereby inhibiting JEV infection. Taken together, these findings provide effective data for studying important host factors of JEV replication and scientific data for selecting antiviral drug targets.

Dual action effects of ethyl-p-methoxycinnamate against dengue virus infection and inflammation via NF-κB pathway suppression

Dengue virus (DENV) infection can lead to severe outcomes through a virus-induced cytokine storm, resulting in vascular leakage and inflammation. An effective treatment strategy should target both virus replication and cytokine storm. This study identified Kaempferia galanga L. (KG) extract as exhibiting anti-DENV activity. The major bioactive compound, ethyl-p-methoxycinnamate (EPMC), significantly reduced DENV-2 infection, virion production, and viral protein synthesis in HepG2 and A549 cells, with half-maximal effective concentration (EC50) values of 22.58 µM and 6.17 µM, and impressive selectivity indexes (SIs) of 32.40 and 173.44, respectively. EPMC demonstrated efficacy against all four DENV serotypes, targeting the replication phase of the virus life cycle. Importantly, EPMC reduced DENV-2-induced cytokines (IL-6 and TNF-α) and chemokines (RANTES and IP-10), as confirmed by immunofluorescence and immunoblot analyses, indicating inhibition of NF-κB activation. EPMC's role in preventing excessive inflammatory responses suggests it as a potential candidate for dengue treatment. Absorption, distribution, metabolism, excretion, and toxicity (ADMET) and drug-likeness for EPMC were predicted using SwissADME and ProTox II servers, showing good drug-like properties without toxicity. These findings highlight KG extract and EPMC as promising candidates for future anti-dengue therapeutics, offering a dual-action approach by inhibiting virus replication and mitigating inflammatory reactions.

A viral assembly inhibitor blocks SARS-CoV-2 replication in airway epithelial cells

The ongoing evolution of SARS-CoV-2 to evade vaccines and therapeutics underlines the need for innovative therapies with high genetic barriers to resistance. Therefore, there is pronounced interest in identifying new pharmacological targets in the SARS-CoV-2 viral life cycle. The small molecule PAV-104, identified through a cell-free protein synthesis and assembly screen, was recently shown to target host protein assembly machinery in a manner specific to viral assembly. In this study, we investigate the capacity of PAV-104 to inhibit SARS-CoV-2 replication in human airway epithelial cells (AECs). We show that PAV-104 inhibits >99% of infection with diverse SARS-CoV-2 variants in immortalized AECs, and in primary human AECs cultured at the air-liquid interface (ALI) to represent the lung microenvironment in vivo. Our data demonstrate that PAV-104 inhibits SARS-CoV-2 production without affecting viral entry, mRNA transcription, or protein synthesis. PAV-104 interacts with SARS-CoV-2 nucleocapsid (N) and interferes with its oligomerization, blocking particle assembly. Transcriptomic analysis reveals that PAV-104 reverses SARS-CoV-2 induction of the type-I interferon response and the maturation of nucleoprotein signaling pathway known to support coronavirus replication. Our findings suggest that PAV-104 is a promising therapeutic candidate for COVID-19 with a mechanism of action that is distinct from existing clinical management approaches.

Research progress on novel antiviral therapeutic drugs for chronic hepatitis B

The ideal goal of hepatitis B treatment is to achieve a functional cure, and the persistent cccDNA in the liver is a barrier to functional cure. Currently, antiviral drugs represented by pegylated interferon-α and nucleos (t) ide analogues cannot eliminate cccDNA, which is difficult to achieve functional cure. With the deepening of the exploration of various mechanisms and drug targets, significant progress has been made in the research and development of several novel drugs targeting the hepatitis B virus's life cycle and immune system, offering hope for a functional cure. This article presents an overview of the new progress in clinical research on antiviral therapy for chronic hepatitis B based on the literature published in recent years and international conference materials.

E-M349E and NS2A/2B–P1(T) are compensatory mutations of rDTMUV-NS2AB-P1P1’(AA), which regain virus proliferation by enhancing the virus package and restoring NS2A/2B cleavage

Duck Tembusu virus (DTMUV) belongs to the Flaviviridae family and mainly infects ducks. The genome of DTMUV is translated into a polyprotein, which is further cleaved into several protein by viral NS2B3 protease and host proteases. Crucially, the cleavage of the NS2A/2B precursor during this process is essential for the formation of replication complexes and viral packaging. Previous research has demonstrated that alanine mutations in NS2A/2B (P1P1’ (AA)) result in an attenuated strain (rDTMUV-NS2A/2B–P1P1’ (AA)) by disrupting NS2A/2B cleavage. In this study, we investigate the effects of the P1P1’ (AA) mutation on the viral life cycle and explore compensatory mutations in rDTMUV-NS2A/2B–P1P1’ (AA). Infected ducklings exhibit similar body weight gain and viral tissue loads to DTMUV-WT. Compensatory mutations E-M349E and P1(T) emerge, restoring proliferation levels to those of rDTMUV-WT. Specifically, E-M349E enhances viral packaging, while P1(T) reinstates NS2A/2B proteolysis in vitro. Thus, our findings reveal novel compensatory sites capable of restoring the attenuated DTMUV during polyprotein cleavage and packaging.

Porcine epidemic diarrhea virus E protein induces formation of stress granules and attenuates protein translation through activation of the PERK/eIF2α signaling pathway

Porcine epidemic diarrhea virus (PEDV) envelope protein (E) has been characterized as an important structural protein that plays critical roles in the interplay with its host to affect the virus life cycle. Stress granules (SGs) are host translationally silent ribonucleoproteins, which are mainly induced by the phosphorylation of eIF2α in the PERK/eIF2α signaling pathway. Our previous study found that PEDV E protein caused endoplasmic reticulum stress response (ERS)-mediated suppression of antiviral proteins’ translation. However, the link and the underlying mechanism by which PEDV induces SGs formation and suppresses host translation remain elusive. In this study, our results showed that PEDV E protein significantly elevated the expression of GRP78, CANX, and phosphorylation of PERK and eIF2α, indicating that the PERK/eIF2α branch of ERS was activated. PEDV E protein localized to the ER and aggregated into puncta to reconstruct ER structure, and further induced SGs formation, which has been caused through upregulating the G3BP1 expression level. In addition, a significant global translational stall and endogenous protein translation attenuation were detected in the presence of E protein overexpression, but the global mRNA transcriptional level remained unchanged, suggesting that the shutoff of protein translation was associated with the translation, not with the transcription process. Collectively, this study demonstrates that PERK/eIF2α activation is required for SGs formation and protein translation stall. This study is beneficial for us to better understand the mechanism by which PEDV E suppresses host protein synthesis, and provides us a new insight into the host translation regulation during virus infection.

Polysaccharides and Lectins: A Natural Complementary Approach against the SARS-CoV-2 Pandemic

Infection with the novel coronavirus SARS-CoV-2, the cause of coronavirus disease (COVID-19), has emerged as a global pandemic, with a high toll on casualties, economic impact, and human lifestyle. Despite the recent approval of various vaccines against the virus, challenges remain, including the limited availability of these vaccines, the prevalent rejection of vaccination by a large proportion of the population, and the recurrent appearance of new variants of the virus due to mutations. This context raises the alarm for scientists and clinicians to seek alternative and complementary therapies. In this context, natural products and their derivatives serve as reservoirs for potential therapeutic compounds that can be exploited in the research and production of antiviral drugs against COVID-19. Among these substances, lectin and polysaccharides isolated from fauna and flora emerge as complementary strategies for treating coronavirus infection. The review objective is to cover and analyze the specific role of polysaccharides and lectins and their synergy in the fight against this deadly SARS-CoV-2 virus. For this purpose, a primary literature search was conducted on Google Scholar, PubMed, and Web of Sciences using relevant keywords like “SARS-CoV-2 Variants”; “Antiviral Strategies”; “Antiviral Polysaccharides”; “Antiviral Lectins”; and “Synergistic effect”. The results demonstrate that lectins and polysaccharides exhibit antiviral activities against SARS-CoV-2 via mechanisms related to binding and steric blocking, the binding of glycan-based decoys, chemical reactions, virus particle disruption strategies, and steric blocking for competitive inhibition to block SARS-CoV-2 and its variants’ entry. In addition, this review analyzes the rationale behind combining polysaccharides and lectins, emphasizing complementary mechanisms of action. By simultaneously targeting multiple stages of the viral life cycle, this dual strategy aims to comprehensively inhibit viral propagation and enhance the durability of antiviral strategies over time.

The topological model of NS4B and its TMD3 in duck TMUV proliferation

Duck Tembusu virus (DTMUV) belongs to the Flaviviridae family and mainly infects ducks. Duck Tembusu virus genome encodes one polyprotein that undergoes cleavage to produce 10 proteins. Among these, NS4B, the largest transmembrane protein, plays a crucial role in the viral life cycle. In this study, we investigated the localization of NS4B and found that it is located in the endoplasmic reticulum, where it co-localizes with DTMUV dsRNA. Subsequently, we confirmed 5 different transmembrane domains of NS4B and discovered that only its transmembrane domain 3 (TMD3) can traverse ER membrane. Then mutations were introduced in the conserved amino acids of NS4B TMD3 of DTMUV replicon and infectious clone. The results showed that V111G, V117G, and I118G mutations enhanced viral RNA replication, while Q104A, T106A, A113L, M116A, H120A, Y121A, and A122G mutations reduced viral replication. Recombinant viruses with these mutations were rescued and studied in BHK21 cells. The findings demonstrated that A113L and H120A mutations led to higher viral titers than the wild-type strain, while Q104A, T106A, V111G, V117G, and Y121A mutations attenuated viral proliferation. Additionally, H120A, M116A, and A122G mutations enhanced viral proliferation. Furthermore, Q104A, T106A, V111G, M116A, V117G, Y121A, and A122G mutants showed reduced viral virulence to 10-d duck embryos. Animal experiments further indicated that all mutation viruses resulted in lower genome copy numbers in the spleen compared to the WT group 5 days postinfection. Our data provide insights into the topological model of DTMUV NS4B, highlighting the essential role of NS4B TMD3 in viral replication and proliferation.