Search Results (26)

The ability of a nucleic acid molecule to self-replicate is the driving force behind the evolution of cellular life and the transition from RNA to DNA as the genetic material. Thus, the physicochemical properties of genome replication, such as the requirement for a terminal hydroxyl group for de novo DNA synthesis, are conserved in all three domains of life: eukaryotes, bacteria, and archaea. Canonical DNA replication is initiated from specific chromosomal sequences termed origins. Early bacterial models of DNA replication proposed origins as regulatory points for spatiotemporal control, with replication factors acting on a single origin on the chromosome. In eukaryotes and archaea, however, replication initiation usually involves multiple origins, with complex spatiotemporal regulation in the former. An alternative replication initiation mechanism, recombination-dependent replication, is observed in every cellular domain (and viruses); DNA synthesis is initiated instead from the 3′ end of a recombination intermediate. In the domain archaea, species including Haloferax volcanii are not only capable of initiating DNA replication without origins but grow faster without them. This raises questions about the necessity and nature of origins. Why have archaea retained such an alternative DNA replication initiation mechanism? Might recombination-dependent replication be the ancestral mode of DNA synthesis that was used during evolution from the primordial RNA world? This review provides a historical overview of major advancements in the study of DNA replication, followed by a comparative analysis of replication initiation systems in the three domains of life. Our current knowledge of origin-dependent and recombination-dependent DNA replication in archaea is summarised. Full article

Background/Objectives: Since its emergence in 2019, the rapid spread of SARS-CoV-2 led to the global pandemic. Recent large-scale dengue fever outbreaks overlapped with the COVID-19 pandemic, leading to increased cases of co-infection and posing severe public health risks. Accordingly, the development of effective combined SARS-CoV-2 and dengue virus (DENV) vaccines is necessary to control the spread and prevalence of both viruses. Methods: In this study, we designed Sindbis virus (SINV) replicon-based SARS-CoV-2 and DENV chimeric vaccines using two delivery strategies: DNA-launched self-replicating RNA replicon (DREP) and viral replicon particle (VRP) systems. Results: Cellular and animal experiments confirmed that the vaccines effectively produced viral proteins and elicited strong immunogenicity. These vaccines induced robust immune responses and neutralizing activity against live SARS-CoV-2, DENV1, and DENV2 viruses. In addition, passively transferred sera from BALB/c mice immunized with these vaccines into AG129 mice provided significant protection against lethal DENV2 challenge. The transferred sera protected the mice from physical symptoms, reduced viral loads in the kidney, spleen, liver, and intestine, and prevented DENV2-induced vascular leakage in these tissues. Conclusions: Therefore, combined vaccines based on the SINV replicon system are promising candidates for pandemic control. These results lay a foundation for further development of a safe and effective combination vaccine against SARS-CoV-2 and DENV. Full article

Autophagy is an important cellular response against intracellular pathogens. However, some viruses have evolved mechanisms to hijack this defensive process to provide favorable conditions for virus replication in host cells. The porcine epidemic diarrhea virus (PEDV) has been shown to alter autophagy pathways; however, it is still unknown through which receptors PEDV induces autophagy in IPEC-J2 cells, whether autophagy facilitates PEDV replication, and which functional domains of PEDV proteins are primarily responsible for inducing autophagy. Here, we found that PEDV infection induces autophagy in host cells via distinct and uncoupled molecular pathways. RNA-seq technology was used to analyze the expression patterns of intracellular genes in PEDV-infected IPEC-J2 cells using transcriptomics. The results demonstrate that PEDV triggers autophagy via the cellular pathogen receptor TLR4 and the AKT-mTOR pathway. As evidenced by autophagosome detection, PEDV infection increases autophagosomes and light chain 3 (LC3)-II as well as downregulated AKT-mTOR phosphorylation. Our study revealed that the binding of the viral protein NSP61-2C (56-151aa) to TLR4 triggers autophagy and inactivates the AKT-mTOR pathway, both of which are critical for facilitating PEDV infection. Through screening and analysis, TLR4 was found to be a key gene involved in PEDV-induced autophagy. The screening of the key functional domains of NSP6 (56-151aa) for their ability to induce autophagy in IPEC-J2 cells provided a basis for the in-depth analysis of the pathogenic mechanism of PEDV infection-induced autophagy and promotion of self-replication and also provided an important target for the study of PEDV antiviral drugs. In conclusion, we elucidated that the PEDV infection of IPEC-J2 cells could induce autophagy and found that PEDV could use autophagy to promote its own replication. Full article

Alphaviruses are known for being model viruses for studying cellular functions related to viral infections but also for causing epidemics in different parts of the world. More recently, alphavirus-based expression systems have demonstrated efficacy as vaccines against infectious diseases and as therapeutic applications for different cancers. Point mutations in the non-structural alphaviral replicase genes have generated enhanced transgene expression and created temperature-sensitive expression vectors. The recently engineered trans-amplifying RNA system can provide higher translational efficiency and eliminate interference with cellular translation. The self-replicating feature of alphaviruses has provided the advantage of extremely high transgene expression of vaccine-related antigens and therapeutic anti-tumor and immunostimulatory genes, which has also permitted significantly reduced doses for prophylactic and therapeutic applications, potentially reducing adverse events. Furthermore, alphaviruses have shown favorable flexibility as they can be delivered as recombinant viral particles, RNA replicons, or DNA-replicon-based plasmids. In the context of infectious diseases, robust immune responses against the surface proteins of target agents have been observed along with protection against challenges with lethal doses of infectious agents in rodents and primates. Similarly, the expression of anti-tumor genes and immunostimulatory genes from alphavirus vectors has provided tumor growth inhibition, tumor regression, and cures in animal cancer models. Moreover, protection against tumor challenges has been observed. In clinical settings, patient benefits have been reported. Alphaviruses have also been considered for the treatment of neurological disorders due to their neurotrophic preference. Full article

Viruses often pose a significant threat to the host through the exploitation of cellular machineries for their own benefit. In the context of immune responses, myriad host factors are deployed to target viral RNAs and inhibit viral protein translation, ultimately hampering viral replication. Understanding how “non-self” RNAs interact with the host translation machinery and trigger immune responses would help in the development of treatment strategies for viral infections. In this review, we explore how interferon-stimulated gene products interact with viral RNA and the translation machinery in order to induce either global or targeted translation inhibition. Full article

The epitranscriptomic modification m⁶A is a prevalent RNA modification that plays a crucial role in the regulation of various aspects of RNA metabolism. It has been found to be involved in a wide range of physiological processes and disease states. Of particular interest is the role of m⁶A machinery and modifications in viral infections, serving as an evolutionary marker for distinguishing between self and non-self entities. In this review article, we present a comprehensive overview of the epitranscriptomic modification m⁶A and its implications for the interplay between viruses and their host, focusing on immune responses and viral replication. We outline future research directions that highlight the role of m⁶A in viral nucleic acid recognition, initiation of antiviral immune responses, and modulation of antiviral signaling pathways. Additionally, we discuss the potential of m⁶A as a prognostic biomarker and a target for therapeutic interventions in viral infections. Full article

Viruses provide vital insights into gene expression control. Viral transactivators, with other viral and cellular proteins, regulate expression of self, other viruses, and host genes with profound effects on infected cells, underlying inflammation, control of immune responses, and pathogenesis. The multifunctional Tat proteins of lentiviruses (HIV-1, HIV-2, and SIV) transactivate gene expression by recruiting host proteins and binding to transacting responsive regions (TARs) in viral and host RNAs. SARS-CoV-2 nucleocapsid participates in early viral transcription, recruits similar cellular proteins, and shares intracellular, surface, and extracellular distribution with Tat. SARS-CoV-2 nucleocapsid interacting with the replication–transcription complex might, therefore, transactivate viral and cellular RNAs in the transcription and reactivation of self and other viruses, acute and chronic pathogenesis, immune evasion, and viral evolution. Here, we show, by using primary and secondary structural comparisons, that the leaders of SARS-CoV-2 and other coronaviruses contain TAR-like sequences in stem-loops 2 and 3. The coronaviral nucleocapsid C-terminal domains harbor a region of similarity to TAR-binding regions of lentiviral Tat proteins, and coronaviral nonstructural protein 12 has a cysteine-rich metal binding, dimerization domain, as do lentiviral Tat proteins. Although SARS-CoV-1 nucleocapsid transactivated gene expression in a replicon-based study, further experimental evidence for coronaviral transactivation and its possible implications is warranted. Full article

SERINC5 is a restriction factor that becomes incorporated into nascent retroviral particles, impairing their ability to infect target cells. In turn, retroviruses have evolved countermeasures against SERINC5. For instance, the primate lentiviruses (HIV and SIV) use Nef, Moloney Murine Leukemia Virus (MLV) uses GlycoGag, and Equine Infectious Anemia Virus (EIAV) uses S2 to remove SERINC5 from the plasma membrane, preventing its incorporation into progeny virions. Recent studies have shown that SERINC5 also restricts other viruses, such as Hepatitis B Virus (HBV) and Classical Swine Fever Virus (CSFV), although through a different mechanism, suggesting that SERINC5 can interfere with multiple stages of the virus life cycle. To investigate whether SERINC5 can also impact other steps of the replication cycle of HIV, the effects of SERINC5 on viral transcripts, proteins, and virus progeny size were studied. Here, we report that SERINC5 causes significant defects in HIV gene expression, which impacts virion production. While the underlying mechanism is still unknown, we found that the restriction occurs at the transcriptional level and similarly affects plasmid and non-integrated proviral DNA (ectopic or non-self-DNA). However, SERINC5 causes no defects in the expression of viral RNA, host genes, or proviral DNA that is integrated in the cellular genome. Hence, our findings reveal that SERINC5’s actions in host defense extend beyond blocking virus entry. Full article

Although conventional vaccine approaches have proven to be successful in preventing infectious diseases in past decades, for vaccine development against emerging/re-emerging viruses, one of the main challenges is rapid response in terms of design and manufacture. mRNA vaccines can be designed and produced within days, representing a powerful approach for developing vaccines. Furthermore, mRNA vaccines can be scaled up and may not have the risk of integration. mRNA vaccines are roughly divided into non-replicating mRNA vaccines and self-amplifying RNA (saRNA) vaccines. In this review, we provide an overview of saRNA vaccines, and discuss future directions and challenges in advancing this promising vaccine platform to combat emerging/re-emerging viruses. Full article

Cancer therapy has experienced a breakthrough with the use of immune checkpoint inhibitors (ICIs) based on monoclonal antibodies (mAbs), which are able to unleash immune responses against tumors refractory to other therapies. Despite the great advancement that ICIs represent, most patients with gastrointestinal tumors have not benefited from this therapy. In addition, ICIs often induce adverse effects that are related to their systemic use. Local administration of ICIs in tumors could concentrate their effect in the malignant tissue and provide a higher safety profile. A new and attractive approach for local delivery of ICIs is the use of gene therapy vectors to express these blocking antibodies in tumor cells. Several vectors have been evaluated in preclinical models of gastrointestinal tumors to express ICIs against PD-1, PD-L1, and CTLA-4, among other immune checkpoints, with promising results. Vectors used in these settings include oncolytic viruses, self-replicating RNA vectors, and non-replicative viral and non-viral vectors. The use of viral vectors, especially when they have replication capacity, provides an additional adjuvant effect that has been shown to enhance antitumor responses. This review covers the most recent studies involving the use of gene therapy vectors to deliver ICIs to gastrointestinal tumors. Full article

DNA-based gene therapy and vaccine development has received plenty of attention lately. DNA replicons based on self-replicating RNA viruses such as alphaviruses and flaviviruses have been of particular interest due to the amplification of RNA transcripts leading to enhanced transgene expression in transfected host cells. Moreover, significantly reduced doses of DNA replicons compared to conventional DNA plasmids can elicit equivalent immune responses. DNA replicons have been evaluated in preclinical animal models for cancer immunotherapy and for vaccines against infectious diseases and various cancers. Strong immune responses and tumor regression have been obtained in rodent tumor models. Immunization with DNA replicons has provided robust immune responses and protection against challenges with pathogens and tumor cells. DNA replicon-based COVID-19 vaccines have shown positive results in preclinical animal models. Full article

Self-replicating RNA viruses have become attractive delivery vehicles for therapeutic applications. They are easy to handle, can be rapidly produced in large quantities, and can be delivered as recombinant viral particles, naked or nanoparticle-encapsulated RNA, or plasmid DNA-based vectors. The self-replication of RNA in infected host cells provides the means for generating much higher transgene expression levels and the possibility to apply substantially reduced amounts of RNA to achieve similar expression levels or immune responses compared to conventional synthetic mRNA. Alphaviruses and flaviviruses, possessing a single-stranded RNA genome of positive polarity, as well as measles viruses and rhabdoviruses with a negative-stranded RNA genome, have frequently been utilized for therapeutic applications. Both naturally and engineered oncolytic self-replicating RNA viruses providing specific replication in tumor cells have been evaluated for cancer therapy. Therapeutic efficacy has been demonstrated in animal models. Furthermore, the safe application of oncolytic viruses has been confirmed in clinical trials. Multiple myeloma patients treated with an oncolytic measles virus (MV-NIS) resulted in increased T-cell responses against the measles virus and several tumor-associated antigen responses and complete remission in one patient. Furthermore, MV-CEA administration to patients with ovarian cancer resulted in a stable disease and more than doubled the median overall survival. Full article

The rolling circle mechanism found in viroids and some RNA viruses is a likely way that replication could have begun in the RNA World. Here, we consider simulations of populations of protocells, each containing multiple copies of rolling circle RNAs that can replicate non-enzymatically. The mechanism requires the presence of short self-cleaving ribozymes such as hammerheads, which can cleave and re-circularize RNA strands. A rolling circle must encode a hammerhead and the complement of a hammerhead, so that both plus and minus strands can cleave. Thus, the minimal functional length is twice the length of the hammerhead sequence. Selection for speed of replication will tend to reduce circles to this minimum length. However, if sequence errors occur when copying the hammerhead sequence, this prevents cleavage at one point, but still allows cleavage on the next passage around the rolling circle. Thus, there is a natural doubling mechanism that creates strands that are multiple times the length of the minimal sequence. This can provide space for the origin of new genes with beneficial functions. We show that if a beneficial gene appears in this new space, the longer sequence with the beneficial function can be selected, even though it replicates more slowly. This provides a route for the evolution of longer circles encoding multiple genes. Full article

Diseases caused by flaviviruses such as dengue virus (DENV) and West Nile Virus (WNV), are a serious threat to public health. The flavivirus single-stranded RNA genome is translated into a polyprotein which is cleaved into three structural proteins and seven non-structural proteins by the viral and cellular proteases. Non-structural (NS) protein 3 is a multifunctional protein that has N-terminal protease and C-terminal helicase domains. The NS3 protease requires co-factor NS2B for enzymatic activity and folding. Due to its essential role in viral replication, NS2B-NS3 protease is an attractive target for antiviral drugs. Despite the availability of crystal structures, dynamic interactions of the N- and C-termini of NS2B co-factor have been elusive due to their flexible fold. In this study, we employ integrative structural approaches combined with biochemical assays to elucidate the dynamic interactions of the flexible DENV4 NS2B and NS3 N- and C-termini. We captured the crystal structure of self-cleaved DENV4 NS2B₄₇NS3 protease in post cleavage state. The intermediate conformation adopted in the reported structure can be targeted by allosteric inhibitors. Comparison of our new findings from DENV4 against previously studied ZIKV NS2B-NS3 proteins reveals differences in NS2B-NS3 function between the two viruses. No inhibition of protease activity was observed for unlinked DENV NS2B-NS3 in presence of the cleavage site while ZIKV NS2B-NS3 cleavage inhibits protease activity. Another difference is that binding of the NS2B C-terminus to DENV4 eNS2B₄₇NS3Pro active site is mediated via interactions with P4-P6 residues while for ZIKV, the binding of NS2B C-terminus to active site is mediated by P1-P3 residues. The mapping of NS2B N- and C-termini with NS3 indicates that these intermolecular interactions occur mainly on the beta-barrel 2 of the NS3 protease domain. Our integrative approach enables a comprehensive understanding of the folding and dynamic interactions of DENV NS3 protease and its cofactor NS2B. Full article

Nodaviruses are small bipartite RNA viruses and are considered animal viruses. Here, we identified two novel noda-like viruses (referred to as rice-associated noda-like virus 1 (RNLV1) and rice-associated noda-like virus 2 (RNLV2)) in field-collected rice plants showing a dwarfing phenotype through RNA-seq. RNLV1 genome consists of 3335 nt RNA1 and 1769 nt RNA2, and RNLV2 genome consists of 3279 nt RNA1 and 1525 nt RNA2. Three conserved ORFs were identified in each genome of the two novel viruses, encoding an RNA-dependent RNA polymerase, an RNA silencing suppressor, and a capsid protein, respectively. The results of sequence alignment, protein domain prediction, and evolutionary analysis indicate that these two novel viruses are clearly different from the known nodaviruses, especially the CPs. We have also determined that the B2 protein encoded by the two new noda-like viruses can suppress RNA silencing in plants. Two reverse genetic systems were constructed and used to show that RNLV1 RNA1 can replicate in plant cells and RNLV1 can replicate in insect Sf9 cells. We have also found two unusual peptidase family A21 domains in the RNLV1 CP, and RNLV1 CP can self-cleave in acidic environments. These findings provide new knowledge of novel nodaviruses. Full article