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Viruses Jun 2024Human cytomegalovirus (CMV) infection is the leading non-genetic cause of congenital malformation in developed countries, causing significant fetal injury, and in some...
Human Cytomegalovirus Dysregulates Cellular Dual-Specificity Tyrosine Phosphorylation-Regulated Kinases and Sonic Hedgehog Pathway Proteins in Neural Astrocyte and Placental Models.
Human cytomegalovirus (CMV) infection is the leading non-genetic cause of congenital malformation in developed countries, causing significant fetal injury, and in some cases fetal death. The pathogenetic mechanisms through which this host-specific virus infects then damages both the placenta and the fetal brain are currently ill-defined. We investigated the CMV modulation of key signaling pathway proteins for these organs including dual-specificity tyrosine phosphorylation-regulated kinases (DYRK) and Sonic Hedgehog (SHH) pathway proteins using human first trimester placental trophoblast (TEV-1) cells, primary human astrocyte (NHA) brain cells, and CMV-infected human placental tissue. Immunofluorescence demonstrated the accumulation and re-localization of SHH proteins in CMV-infected TEV-1 cells with Gli2, Ulk3, and Shh re-localizing to the CMV cytoplasmic virion assembly complex (VAC). In CMV-infected NHA cells, DYRK1A re-localized to the VAC and DYRK1B re-localized to the CMV nuclear replication compartments, and the SHH proteins re-localized with a similar pattern as was observed in TEV-1 cells. Western blot analysis in CMV-infected TEV-1 cells showed the upregulated expression of Rb, Ulk3, and Shh, but not Gli2. In CMV-infected NHA cells, there was an upregulation of DYRK1A, DYRK1B, Gli2, Rb, Ulk3, and Shh. These in vitro monoculture findings are consistent with patterns of protein upregulation and re-localization observed in naturally infected placental tissue and CMV-infected ex vivo placental explant histocultures. This study reveals CMV-induced changes in proteins critical for fetal development, and identifies new potential targets for CMV therapeutic development.
Topics: Humans; Hedgehog Proteins; Cytomegalovirus; Pregnancy; Placenta; Astrocytes; Female; Protein-Tyrosine Kinases; Cytomegalovirus Infections; Signal Transduction; Protein Serine-Threonine Kinases; Phosphorylation; Trophoblasts; Dyrk Kinases; Cell Line; Cells, Cultured
PubMed: 38932210
DOI: 10.3390/v16060918 -
Viruses May 2024DNA viruses that produce persistent infections have been proposed as potential causes for the extinction of Neanderthals, and, therefore, the identification of viral...
DNA viruses that produce persistent infections have been proposed as potential causes for the extinction of Neanderthals, and, therefore, the identification of viral genome remnants in Neanderthal sequence reads is an initial step to address this hypothesis. Here, as proof of concept, we searched for viral remnants in sequence reads of Neanderthal genome data by mapping to adenovirus, herpesvirus and papillomavirus, which are double-stranded DNA viruses that may establish lifelong latency and can produce persistent infections. The reconstructed ancient viral genomes of adenovirus, herpesvirus and papillomavirus revealed conserved segments, with nucleotide identity to extant viral genomes and variable regions in coding regions with substantial divergence to extant close relatives. Sequence reads mapped to extant viral genomes showed deamination patterns of ancient DNA, and these ancient viral genomes showed divergence consistent with the age of these samples (≈50,000 years) and viral evolutionary rates (10 to 10 substitutions/site/year). Analysis of random effects showed that the Neanderthal mapping to genomes of extant persistent viruses is above what is expected by random similarities of short reads. Also, negative control with a nonpersistent DNA virus does not yield statistically significant assemblies. This work demonstrates the feasibility of identifying viral genome remnants in archaeological samples with signal-to-noise assessment.
Topics: Animals; Neanderthals; Genome, Viral; DNA, Ancient; Evolution, Molecular; DNA, Viral; Sequence Analysis, DNA; Humans; Phylogeny; DNA Viruses; Fossils
PubMed: 38932149
DOI: 10.3390/v16060856 -
Viruses May 2024Rotaviruses (RVs) are 11-segmented, double-stranded (ds) RNA viruses and important causes of acute gastroenteritis in humans and other animal species. Early RV particle... (Review)
Review
Rotaviruses (RVs) are 11-segmented, double-stranded (ds) RNA viruses and important causes of acute gastroenteritis in humans and other animal species. Early RV particle assembly is a multi-step process that includes the assortment, packaging and replication of the 11 genome segments in close connection with capsid morphogenesis. This process occurs inside virally induced, cytosolic, membrane-less organelles called viroplasms. While many viral and cellular proteins play roles during early RV assembly, the octameric nonstructural protein 2 (NSP2) has emerged as a master orchestrator of this key stage of the viral replication cycle. NSP2 is critical for viroplasm biogenesis as well as for the selective RNA-RNA interactions that underpin the assortment of 11 viral genome segments. Moreover, NSP2's associated enzymatic activities might serve to maintain nucleotide pools for use during viral genome replication, a process that is concurrent with early particle assembly. The goal of this review article is to summarize the available data about the structures, functions and interactions of RV NSP2 while also drawing attention to important unanswered questions in the field.
Topics: Rotavirus; Virus Assembly; Viral Nonstructural Proteins; Humans; Animals; Virus Replication; Genome, Viral; RNA, Viral; Capsid; RNA-Binding Proteins
PubMed: 38932107
DOI: 10.3390/v16060814 -
Microorganisms May 2024Brucellosis is an important bacterial disease of livestock and the most common zoonotic disease. The current vaccines are effective but unsafe, as they result in animal...
Brucellosis is an important bacterial disease of livestock and the most common zoonotic disease. The current vaccines are effective but unsafe, as they result in animal abortions and are pathogenic to humans. Virus-like particles are being investigated as molecular scaffolds for foreign antigen presentation to the immune system. Here, we sought to develop a new-generation vaccine by presenting selected T cell epitopes on the surface of Orbivirus core-like particles (CLPs) and transiently expressing these chimeric particles in plants. We successfully demonstrated the assembly of five chimeric CLPs in plants, with each CLP presenting a different T cell epitope. The safety and protective efficacy of three of the highest-yielding CLPs was investigated in a mouse model of brucellosis. All three plant-expressed chimeric CLPs were safe when inoculated into BALB/c mice at specific antigen doses. However, only one chimeric CLP induced protection against the virulent strain challenge equivalent to the protection induced by the commercial Rev1 vaccine. Here, we have successfully shown the assembly, safety and protective efficacy of plant-expressed chimeric CLPs presenting T cell epitopes. This is the first step in the development of a safe and efficacious subunit vaccine against brucellosis.
PubMed: 38930429
DOI: 10.3390/microorganisms12061047 -
International Journal of Molecular... Jun 2024The maturation of HIV-1 virions is a crucial process in viral replication. Although T-cells are a primary source of virus production, much of our understanding of virion...
The maturation of HIV-1 virions is a crucial process in viral replication. Although T-cells are a primary source of virus production, much of our understanding of virion maturation comes from studies using the HEK293T human embryonic kidney cell line. Notably, there is a lack of comparative analyses between T-cells and HEK293T cells in terms of virion maturation efficiency in existing literature. We previously developed an advanced virion visualization system based on the FRET principle, enabling the effective distinction between immature and mature virions via fluorescence microscopy. In this study, we utilized pseudotyped, single-round infectious viruses tagged with FRET labels (HIV-1 Gag-iFRET∆Env) derived from Jurkat (a human T-lymphocyte cell line) and HEK293T cells to evaluate their virion maturation rates. HEK293T-derived virions demonstrated a maturity rate of 81.79%, consistent with other studies and our previous findings. However, virions originating from Jurkat cells demonstrated a significantly reduced maturation rate of 68.67% ( < 0.0001). Correspondingly, viruses produced from Jurkat cells exhibited significantly reduced infectivity compared to those derived from HEK293T cells, with the relative infectivity measured at 65.3%. This finding is consistent with the observed relative maturation rate of viruses produced by Jurkat cells. These findings suggest that initiation of virion maturation directly correlates with viral infectivity. Our observation highlights the dynamic nature of virus-host interactions and their implications for virion production and infectivity.
Topics: Humans; HIV-1; HEK293 Cells; Virion; Jurkat Cells; Fluorescence Resonance Energy Transfer; Virus Replication; Virus Assembly; HIV Infections
PubMed: 38928103
DOI: 10.3390/ijms25126396 -
Biomolecules Jun 2024The Ebola virus (EBOV) is a lethal pathogen causing hemorrhagic fever syndrome which remains a global health challenge. In the EBOV, two multifunctional proteins, VP35...
The Ebola virus (EBOV) is a lethal pathogen causing hemorrhagic fever syndrome which remains a global health challenge. In the EBOV, two multifunctional proteins, VP35 and VP40, have significant roles in replication, virion assembly, and budding from the cell and have been identified as druggable targets. In this study, we employed in silico methods comprising molecular docking, molecular dynamic simulations, and pharmacological properties to identify prospective drugs for inhibiting VP35 and VP40 proteins from the myxobacterial bioactive natural product repertoire. Cystobactamid 934-2, Cystobactamid 919-1, and Cittilin A bound firmly to VP35. Meanwhile, 2-Hydroxysorangiadenosine, Enhypyrazinone B, and Sorangiadenosine showed strong binding to the matrix protein VP40. Molecular dynamic simulations revealed that, among these compounds, Cystobactamid 919-1 and 2-Hydroxysorangiadenosine had stable interactions with their respective targets. Similarly, molecular mechanics Poisson-Boltzmann surface area (MMPBSA) calculations indicated close-fitting receptor binding with VP35 or VP40. These two compounds also exhibited good pharmacological properties. In conclusion, we identified Cystobactamid 919-1 and 2-Hydroxysorangiadenosine as potential ligands for EBOV that target VP35 and VP40 proteins. These findings signify an essential step in vitro and in vivo to validate their potential for EBOV inhibition.
Topics: Ebolavirus; Molecular Docking Simulation; Biological Products; Molecular Dynamics Simulation; Antiviral Agents; Myxococcales; Humans; Viral Regulatory and Accessory Proteins; Viral Matrix Proteins; Nucleocapsid Proteins
PubMed: 38927063
DOI: 10.3390/biom14060660 -
Veterinary Sciences Jun 2024Porcine circovirus type 3 (PCV3) infection can cause symptoms similar to those of porcine circovirus type 2 (PCV2) infection, and coinfections with both PCV2 and PCV3...
Porcine circovirus type 3 (PCV3) infection can cause symptoms similar to those of porcine circovirus type 2 (PCV2) infection, and coinfections with both PCV2 and PCV3 are observed in the swine industry. Consequently, developing chimeric vaccines is essential to prevent and control porcine circovirus infections. In this study, we used both and mammalian expression systems to express PCV3 Cap (Cap3) and a chimeric gene containing the PCV2-neutralizing epitope within the PCV3 Cap (Cap3-Cap2E), which were assembled into virus-like particle (VLP) vaccines. We found that Cap3 lacking nuclear localization signal (NLS) could not form VLPs, while Cap3 with a His-tag successfully assembled into VLPs. Additionally, the chimeric of PCV2-neutralizing epitopes did not interfere with the assembly process of VLPs. Various immunization approaches revealed that pCap3-Cap2E VLP vaccines were capable of activating high PCV3 Cap-specific antibody levels and effectively neutralizing both PCV3 and PCV2. Furthermore, pCap3-Cap2E VLPs demonstrated a potent ability to activate cellular immunity, protecting against PCV3 infection and preventing lung damage in mice. In conclusion, this study successfully developed a PCV3 Cap VLP vaccine incorporating chimeric PCV2-neutralizing epitope genes, providing new perspectives for PCV3 vaccine development.
PubMed: 38922011
DOI: 10.3390/vetsci11060264 -
ACS Infectious Diseases Jun 2024Human immunodeficiency virus (HIV) assembly at an infected cell's plasma membrane requires membrane deformation to organize the near-spherical shape of an immature...
Human immunodeficiency virus (HIV) assembly at an infected cell's plasma membrane requires membrane deformation to organize the near-spherical shape of an immature virus. While the cellular expression of HIV Gag is sufficient to initiate budding of virus-like particles, how Gag generates membrane curvature is not fully understood. Using highly curved lipid nanotubes, we have investigated the physicochemical basis of the membrane activity of recombinant nonmyristoylated Gag-Δp6. Gag protein, upon adsorption onto the membrane, resulted in the shape changes of both charged and uncharged nanotubes. This shape change was more pronounced in the presence of charged lipids, especially phosphatidylinositol bisphosphate (PI(4,5)P). We found that Gag modified the interfacial tension of phospholipid bilayer membranes, as judged by comparison with the effects of amphipathic peptides and nonionic detergent. Bioinformatic analysis demonstrated that a region of the capsid and SP1 domains junction of Gag is structurally similar to the amphipathic peptide magainin-1. This region accounts for integral changes in the physical properties of the membrane upon Gag adsorption, as we showed with the synthetic CA-SP1 junction peptide. Phenomenologically, membrane-adsorbed Gag could diminish the energetic cost of increasing the membrane area in a way similar to foam formation. We propose that Gag acts as a surface-active substance at the HIV budding site that softens the membrane at the place of Gag adsorption, lowering the energy for membrane bending. Finally, our experimental data and theoretical considerations give a lipid-centric view and common mechanism by which proteins could bend membranes, despite not having intrinsic curvature in their molecular surfaces or assemblies.
PubMed: 38917054
DOI: 10.1021/acsinfecdis.4c00251 -
BMC Bioinformatics Jun 2024Pan-virus detection, and virome investigation in general, can be challenging, mainly due to the lack of universally conserved genetic elements in viruses. Metagenomic...
BACKGROUND
Pan-virus detection, and virome investigation in general, can be challenging, mainly due to the lack of universally conserved genetic elements in viruses. Metagenomic next-generation sequencing can offer a promising solution to this problem by providing an unbiased overview of the microbial community, enabling detection of any viruses without prior target selection. However, a major challenge in utilising metagenomic next-generation sequencing for virome investigation is that data analysis can be highly complex, involving numerous data processing steps.
RESULTS
Here, we present Entourage to address this challenge. Entourage enables short-read sequence assembly, viral sequence search with or without reference virus targets using contig-based approaches, and intrasample sequence variation quantification. Several workflows are implemented in Entourage to facilitate end-to-end virus sequence detection analysis through a single command line, from read cleaning, sequence assembly, to virus sequence searching. The results generated are comprehensive, allowing for thorough quality control, reliability assessment, and interpretation. We illustrate Entourage's utility as a streamlined workflow for virus detection by employing it to comprehensively search for target virus sequences and beyond in raw sequence read data generated from HeLa cell culture samples spiked with viruses. Furthermore, we showcase its flexibility and performance on a real-world dataset by analysing a preassembled Tara Oceans dataset. Overall, our results show that Entourage performs well even with low virus sequencing depth in single digits, and it can be used to discover novel viruses effectively. Additionally, by using sequence data generated from a patient with chronic SARS-CoV-2 infection, we demonstrate Entourage's capability to quantify virus intrasample genetic variations, and generate publication-quality figures illustrating the results.
CONCLUSIONS
Entourage is an all-in-one, versatile, and streamlined bioinformatics software for virome investigation, developed with a focus on ease of use. Entourage is available at https://codeberg.org/CENMIG/Entourage under the MIT license.
Topics: Software; Genome, Viral; Humans; High-Throughput Nucleotide Sequencing; SARS-CoV-2; Metagenomics; Viruses; COVID-19; Virome; HeLa Cells
PubMed: 38914932
DOI: 10.1186/s12859-024-05846-y -
MBio Jun 2024We have investigated the function of inositol hexakisphosphate (IP6) and inositol pentakisphosphate (IP5) in the replication of murine leukemia virus (MLV). While IP6 is...
We have investigated the function of inositol hexakisphosphate (IP6) and inositol pentakisphosphate (IP5) in the replication of murine leukemia virus (MLV). While IP6 is known to be critical for the life cycle of HIV-1, its significance in MLV remains unexplored. We find that IP6 is indeed important for MLV replication. It significantly enhances endogenous reverse transcription (ERT) in MLV. Additionally, a pelleting-based assay reveals that IP6 can stabilize MLV cores, thereby facilitating ERT. We find that IP5 and IP6 are packaged in MLV particles. However, unlike HIV-1, MLV depends upon the presence of IP6 and IP5 in target cells for successful infection. This IP6/5 requirement for infection is reflected in impaired reverse transcription observed in IP6/5-deficient cell lines. In summary, our findings demonstrate the importance of capsid stabilization by IP6/5 in the replication of diverse retroviruses; we suggest possible reasons for the differences from HIV-1 that we observed in MLV.IMPORTANCEInositol hexakisphosphate (IP6) is crucial for the assembly and replication of HIV-1. IP6 is packaged in HIV-1 particles and stabilizes the viral core enabling it to synthesize viral DNA early in viral infection. While its importance for HIV-1 is well established, its significance for other retroviruses is unknown. Here we report the role of IP6 in the gammaretrovirus, murine leukemia virus (MLV). We found that like HIV-1, MLV packages IP6, and as in HIV-1, IP6 stabilizes the MLV core thus promoting reverse transcription. Interestingly, we discovered a key difference in the role of IP6 in MLV versus HIV-1: while HIV-1 is not dependent upon IP6 levels in target cells, MLV replication is significantly reduced in IP6-deficient cell lines. We suggest that this difference in IP6 requirements reflects key differences between HIV-1 and MLV replication.
PubMed: 38912776
DOI: 10.1128/mbio.01158-24