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Current Publisher: MDPI (10.3390)
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Proceedings, Volume 50; doi:10.3390/proceedings2020050106
Plotka, M.; Kapusta, M.; Dorawa, S.; Kaczorowska, A.-K.; Kaczorowski, T. Ts2631 Endolysin from the Extremophilic Thermus scotoductus Bacteriophage vB_Tsc2631 as an Antimicrobial Agent against Gram-Negative Multidrug-Resistant Bacteria. Viruses 2019, 11, 657. doi: 10.3390/v11070657
Proceedings, Volume 50; doi:10.3390/proceedings2020050097
Background: A Src kinase-activating phosphatase (PTPRE) is targeted by a genome-derived yellow fever virus (YFV) short noncoding RNA (vsRNA) in vitro. The vsRNA reduces PTPRE translation, which leads to reduced TCR signaling. vsRNA point mutations restore PTPRE expression and T cell function. We examined TCR signaling and PTPRE levels in individuals before and after YFV vaccination (YFVax). Methods: Fourteen individuals receiving YFVax (104.7–5.6) IM for travel prophylaxis provided written informed consent for these studies. Blood was obtained once before vaccination and four times after vaccination (days 3 to 28). Serum and PBMCs were purified and YFV was quantified by RNA and infectivity. PBMCs were assessed for activation following anti-CD3 stimulation by measuring phospho-tyrosine-394-Lck and IL-2 release. PBMC PTPRE levels were determined by immunoblot analyses (normalized to actin). A YFV-neutralizing antibody was determined by PRNT. Results: YFVax was administered alone (six out of 14 subjects) or in combination with other vaccines (eight out of 14). All subjects demonstrated reduced resting PBMC PTPRE levels and post-TCR stimulation had reduced IL-2 release between days 4 and 21 compared to pre- and day 28 samples. Phospho-Lck was reduced in all but two subjects on the same days, and both of these subjects also received an influenza vaccine. Low-level viremia was detected in 10/14 subjects, with infectious titers of 100/mL. Viremia was not detected in four out of 14 subjects. All recipients developed neutralizing antibodies by day 21. Conclusion: YFV vaccination regulates PBMC PTPRE levels 4–21 days after infection, despite the low to absent infectious YFV detected in serum, suggesting that enough YFV vsRNA is produced and released from cells to have a functional (and measurable) effect on T cell function. Studies are underway to determine if this is mediated by exosomes or defective particles containing the vsRNA that targets PTPRE. Furthermore, the association between PTPRE and TCR signaling confirms a role for PTPRE in TCR function.
Proceedings, Volume 50; doi:10.3390/proceedings2020050102
The retroviral Gag protein targets the plasma membrane of infected cells for viral particle formation and release. The matrix domain (MA) of Gag is myristoylated for membrane anchoring but also contains a highly basic region that recognizes acidic phospholipids. Gag targets lipid molecules at the inner leaflet of the plasma membrane including phosphatidylinositol (4,5) bisphosphate (PI(4,5)P2) and cholesterol. Here, we addressed the question whether HIV-1 Gag was able to trap PI(4,5)P2 and/or other lipids during HIV-1 assembly in silico, in vitro on reconstituted membranes and in cellulo at the plasma membrane of the host CD4+ T cells. In silico, we could observe the first PI(4,5)P2 preferential recruitment by HIV-1 MA or Gag while protein docked on artificial membranes. In vitro, using biophysical techniques, we observed the specific trapping of PI(4,5)P2, and, to a lesser extent, cholesterol and the exclusion of sphingomyelin, during HIV-1 myr(-)Gag self-assembly on LUVs and SLBs. Finally, in infected living CD4+ T cells, we measured lipid dynamics within and away from HIV-1 assembly sites using super-resolution stimulated emission depletion (STED) microscopy coupled with scanning Fluorescence Correlation Spectroscopy (sSTED-FCS). The analysis of HIV-1 infected CD4+ T lymphocytes revealed that, upon virus assembly, HIV-1 is able to specifically trap PI(4,5)P2, and cholesterol but not phosphatidylethanolamine (PE) or sphingomyelin (SM) at the cellular membrane. Furthermore, analyzing CD4+ T cells expressing only HIV-1 Gag protein showed that Gag is the main driving force restricting the mobility of PI(4,5)P2 and cholesterol at the cell plasma membrane. Our data provide the first direct evidence showing that HIV-1 Gag creates its own specific lipid environment for virus assembly by selectively recruiting lipids to generate PI(4,5)P2/cholesterol-enriched nanodomains favoring virus assembly, and that HIV-1 does not assemble on pre-existing lipid domains.
Proceedings, Volume 50; doi:10.3390/proceedings2020050098
Usutu virus (USUV) is a mosquito-borne arbovirus that has rapidly propagated in birds across several European countries over the last two decades, leading to substantial avian mortalities. USUV infection in humans has been associated with a growing number of cases of neurological disease in the last years, underlining the need for increased awareness and suitable treatments. Our group is working on the characterization of the NS5 protein of USUV. This protein is responsible for the replication activity of the viral genome and can be a suitable viral target to treat the infection. NS5 contains a RNA-dependent RNA polymerase (RdRpD) and a methyltransferase domains. Recombinant NS5 and RdRpD proteins expressed in bacteria were purified and biochemically characterized to determine the best conditions for their polymerase activities. Both proteins showed de novo and primer extension activities. Optimal RNA–polymerase reaction conditions included low NaCl (less than 20 mM), 2.5 mM MgCl2 and 5 mM MnCl2, 30 °C, and pH 7.25. Polymerase activity was cooperative for the polymerase domain (Hill coefficient = 5.8) but not for the complete NS5 (Hill coefficient = 1.2). To study their subcellular location, suitable sequences were cloned into a pcDNA3 vector and expressed in Huh7.5 and HEK293T cells. Both proteins were preferentially located in the cytoplasmic region, although a significant amount was found in the nucleus. Preliminary results showed that the concentration of sofosbuvir (SOFTP) necessary to achieve its incorporation by NS5 in 50% of the nascent RNA is higher than 100 µM, as already observed for dengue virus DENV. In this work, we describe the main features of the full-length USUV NS5, including the polymerase activity as well as the effect of protein–protein interactions and subcellular localization. Our results will be very useful for the study of this viral enzyme as a suitable target against the infection and the effect of antiviral drugs.
Proceedings, Volume 50; doi:10.3390/proceedings2020050099
Bacterial viruses (phages) are amongst the smallest, most powerful biological entities on Earth. Through infection, phages impact host metabolism, bacterial mortality, and evolution. In the oceans, 20–40% of surface microbes are infected, with 1023 new infections each second. Yet, infections remain virtually uncharacterized, as the available phage isolates underrepresent the diversity of marine phage–host interactions. Additionally, while sequencing efforts reveal “who is there?”, a gap between sequence and function prevents answering “what are they doing?” and “how?”. We have developed new Bacteroidetes and Proteobacteria marine phage–host model systems with which to connect genomes, infection strategies, and functions using both traditional and genome-wide “-omics” experiments. We ask: How do infections by genomically divergent phages compare? Are there links between phage–host genomes and infection strategies? Our findings are as follows. In Bacteroidetes, a phage infecting two nearly identical strains (host38 and host18) under identical conditions is more fit and efficient on host38. By contrast, on host18, it is less fit and, except for phage transcription, it fails at efficiently mastering all stages of the infection: from adsorption through to cell lysis. In Proteobacteria, genomically unrelated podovirus and siphovirus phages infecting the same strain reprogram host metabolisms very differently. Namely, siphovirus-infected cells hardly differ from uninfected and mainly repress energy-consuming processes such as motility and translation. By contrast, podovirus-infected cells greatly differ from uninfected cells in transcription and in uniquely shifting central carbon and energy metabolism. Additionally, the siphovirus is more complementary to the host than the podovirus in %GC, amino acids, and codon usage. We found that phage–host genome complementarity may drive the resource demand and fitness of a phage: the phage most complementary to its host easily accesses intracellular resources, infects with little reprogramming, and accomplishes the largest fitness, which has not previously been shown. Together, this work helps to uncover infection efficiency strategies, and connect genomes with metabolisms in marine phage–host systems.
Proceedings, Volume 50; doi:10.3390/proceedings2020050096
TRIM5α is a cytoplasmic antiviral effector induced by type I interferons (IFN-I) that has the potential to intercept incoming retroviruses by interacting with their capsid core, leading to uncoating induction and the partial degradation of core components. Most HIV-1 strains escape restriction by human TRIM5α due to a lack of interaction between TRIM5α and its viral molecular target. We previously showed, however, that two point mutations, R332G/R335G, in the capsid-binding region confer human TRIM5α with the capacity to target and strongly restrict HIV-1 upon the overexpression of the mutated protein. Here, we explored the possibility to introduce these two mutations in the endogenous human TRIM5 gene by CRISPR-Cas9-mediated gene editing. For this, we electroporated CRISPR ribonucleoproteins (RNPs) and the donor DNA into Jurkat T lymphocytic cells and isolated clones by limiting dilution. We analyzed 47 clones using specific PCR assays, and found that six clones (13%) contained at least one gene-edited allele. One clone (clone 6) had both alleles edited for R332G, but only one of the two alleles was edited for R335G. Upon challenge with an HIV-1 vector, clone 6 was significantly less permissive compared to unmodified cells, whereas the cell clones with monoallelic modifications were only slightly less permissive. Following IFN-β treatment, the inhibition of HIV-1 infection in clone 6 was significantly enhanced (~50-fold inhibition), whereas IFN-β treatment had no effect on TRIM5α overexpressed by retroviral transduction. Knockdown experiments confirmed that HIV-1 was inhibited by the edited TRIM5 gene products, whereas quantification of HIV-1 reverse transcription products confirmed that inhibition occurred through the expected mechanism. In conclusion, we demonstrate the feasibility of potently inhibiting a viral infection through the editing of innate effector genes, but our results also emphasize the importance of biallelic modification in order to reach significant levels of inhibition by TRIM5α.
Proceedings, Volume 50; doi:10.3390/proceedings2020050101
During herpesvirus infection, nascent viral capsids egress the nucleus into the cytoplasm by an unusual mechanism whereby capsids bud at the inner nuclear membrane. This process is mediated by the conserved heterodimeric nuclear egress complex (NEC), anchored to the inner nuclear membrane, that deforms the membrane around the capsid by forming a hexagonal array. However, how the NEC coat interacts with the capsid and how proper curvature of the coat is achieved to enable budding are yet unclear. Here, we show that the binding of a capsid protein, UL25, promotes the formation of a pentagonal rather than hexagonal NEC arrangement. Our results suggest that during nuclear budding interactions between the UL25 bound to the pentagonal capsid vertices and the NEC introduce pentagonal insertions into the hexagonal NEC array to yield an NEC coat of the appropriate size and curvature, leading to the productive budding and egress of UL25-decorated capsids.
Proceedings, Volume 50; doi:10.3390/proceedings2020050104
Severe fever with thrombocytopenia syndrome (SFTS) is an emerging tick-borne disease caused by SFTS virus (SFTSV) infection. Despite the gradual increase in SFTS cases and high mortality in the endemic region, no specific viral therapy nor vaccine is available. Here, we developed single recombinant plasmid DNA encoding SFTSV genes Gn and Gc together with the NP–NS fusion antigen as a vaccine candidate. The viral antigens were fused with Fms-like tyrosine kinase-3 ligand (Flt3L) and the IL-12 gene was incorporated into the plasmid to enhance cell-mediated immunity. Vaccination with the DNA can provide complete protection of IFNAR KO mice upon lethal SFTSV challenge, whereas immunization with a plasmid lacking the IL-12 gene resulted in partial protection. Since we failed to detect antibodies against the surface glycoproteins Gn and Gc in the immunized mice, antigen-specific cellular immunity, as confirmed by enhanced antigen-specific T cell responses, might play a major role in protection. Finally, we evaluated the degree of protective immunity after protein immunization that was provided by individual glycoproteins Gn or Gc. Although both protein antigens induced significant levels of neutralizing activity against SFTSV, Gn vaccination resulted in relatively higher neutralizing activity and better protection than Gc vaccination. However, both antigens failed to provide complete protection. Given that the DNA vaccines have failed to induce sufficient immunogenicity in human trials when compared with protein vaccines, optimal combinations of DNA and protein vaccine, proper selection of target antigens, and incorporation of an efficient adjuvant need to be further investigated for SFTS vaccine development.
Proceedings, Volume 50; doi:10.3390/proceedings2020050105
Cyanophages are viruses that infect cyanobacteria (also known as blue-green algae) and are ubiquitious in marine and freshwater environments. In recent years, freshwater cyanophages have attracted more attention because they can affect global freshwater ecosystems. The spatial distribution and morphological diversity of cyanophage populations were examined in Lake Donghu with three trophic regions: hypertrophic, eutrophic, and mesotrophic regions. The surprisingly high viral abundance (ranging from 108 to 109 phage mL−1) and morphological diversity were detected. Most of them have tails and belong to the families Siphoviridae, Myoviridae, and Podoviridae. Various morphotypes were observed, such as prolate-headed virus-like particles and lemon-shaped virus-like particles. In addition, some cyanophages were studied by virological experiments and whole-genome analyses, combined with morphological observation. For example, three cyanophages were isolated and their whole genomes were sequenced. Contractile tail myonophage MaMV-DC infects bloom-forming cyanobacterium Microcystis aeruginosa. Tailless cyanophage Planktothrix agardhii virus isolated from Lake Donghu (PaV-LD) infects filamentous cyanobacterium. Short-tail podovirus A-4L can infect the model cyanobacterium Anabaena sp. strain PCC 7120. The MaMV-DC genome contains 169,223 bp encoding 170 putative open reading frames (ORFs). The PaV-LD genome posseses 95,299 bp encoding 142 putative ORFs. The genome of short-tail podovirus A-4L has 41,750 bp encoding 38 putative ORFs. There are significant differences in their genomic size and encoded tail proteins, but all three cyanophages contain genes that are not commonly found in phages. By studying the vast biodiversity of viruses in freshwater environments, these novel findings of cyanophages broaden our insights, and allow us to gain more useful knowledge about the global impact of these viruses in freshwater ecosystems.
Proceedings, Volume 50; doi:10.3390/proceedings2020050100
Before its recent spread, serological investigations conducted between the 1960s and the 1990s showed the wide presence of Zika virus in Africa. According to the World Health Organization, the entire Africa continent is at risk of Zika outbreak due to the presence of the virus, competent vectors, and the low capacity for surveillance and containment of an epidemic. However, limited data are available on the recent prevalence in the African population. The aim of this study was to evaluate the immunity against Zika virus in samples of a selected cohort from West Africa, in order to investigate the circulation of the virus in the region during the first years of its emergence in the Pacific. Human serum samples were collected in 2007 and between 2011 and 2012 from a cohort of subjects from Mali, Senegal, and The Gambia. The samples were tested using an enzyme-linked immunosorbent assay (ELISA) detection kit and positives were further confirmed by microneutralization test. The results indicate that Zika virus is present and actively circulating in Senegal and The Gambia, with prevalence values of 13.7% and 6.9% in 2012, respectively. Although no significant differences in prevalence were found for the considered time period, seroconversion of some subjects showed the active circulation of Zika virus in the West African area. Analysis by age showed an increase in immunity in relation to increasing age, demonstrating that the population is consistently exposed to the virus throughout life and with a high possibility of being infected during reproductive age. In conclusion, the obtained results allow for better knowledge of the circulation of Zika virus within three different ecological and demographic contexts, and represent an update to the limited data currently available.