The Mechanism of Action of L-Tyrosine Derivatives against Chikungunya Virus Infection In Vitro Depends on Structural Changes.

Although the disease caused by chikungunya virus (CHIKV) is of great interest to public health organizations around the world, there are still no authorized antivirals for its treatment. Previously, dihalogenated anti-CHIKV compounds derived from L-tyrosine (dH-Y) were identified as being effective against in vitro infection by this virus, so the objective of this study was to determine the mechanisms of its antiviral action. Six dH-Y compounds (C1 to C6) dihalogenated with bromine or chlorine and modified in their amino groups were evaluated by different in vitro antiviral strategies and in silico tools. When the cells were exposed before infection, all compounds decreased the expression of viral proteins; only C4, C5 and C6 inhibited the genome; and C1, C2 and C3 inhibited infectious viral particles (IVPs). Furthermore, C1 and C3 reduce adhesion, while C2 and C3 reduce internalization, which could be related to the in silico interaction with the fusion peptide of the E1 viral protein. Only C3, C4, C5 and C6 inhibited IVPs when the cells were exposed after infection, and their effect occurred in late stages after viral translation and replication, such as assembly, and not during budding. In summary, the structural changes of these compounds determine their mechanism of action. Additionally, C3 was the only compound that inhibited CHIKV infection at different stages of the replicative cycle, making it a compound of interest for conversion as a potential drug.

Citrus psorosis virus 24K protein inhibits the processing of miRNA precursors by interacting with components of the biogenesis machinery.

Sweet orange (Citrus sinensis) is one of the most important fruit crops worldwide. Virus infections in this crop can interfere with cellular processes, causing dramatic economic losses. By performing RT-qPCR analyses, we demonstrated that citrus psorosis virus (CPsV)-infected orange plants exhibited higher levels of unprocessed microRNA (miRNA) precursors than healthy plants. This result correlated with the reported reduction of mature miRNAs species. The protein 24K, the CPsV suppressor of RNA silencing (VSR), interacts with miRNA precursors in vivo. Thus, this protein becomes a candidate responsible for the increased accumulation of unprocessed miRNAs. We analyzed 24K RNA-binding and protein-protein interaction domains and described patterns of its subcellular localization. We also showed that 24K colocalizes within nuclear D-bodies with the miRNA biogenesis proteins DICER-LIKE 1 (DCL1), HYPONASTIC LEAVES 1 (HYL1), and SERRATE (SE). According to the results of bimolecular fluorescence complementation and co-immunoprecipitation assays, the 24K protein interacts with HYL1 and SE. Thus, 24K may inhibit miRNA processing in CPsV-infected citrus plants by direct interaction with the miRNA processing complex. This work contributes to the understanding of how a virus can alter the regulatory mechanisms of the host, particularly miRNA biogenesis and function.IMPORTANCESweet oranges can suffer from disease symptoms induced by virus infections, thus resulting in drastic economic losses. In sweet orange plants, CPsV alters the accumulation of some precursors from the regulatory molecules called miRNAs. This alteration leads to a decreased level of mature miRNA species. This misregulation may be due to a direct association of one of the viral proteins (24K) with miRNA precursors. On the other hand, 24K may act with components of the cell miRNA processing machinery through a series of predicted RNA-binding and protein-protein interaction domains.

Combined effect of SAR-endolysin LysKpV475 with polymyxin B and Salmonella bacteriophage phSE-5.

Endolysins are bacteriophage (or phage)-encoded enzymes that catalyse the peptidoglycan breakdown in the bacterial cell wall. The exogenous action of recombinant phage endolysins against Gram-positive organisms has been extensively studied. However, the outer membrane acts as a physical barrier when considering the use of recombinant endolysins to combat Gram-negative bacteria. This study aimed to evaluate the antimicrobial activity of the SAR-endolysin LysKpV475 against Gram-negative bacteria as single or combined therapies, using an outer membrane permeabilizer (polymyxin B) and a phage, free or immobilized in a pullulan matrix. In the first step, the endolysin LysKpV475 in solution, alone and combined with polymyxin B, was tested in vitro and in vivo against ten Gram-negative bacteria, including highly virulent strains and multidrug-resistant isolates. In the second step, the lyophilized LysKpV475 endolysin was combined with the phage phSE-5 and investigated, free or immobilized in a pullulan matrix, against Salmonella enterica subsp. enterica serovar Typhimurium ATCC 13311. The bacteriostatic action of purified LysKpV475 varied between 8.125 µg ml-1 against Pseudomonas aeruginosa ATCC 27853, 16.25 µg ml-1 against S. enterica Typhimurium ATCC 13311, and 32.50 µg ml-1 against Klebsiella pneumoniae ATCC BAA-2146 and Enterobacter cloacae P2224. LysKpV475 showed bactericidal activity only for P. aeruginosa ATCC 27853 (32.50 µg ml-1) and P. aeruginosa P2307 (65.00 µg ml-1) at the tested concentrations. The effect of the LysKpV475 combined with polymyxin B increased against K. pneumoniae ATCC BAA-2146 [fractional inhibitory concentration index (FICI) 0.34; a value lower than 1.0 indicates an additive/combined effect] and S. enterica Typhimurium ATCC 13311 (FICI 0.93). A synergistic effect against S. enterica Typhimurium was also observed when the lyophilized LysKpV475 at ⅔ MIC was combined with the phage phSE-5 (m.o.i. of 100). The lyophilized LysKpV475 immobilized in a pullulan matrix maintained a significant Salmonella reduction of 2 logs after 6 h of treatment. These results demonstrate the potential of SAR-endolysins, alone or in combination with other treatments, in the free form or immobilized in solid matrices, which paves the way for their application in different areas, such as in biocontrol at the food processing stage, biosanitation of food contact surfaces and biopreservation of processed food in active food packing.

Viroporins Manipulate Cellular Powerhouses and Modulate Innate Immunity.

Viruses have a wide repertoire of molecular strategies that focus on their replication or the facilitation of different stages of the viral cycle. One of these strategies is mediated by the activity of viroporins, which are multifunctional viral proteins that, upon oligomerization, exhibit ion channel properties with mild ion selectivity. Viroporins facilitate multiple processes, such as the regulation of immune response and inflammasome activation through the induction of pore formation in various cell organelle membranes to facilitate the escape of ions and the alteration of intracellular homeostasis. Viroporins target diverse membranes (such as the cellular membrane), endoplasmic reticulum, and mitochondria. Cumulative data regarding the importance of mitochondria function in multiple processes, such as cellular metabolism, energy production, calcium homeostasis, apoptosis, and mitophagy, have been reported. The direct or indirect interaction of viroporins with mitochondria and how this interaction affects the functioning of mitochondrial cells in the innate immunity of host cells against viruses remains unclear. A better understanding of the viroporin-mitochondria interactions will provide insights into their role in affecting host immune signaling through the mitochondria. Thus, in this review, we mainly focus on descriptions of viroporins and studies that have provided insights into the role of viroporins in hijacked mitochondria.

Preclinical assessment of an anti-HTLV-1 heterologous DNA/MVA vaccine protocol expressing a multiepitope HBZ protein.

BACKGROUND: Human T-lymphotropic virus 1 (HTLV-1) is associated with the development of several pathologies and chronic infection in humans. The inefficiency of the available treatments and the challenge in developing a protective vaccine highlight the need to produce effective immunotherapeutic tools. The HTLV-1 basic leucine zipper (bZIP) factor (HBZ) plays an important role in the HTLV-1 persistence, conferring a survival advantage to infected cells by reducing the HTLV-1 proteins expression, allowing infected cells to evade immune surveillance, and enhancing cell proliferation leading to increased proviral load. METHODS: We have generated a recombinant Modified Virus Vaccinia Ankara (MVA-HBZ) and a plasmid DNA (pcDNA3.1(+)-HBZ) expressing a multiepitope protein based on peptides of HBZ to study the immunogenic potential of this viral-derived protein in BALB/c mice model. Mice were immunized in a prime-boost heterologous protocol and their splenocytes (T CD4+ and T CD8+) were immunophenotyped by flow cytometry and the humoral response was evaluated by ELISA using HBZ protein produced in prokaryotic vector as antigen. RESULTS: T CD4+ and T CD8+ lymphocytes cells stimulated by HBZ-peptides (HBZ42-50 and HBZ157-176) showed polyfunctional double positive responses for TNF-α/IFN-γ, and TNF-α/IL-2. Moreover, T CD8+ cells presented a tendency in the activation of effector memory cells producing granzyme B (CD44+High/CD62L-Low), and the activation of Cytotoxic T Lymphocytes (CTLs) and cytotoxic responses in immunized mice were inferred through the production of granzyme B by effector memory T cells and the expression of CD107a by CD8+ T cells. The overall data is consistent with a directive and effector recall response, which may be able to operate actively in the elimination of HTLV-1-infected cells and, consequently, in the reduction of the proviral load. Sera from immunized mice, differently from those of control animals, showed IgG-anti-HBZ production by ELISA. CONCLUSIONS: Our results highlight the potential of the HBZ multiepitope protein expressed from plasmid DNA and a poxviral vector as candidates for therapeutic vaccine.

Oropouche Virus Glycoprotein Topology and Cellular Requirements for Glycoprotein Secretion.

Oropouche virus (OROV; genus Orthobunyavirus) is the etiological agent of Oropouche fever, a debilitating febrile illness common in South America. We used recombinant expression of the OROV M polyprotein, which encodes the surface glycoproteins Gn and Gc plus the nonstructural protein NSm, to probe the cellular determinants for OROV assembly and budding. Gn and Gc self-assemble and are secreted independently of NSm. Mature OROV Gn has two predicted transmembrane domains that are crucial for glycoprotein translocation to the Golgi complex and glycoprotein secretion, and unlike related orthobunyaviruses, both transmembrane domains are retained during Gn maturation. Disruption of Golgi function using the drugs brefeldin A and monensin inhibits glycoprotein secretion. Infection studies have previously shown that the cellular endosomal sorting complexes required for transport (ESCRT) machinery is recruited to Golgi membranes during OROV assembly and that ESCRT activity is required for virus secretion. A dominant-negative form of the ESCRT-associated ATPase VPS4 significantly reduces recombinant OROV glycoprotein secretion and blocks virus release from infected cells, and VPS4 partly colocalizes with OROV glycoproteins and membranes costained with Golgi markers. Furthermore, immunoprecipitation and fluorescence microscopy experiments demonstrate that OROV glycoproteins interact with the ESCRT-III component CHMP6, with overexpression of a dominant-negative form of CHMP6 significantly reducing OROV glycoprotein secretion. Taken together, our data highlight differences in M polyprotein processing across orthobunyaviruses, indicate that Golgi and ESCRT function are required for glycoprotein secretion, and identify CHMP6 as an ESCRT-III component that interacts with OROV glycoproteins. IMPORTANCE Oropouche virus causes Oropouche fever, a debilitating illness common in South America that is characterized by high fever, headache, myalgia, and vomiting. The tripartite genome of this zoonotic virus is capable of reassortment, and there have been multiple epidemics of Oropouche fever in South America over the last 50 years, making Oropouche virus infection a significant threat to public health. However, the molecular characteristics of this arbovirus are poorly understood. We developed a recombinant protein expression system to investigate the cellular determinants of OROV glycoprotein maturation and secretion. We show that the proteolytic processing of the M polypeptide, which encodes the surface glycoproteins (Gn and Gc) plus a nonstructural protein (NSm), differs between OROV and its close relative Bunyamwera virus. Furthermore, we demonstrate that OROV M glycoprotein secretion requires the cellular endosomal sorting complexes required for transport (ESCRT) membrane-remodeling machinery and identify that the OROV glycoproteins interact with the ESCRT protein CHMP6.

Subcellular localization and interactions among TGB proteins of cowpea mild mottle virus.

Cowpea mild mottle virus (CPMMV) is a flexuous filamentous virus that belongs to the genus Carlavirus (family Betaflexiviridae). The CPMMV genome contains six open reading frames (ORFs), among which the triple gene block (TGB), encoded by ORFs 2 to 4, has been reported to encode movement proteins for different viruses. The subcellular localization of the TGB proteins of CPMMV isolate CPMMV:BR:MG:09:2 was analysed by transient expression of each protein fused to a fluorophore. Overall, the accumulation pattern and interactions among CPMMV TGB proteins (TGBp) were similar to those of their counterparts from the potex-like group. Considering these similarities, we evaluated the potential interactions between the TGB proteins of CPMMV and of potato virus X, which could complement cell-to-cell movement. The TGBp2 and TGBp3 of PVX had an effect on CPMMV TGBp1, directing it to the plasmodesmata, but the reverse was not true.

Structural aspects of hepatitis E virus.

Hepatitis E virus (HEV) is a leading cause of acute hepatitis worldwide. Hepatitis E is an enterically transmitted zoonotic disease that causes large waterborne epidemic outbreaks in developing countries and has become an increasing public-health concern in industrialized countries. In this setting, the infection is usually acute and self-limiting in immunocompetent individuals, although chronic cases in immunocompromised patients have been reported, frequently associated with several extrahepatic manifestations. Moreover, extrahepatic manifestations have also been reported in immunocompetent individuals with acute HEV infection. HEV belongs to the alphavirus-like supergroup III of single-stranded positive-sense RNA viruses, and its genome contains three partially overlapping open reading frames (ORFs). ORF1 encodes a nonstructural protein with eight domains, most of which have not been extensively characterized: methyltransferase, Y domain, papain-like cysteine protease, hypervariable region, proline-rich region, X domain, Hel domain, and RNA-dependent RNA polymerase. ORF2 and ORF3 encode the capsid protein and a multifunctional protein believed to be involved in virion release, respectively. The novel ORF4 is only expressed in HEV genotype 1 under endoplasmic reticulum stress conditions, and its exact function has not yet been elucidated. Despite important advances in recent years, the biological and molecular processes underlying HEV replication remain poorly understood, primarily due to a lack of detailed information about the functions of the viral proteins and the mechanisms involved in host-pathogen interactions. This review summarizes the current knowledge concerning HEV proteins and their biological properties, providing updated detailed data describing their function and focusing in detail on their structural characteristics. Furthermore, we review some unclear aspects of the four proteins encoded by the ORFs, highlighting the current key information gaps and discussing potential novel experimental strategies for shedding light on those issues.

A Review of In Silico Research, SARS-CoV-2, and Neurodegeneration: Focus on Papain-Like Protease.

Since the appearance of SARS-CoV-2 and the COVID-19 pandemic, the search for new approaches to treat this disease took place in the scientific community. The in silico approach has gained importance at this moment, once the methodologies used in this kind of study allow for the identification of specific protein-ligand interactions, which may serve as a filter step for molecules that can act as specific inhibitors. In addition, it is a low-cost and high-speed technology. Molecular docking has been widely used to find potential viral protein inhibitors for structural and non-structural proteins of the SARS-CoV-2, aiming to block the infection and the virus multiplication. The papain-like protease (PLpro) participates in the proteolytic processing of SARS-CoV-2 and composes one of the main targets studied for pharmacological intervention by in silico methodologies. Based on that, we performed a systematic review about PLpro inhibitors from the perspective of in silico research, including possible therapeutic molecules in relation to this viral protein. The neurological problems triggered by COVID-19 were also briefly discussed, especially relative to the similarities of neuroinflammation present in Alzheimer's disease. In this context, we focused on two molecules, curcumin and glycyrrhizinic acid, given their PLpro inhibitory actions and neuroprotective properties and potential therapeutic effects on COVID-19.

The impact of the suppression of highly connected protein interactions on the corona virus infection.

Several highly effective Covid-19 vaccines are in emergency use, although more-infectious coronavirus strains, could delay the end of the pandemic even further. Because of this, it is highly desirable to develop fast antiviral drug treatments to accelerate the lasting immunity against the virus. From a theoretical perspective, computational approaches are useful tools for antiviral drug development based on the data analysis of gene expression, chemical structure, molecular pathway, and protein interaction mapping. This work studies the structural stability of virus-host interactome networks based on the graphical representation of virus-host protein interactions as vertices or nodes connected by commonly shared proteins. These graphical network visualization methods are analogous to those use in the design of artificial neural networks in neuromorphic computing. In standard protein-node-based network representation, virus-host interaction merges with virus-protein and host-protein networks, introducing redundant links associated with the internal virus and host networks. On the contrary, our approach provides a direct geometrical representation of viral infection structure and allows the effective and fast detection of the structural robustness of the virus-host network through proteins removal. This method was validated by applying it to H1N1 and HIV viruses, in which we were able to pinpoint the changes in the Interactome Network produced by known vaccines. The application of this method to the SARS-CoV-2 virus-host protein interactome implies that nonstructural proteins nsp4, nsp12, nsp16, the nuclear pore membrane glycoprotein NUP210, and ubiquitin specific peptidase USP54 play a crucial role in the viral infection, and their removal may provide an efficient therapy. This method may be extended to any new mutations or other viruses for which the Interactome Network is experimentally determined. Since time is of the essence, because of the impact of more-infectious strains on controlling the spread of the virus, this method may be a useful tool for novel antiviral therapies.

Inhibitory effect of thymoquinone from Nigella sativa against SARS-CoV-2 main protease. An in-silico study.

Nigella sativa is known for the safety profile, containing a wealth of useful antiviral compounds. The main protease (Mpro, 3CLpro) of severe acute respiratory syndrome 2 (SARS-CoV-2) is being considered as one of the most attractive viral target, processing the polyproteins during viral pathogenesis and replication. In the current investigation we analyzed the potency of active component, thymoquinone (TQ) of Nigella sativa against SARS-CoV-2 Mpro. The structures of TQ and Mpro was retrieved from PubChem (CID10281) and Protein Data Bank (PDB ID 6MO3) respectively. The Mpro and TQ were docked and the complex was subjected to molecular dynamic (MD) simulations for a period 50ns. Protein folding effect was analyzed using radius of gyration (Rg) while stability and flexibility was measured, using root means square deviations (RMSD) and root means square fluctuation (RMSF) respectively. The simulation results shows that TQ is exhibiting good binding activity against SARS-CoV-2 Mpro, interacting many residues, present in the active site (His41, Cys145) and also the Glu166, facilitating the pocket shape. Further, experimental approaches are needed to validate the role of TQ against virus infection. The TQ is interfering with pocket maintaining residues as well as active site of virus Mpro which may be used as a potential inhibitor against SARS-CoV-2 for better management of COVID-19.

The RNA-dependent RNA polymerase of the infectious pancreatic necrosis virus is linked to viral mRNA acting as a cap substitute.

The infectious pancreatic necrosis virus (IPNV) is responsible for significant economic losses in the aquaculture industry. It is an unenveloped virus with an icosahedral capsid. Its viral genome comprises two dsRNA segments, A and B. Segment A contains a small ORF, which encodes VP5, and a large ORF, which encodes a polyprotein that generates the structural proteins and the viral protease. Segment B encodes the RNA-dependent RNA polymerase (RdRp), called VP1 in this free form, or Vpg when it covalently attaches to the viral RNA. The viral genome does not have cap or poly(A). Instead, each 5' end is linked to the Vpg. Recently, we demonstrated that mRNA-A contains an internal ribosome entry site (IRES) to command polyprotein synthesis. However, the presence of Vpg on IPNV mRNAs and its impact on cellular translation has not been investigated. This research demonstrates that IPNV mRNAs are linked to Vpg and that this protein inhibits cap-dependent translation on infected cells. Also, it is demonstrated that Vpg interacts with eIF4E and that rapamycin treatment partially diminishes the viral protein synthesis. In addition, we determined that an IRES does not command translation of IPNV mRNA-B. We show that VPg serves as a cap substitute during the initiation of IPNV translation, contributing to understanding the replicative cycle of Birnaviruses. Our results indicate that the viral protein VP1/Vpg is multifunctional, having a significant role during IPNV RNA synthesis as the RdRp and the primer for IPNV RNA synthesis and translation as the viral protein genome, acting as a cap substitute.

Mutation profile of SARS-CoV-2 genome in a sample from the first year of the pandemic in Colombia.

The severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) is the etiopathogenic agent of COVID-19, a condition that has led to a formally recognized pandemic by March 2020 (World Health Organization -WHO). The SARS-CoV-2 genome is constituted of 29,903 base pairs, that code for four structural proteins (N, M, S, and E) and more than 20 non-structural proteins. Mutations in any of these regions, especially in those that encode for the structural proteins, have allowed the identification of diverse lineages around the world, some of them named as Variants of Concern (VOC) and Variants of Interest (VOI), according to the WHO and CDC. In this study, by using Next Generation Sequencing (NGS) technology, we sequenced the SARS-CoV-2 genome of 422 samples from Colombian residents, all of them collected between April 2020 and January 2021. We obtained genetic information from 386 samples, leading us to the identification of 14 new lineages circulating in Colombia, 13 of which were identified for the first time in South America. GH was the predominant GISAID clade in our sample. Most mutations were either missense (53.6%) or synonymous mutations (37.4%), and most genetic changes were located in the ORF1ab gene (63.9%), followed by the S gene (12.9%). In the latter, we identified mutations E484K, L18F, and D614G. Recent evidence suggests that these mutations concede important particularities to the virus, compromising host immunity, the diagnostic test performance, and the effectiveness of some vaccines. Some important lineages containing these mutations are the Alpha, Beta, and Gamma (WHO Label). Further genomic surveillance is important for the understanding of emerging genomic variants and their correlation with disease severity.

In Silico Analysis of Dengue Virus Serotype 2 Mutations Detected at the Intrahost Level in Patients with Different Clinical Outcomes.

Intrahost genetic diversity is thought to facilitate arbovirus adaptation to changing environments and hosts, and it may also be linked to viral pathogenesis. Intending to shed light on the viral determinants for severe dengue pathogenesis, we previously analyzed the DENV-2 intrahost genetic diversity in 68 patients clinically classified as dengue fever (n = 31), dengue with warning signs (n = 19), and severe dengue (n = 18), performing viral whole-genome deep sequencing from clinical samples with an amplicon-free approach. From it, we identified a set of 141 relevant mutations distributed throughout the viral genome that deserved further attention. Therefore, we employed molecular modeling to recreate three-dimensional models of the viral proteins and secondary RNA structures to map the mutations and assess their potential effects. Results showed that, in general lines, disruptive variants were identified primarily among dengue fever cases. In contrast, potential immune-escape variants were associated mainly with warning signs and severe cases, in line with the latter's longer intrahost evolution times. Furthermore, several mutations were located on protein-surface regions, with no associated function. They could represent sites of further investigation, as the interaction of viral and host proteins is critical for both host immunomodulation and virus hijacking of the cellular machinery. The present analysis provides new information about the implications of the intrahost genetic diversity of DENV-2, contributing to the knowledge about the viral factors possibly involved in its pathogenesis within the human host. Strengthening our results with functional studies could allow many of these variants to be considered in the design of therapeutic or prophylactic compounds and the improvement of diagnostic assays. IMPORTANCE Previous evidence showed that intrahost genetic diversity in arboviruses may be linked to viral pathogenesis and that one or a few amino acid replacements within a single protein are enough to modify a biological feature of an RNA virus. To assess dengue virus serotype 2 determinants potentially involved in pathogenesis, we previously analyzed the intrahost genetic diversity of the virus in patients with different clinical outcomes and identified a set of 141 mutations that deserved further study. Thus, through a molecular modeling approach, we showed that disruptive variants were identified primarily among cases with mild dengue fever, while potential immune-escape variants were mainly associated with cases of greater severity. We believe that some of the variants pointed out in this study were attractive enough to be potentially considered in future intelligent designs of therapeutic or prophylactic compounds or the improvement of diagnostic tools. The present analysis provides new information about DENV-2 viral factors possibly involved in its pathogenesis within the human host.

Evidence That the Adenovirus Single-Stranded DNA Binding Protein Mediates the Assembly of Biomolecular Condensates to Form Viral Replication Compartments.

A common viral replication strategy is characterized by the assembly of intracellular compartments that concentrate factors needed for viral replication and simultaneously conceal the viral genome from host-defense mechanisms. Recently, various membrane-less virus-induced compartments and cellular organelles have been shown to represent biomolecular condensates (BMCs) that assemble through liquid-liquid phase separation (LLPS). In the present work, we analyze biophysical properties of intranuclear replication compartments (RCs) induced during human adenovirus (HAdV) infection. The viral ssDNA-binding protein (DBP) is a major component of RCs that contains intrinsically disordered and low complexity proline-rich regions, features shared with proteins that drive phase transitions. Using fluorescence recovery after photobleaching (FRAP) and time-lapse studies in living HAdV-infected cells, we show that DBP-positive RCs display properties of liquid BMCs, which can fuse and divide, and eventually form an intranuclear mesh with less fluid-like features. Moreover, the transient expression of DBP recapitulates the assembly and liquid-like properties of RCs in HAdV-infected cells. These results are of relevance as they indicate that DBP may be a scaffold protein for the assembly of HAdV-RCs and should contribute to future studies on the role of BMCs in virus-host cell interactions.

Biochemical characterization of LysVpKK5 endolysin from a marine vibriophage.

Endolysins have been proposed as a potential antibacterial alternative for aquaculture, especially against Vibrio; the bacterial-agents that most frequently cause disease. Although multiple marine vibriophages have been characterized to date, research on vibriophage endolysins is recent. In this study, biochemical characterization of LysVpKK5 endolysin encoded by Vibrio parahaemolyticus-infecting VpKK5 phage was performed. In silico analysis revealed that LysVpKK5 possesses a conserved amidase_2 domain with a zinc-binding motif of high structural similarity to T7 lysozyme (RMSD = 0.107 Å). Contrary to expectations, the activity was inhibited with Zn2+ and was improved with other divalent cations, especially Ca2+. It showed optimal muralytic activity at pH 10, and curiously, no lytic activity at pH ≤ 7 was recorded. As for the thermal stability test, the optimal activity was recorded at 30 °C; the higher residual activity was recorded at 4 °C, and was lost at ≥ 50 °C. On the other hand, increasing NaCl concentrations reduced the activity gradually; the optimal activity was recorded at 50 mM NaCl. On the other hand, the enzymatic activity at 0.5 M NaCl was approx 30% and of approx 50% in seawater. LysVpKK5 endolysin exhibited a higher activity on V. parahaemolyticus ATCC-17802 strain, in comparison with AHPND + strains.

A Crystallographic Snapshot of SARS-CoV-2 Main Protease Maturation Process.

SARS-CoV-2 is the causative agent of COVID-19. The dimeric form of the viral Mpro is responsible for the cleavage of the viral polyprotein in 11 sites, including its own N and C-terminus. The lack of structural information for intermediary forms of Mpro is a setback for the understanding its self-maturation process. Herein, we used X-ray crystallography combined with biochemical data to characterize multiple forms of SARS-CoV-2 Mpro. For the immature form, we show that extra N-terminal residues caused conformational changes in the positioning of domain-three over the active site, hampering the dimerization and diminishing its activity. We propose that this form preludes the cis and trans-cleavage of N-terminal residues. Using fragment screening, we probe new cavities in this form which can be used to guide therapeutic development. Furthermore, we characterized a serine site-directed mutant of the Mpro bound to its endogenous N and C-terminal residues during dimeric association stage of the maturation process. We suggest this form is a transitional state during the C-terminal trans-cleavage. This data sheds light in the structural modifications of the SARS-CoV-2 main protease during its self-maturation process.

The morphogenesis of different giant viruses as additional evidence for a common origin of Nucleocytoviricota.

Large and giant DNA viruses are a monophyletic group constituting the recently established phylum Nucleocytoviricota. The virus particle morphogenesis of these viruses exhibit striking similarities. Viral factories are established in the host cells where new virions are assembled by recruiting host membranes, forming an inner lipid layer. An outer protein layer starts as a lamellar structure, commonly referred to as viral crescents, coded by the major capsid protein gene. Also, these viruses have a conserved ATPase-coding gene related to genome encapsidation. Similar properties are described for tectiviruses, putative small ancestors of giant viruses. Here we review the morphogenesis of giant viruses and discuss how the process similarities constitute additional evidence to the common origin of Nucleocytoviricota.

Structure and physiology of giant DNA viruses.

Although giant viruses have existed for millennia and possibly exerted great evolutionary influence in their environment. Their presence has only been noticed by virologists recently with the discovery of Acanthamoeba polyphaga mimivirus in 2003. Its virion with a diameter of 500 nm and its genome larger than 1 Mpb shattered preconceived standards of what a virus is and triggered world-wide prospection studies. Thanks to these investigations many giant virus families were discovered, each with its own morphological peculiarities and genomes ranging from 0.4 to 2.5 Mpb that possibly encode more than 400 viral proteins. This review aims to present the morphological diversity, the different aspects observed in host-virus interactions during replication, as well as the techniques utilized during their investigation.

A Sanger-based approach for scaling up screening of SARS-CoV-2 variants of interest and concern.

The global spread of new SARS-CoV-2 variants of concern underscore an urgent need of simple deployed molecular tools that can differentiate these lineages. Several tools and protocols have been shared since the beginning of the COVID-19 pandemic, but they need to be timely adapted to cope with SARS-CoV-2 evolution. Although whole-genome sequencing (WGS) of the virus genetic material has been widely used, it still presents practical difficulties such as high cost, shortage of available reagents in the global market, need of a specialized laboratorial infrastructure and well-trained staff. These limitations result in SARS-CoV-2 surveillance blackouts across several countries. Here we propose a rapid and accessible protocol based on Sanger sequencing of a single PCR fragment that is able to identify and discriminate all SARS-CoV-2 variants of concern (VOCs) identified so far, according to each characteristic mutational profile at the Spike-RBD region (K417N/T, E484K, N501Y, A570D). Twelve COVID-19 samples from Brazilian patients were evaluated for both WGS and Sanger sequencing: three P.2, two P.1, six B.1.1 and one B.1.1.117 lineage. All results from the Sanger sequencing method perfectly matched the mutational profile of VOCs and non-VOCs RBD's characterized by WGS. In summary, this approach allows a much broader network of laboratories to perform molecular surveillance of SARS-CoV-2 VOCs and report results within a shorter time frame, which is of utmost importance in the context of rapid public health decisions in a fast evolving worldwide pandemic.