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To gain insight into the functional relationship between the nucleocapsid (NC) domains of the Gag polyproteins of feline and simian immunodeficiency viruses, FIV and SIV, respectively, we generated two FIV Gag chimeric proteins containing different SIV NC and gag sequences. A chimeric FIV Gag protein (NC1) containing the SIV two zinc fingers motifs was incapable of assembling into virus-like particles. By contrast, another Gag chimera (NC2) differing from NC1 by the replacement of the C-terminal region of the FIV NC with SIV SP2 produced particles as efficiently as wild-type FIV Gag. Of note, when the chimeric NC2 Gag polyprotein was expressed in the context of the proviral DNA in feline CrFK cells, wild-type levels of virions were produced which encapsidated 50% of genomic RNA when compared to the wild-type virus.

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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)P2). 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.

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The Gag-Pol polyprotein in human immunodeficiency virus type I (HIV-1) encodes enzymes that are essential for virus replication: protease (PR), reverse transcriptase (RT), and integrase (IN). The mature forms of PR, RT and IN are homodimer, heterodimer and tetramer, respectively. The precise mechanism underlying the formation of dimer or tetramer is not yet understood. Here, to gain insight into the dimerization of PR and RT in the precursor, we prepared a model precursor, PR-RT, incorporating an inactivating mutation at the PR active site, D25A, and including two residues in the p6* region, fused to a SUMO-tag, at the N-terminus of the PR region. We also prepared two mutants of PR-RT containing a dimer dissociation mutation either in the PR region, PR(T26A)-RT, or in the RT region, PR-RT(W401A). Size exclusion chromatography showed both monomer and dimer fractions in PR-RT and PR(T26A)-RT, but only monomer in PR-RT(W401A). SEC experiments of PR-RT in the presence of protease inhibitor, darunavir, significantly enhanced the dimerization. Additionally, SEC results suggest an estimated PR-RT dimer dissociation constant that is higher than that of the mature RT heterodimer, p66/p51, but slightly lower than the premature RT homodimer, p66/p66. Reverse transcriptase assays and RT maturation assays were performed as tools to assess the effects of the PR dimer-interface on these functions. Our results consistently indicate that the RT dimer-interface plays a crucial role in the dimerization in PR-RT, whereas the PR dimer-interface has a lesser role.

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The main protease (Mpro) remains an essential therapeutic target for COVID-19 post infection intervention given its critical role in processing the majority of viral proteins encoded by the genome of severe acute respiratory syndrome related coronavirus 2 (SARS-CoV-2). Upon viral entry, the +ssRNA genome is translated into two long polyproteins (pp1a or the frameshift-dependent pp1ab) containing all the nonstructural proteins (nsps) required by the virus for immune modulation, replication, and ultimately, virion assembly. Included among these nsps is the cysteine protease Mpro (nsp5) which self-excises from the polyprotein, dimerizes, then sequentially cleaves 11 of the 15 cut-site junctions found between each nsp within the polyprotein. Many structures of Mpro (often bound to various small molecule inhibitors or peptides) have been detailed recently, including structures of Mpro bound to each of the polyprotein cleavage sequences, showing that Mpro can accommodate a wide range of targets within its active site. However, to date, kinetic characterization of the interaction of Mpro with each of its native cleavage sequences remains incomplete. Here, we present a robust and cost-effective FRET based system that benefits from a more consistent presentation of the substrate that is also closer in organization to the native polyprotein environment compared to previously reported FRET systems that use chemically modified peptides. Using this system, we were able to show that while each site maintains a similar Michaelis constant, the catalytic efficiency of Mpro varies greatly between cut-site sequences, suggesting a clear preference for the order of nsp processing.

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Amino acid preferences at a protein site depend on the role of this site in protein function and structure as well as on external constraints. All these factors can change in the course of evolution, making amino acid propensities of a site time-dependent. When viral subtypes divergently evolve in different host subpopulations, such changes may depend on genetic, medical, and sociocultural differences between these subpopulations. Here, using our previously developed phylogenetic approach, we describe sixty-nine amino acid sites of the Gag protein of human immunodeficiency virus type 1 (HIV-1) where amino acids have different impact on viral fitness in six major subtypes of the type M. These changes in preferences trigger adaptive evolution; indeed, 32 (46 per cent) of these sites experienced strong positive selection at least in one of the subtypes. At some of the sites, changes in amino acid preferences may be associated with differences in immune escape between subtypes. The prevalence of an amino acid in a protein site within a subtype is only a poor predictor for whether this amino acid is preferred in this subtype according to the phylogenetic analysis. Therefore, attempts to identify the factors of viral evolution from comparative genomics data should integrate across multiple sources of information.

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Over the course of the COVID-19 pandemic, several SARS-CoV-2 variants have emerged that may exhibit different etiological effects such as enhanced transmissibility and infectivity. However, genetic variations that reduce virulence and deteriorate viral fitness have not yet been thoroughly investigated. The present study sought to evaluate the effects of viral genetic makeup on COVID-19 epidemiology in Pakistan, where the infectivity and mortality rate was comparatively lower than other countries during the first pandemic wave. For this purpose, we focused on the comparative analyses of 7096 amino-acid long polyprotein pp1ab. Comparative sequence analysis of 203 SARS-CoV-2 genomes, sampled from Pakistan during the first wave of the pandemic revealed 179 amino acid substitutions in pp1ab. Within this set, 38 substitutions were identified within the Nsp3 region of the pp1ab polyprotein. Structural and biophysical analysis of proteins revealed that amino acid variations within Nsp3's macrodomains induced conformational changes and modified protein-ligand interactions, consequently diminishing the virulence and fitness of SARS-CoV-2. Additionally, the epistatic effects resulting from evolutionary substitutions in SARS-CoV-2 proteins may have unnoticed implications for reducing disease burden. In light of these findings, further characterization of such deleterious SARS-CoV-2 mutations will not only aid in identifying potential therapeutic targets but will also provide a roadmap for maintaining vigilance against the genetic variability of diverse SARS-CoV-2 strains circulating globally. Furthermore, these insights empower us to more effectively manage and respond to potential viral-based pandemic outbreaks of a similar nature in the future.

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Millions of people's lives are being devastated by dengue virus (DENV), a severe tropical and subtropical illness spread by mosquitoes and other vectors. Dengue fever may be self-limiting like a common cold or can rapidly progress to catastrophic dengue hemorrhagic fever or dengue shock syndrome. With four distinct dengue serotypes (DENV1-4), each with the potential to contain antibody-boosting complicated mechanisms, developing a dengue vaccine has been an ambitious challenge. Here, we used a computational pan-vaccinomics-based vaccine design strategy (reverse vaccinology) for all 4 DENV serotypes acquired from different regions of the world to develop a new and safe vaccine against DENV. Consequently, only five mapped epitopes from all the 4 serotypes were shown to be extremely effective for the construction of multi-epitope vaccine constructs. The suggested vaccine construct V5 from eight vaccine models was thus classified as an antigenic, non-allergenic, and stable vaccine model. Moreover, molecular docking and molecular dynamics simulation was performed for the V5 vaccine candidate against the HLAs and TRL2 and 4 immunological receptors. Later, the vaccine sequence was transcribed into the cDNA to generate an expression vector for the Escherichia coli K12 strain. Our research suggests that this vaccine design (V5) has promising potential as a dengue vaccine. However, further experimental analysis into the vaccine's efficacy might be required for the V5 proper validation to combat all DENV serotypes.

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Abstract In recent years, the development of high-throughput technologies for obtaining sequence data leveraged the possibility of analysis of protein data in silico. However, when it comes to viral polyprotein interaction studies, there is a gap in the representation of those proteins, given their size and length. The prepare for studies using state-of-the-art techniques such as Machine Learning, a good representation of such proteins is a must. We present an alternative to this problem, implementing a fragmentation and modeling protocol to prepare those polyproteins in the form of peptide fragments. Such procedure is made by several scripts, implemented together on the workflow we call PolyPRep, a tool written in Python script and available in GitHub. This software is freely available only for noncommercial users.

Resumo Nos últimos anos, o desenvolvimento de tecnologias de alto rendimento para obtenção de dados sequenciais potencializou a possibilidade de análise de dados proteicos in silico. No entanto, quando se trata de estudos de interação de poliproteínas virais, existe uma lacuna na representação dessas proteínas, devido ao seu tamanho e comprimento. Para estudos utilizando técnicas de ponta como o Aprendizado de Máquina, uma boa representação dessas proteínas é imprescindível. Apresentamos uma alternativa para este problema, implementando um protocolo de fragmentação e modelagem para preparar essas poliproteínas na forma de fragmentos de peptídeos. Tal procedimento é feito por diversos scripts, implementados em conjunto no workflow que chamamos de PolyPRep, uma ferramenta escrita em script Python e disponível no GitHub. Este software está disponível gratuitamente apenas para usuários não comerciais.

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Abstract In recent years, the development of high-throughput technologies for obtaining sequence data leveraged the possibility of analysis of protein data in silico. However, when it comes to viral polyprotein interaction studies, there is a gap in the representation of those proteins, given their size and length. The prepare for studies using state-of-the-art techniques such as Machine Learning, a good representation of such proteins is a must. We present an alternative to this problem, implementing a fragmentation and modeling protocol to prepare those polyproteins in the form of peptide fragments. Such procedure is made by several scripts, implemented together on the workflow we call PolyPRep, a tool written in Python script and available in GitHub. This software is freely available only for noncommercial users.

Resumo Nos últimos anos, o desenvolvimento de tecnologias de alto rendimento para obtenção de dados sequenciais potencializou a possibilidade de análise de dados proteicos in silico. No entanto, quando se trata de estudos de interação de poliproteínas virais, existe uma lacuna na representação dessas proteínas, devido ao seu tamanho e comprimento. Para estudos utilizando técnicas de ponta como o Aprendizado de Máquina, uma boa representação dessas proteínas é imprescindível. Apresentamos uma alternativa para este problema, implementando um protocolo de fragmentação e modelagem para preparar essas poliproteínas na forma de fragmentos de peptídeos. Tal procedimento é feito por diversos scripts, implementados em conjunto no workflow que chamamos de PolyPRep, uma ferramenta escrita em script Python e disponível no GitHub. Este software está disponível gratuitamente apenas para usuários não comerciais.

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IMPORTANCE: Most protease-targeted antiviral development evaluates the ability of small molecules to inhibit the cleavage of artificial substrates. However, before they can cleave any other substrates, viral proteases need to cleave themselves out of the viral polyprotein in which they have been translated. This can occur either intra- or inter-molecularly. Whether this process occurs intra- or inter-molecularly has implications for the potential for precursors to accumulate and for the effectiveness of antiviral drugs. We argue that evaluating candidate antivirals for their ability to block these cleavages is vital to drug development because the buildup of uncleaved precursors can be inhibitory to the virus and potentially suppress the selection of drug-resistant variants.

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The deformed wing virus (DWV) belongs to the genus Iflavirus and the family Iflaviridae within the order Picornavirales. It is an important pathogen of the Western honey bee, Apis mellifera, causing major losses among honey bee colonies in association with the ectoparasitic mite Varroa destructor. Although DWV is one of the best-studied insect viruses, the mechanisms of viral replication and polyprotein processing have been poorly studied in the past. We investigated the processing of the protease-polymerase region at the C-terminus of the polyprotein in more detail using recombinant expression, novel serological reagents, and virus clone mutagenesis. Edman degradation of purified maturated polypeptides uncovered the C- and N-termini of the mature 3C-like (3CL) protease and RNA-dependent RNA polymerase (3DL, RdRp), respectively. Autocatalytic processing of the recombinant DWV 3CL protease occurred at P1 Q2118 and P1' G2119 (KPQ/GST) as well as P1 Q2393 and P1' S2394 (HAQ/SPS) cleavage sites. New monoclonal antibodies (Mab) detected the mature 3CL protease with an apparent molecular mass of 32 kDa, mature 3DL with an apparent molecular mass of 55 kDa as well as a dominant 3CDL precursor of 90 kDa in DWV infected honey bee pupae. The observed pattern corresponds well to data obtained via recombinant expression and N-terminal sequencing. Finally, we were able to show that 3CL protease activity and availability of the specific protease cleavage sites are essential for viral replication, protein synthesis, and establishment of infection using our molecular clone of DWV-A.

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Porcine kobuvirus (PKV) is an enteric virus commonly detected in both diarrheic and healthy pigs. Little is known about the role of PKV in enteric diseases. In this study, an epidemiological investigation based on 324 intestinal samples collected from six provinces of China during the period of 2018 to 2022 was performed, and showed that PKV has an overall 65.43% (212/324) positive rate. Noticeably, 89.47% (17/19) of PKV and porcine epidemic diarrhea virus (PEDV) double-positive pigs were clinically diseased, while 91.71% (177/193) of PKV-positive but PEDV-negative pigs were clinically healthy, suggesting that PKV infection in itself is unlikely to cause enteric diseases. In addition, three PKV genomes were obtained from both diseased and healthy pigs. Phylogenetic analysis showed that Chinese PKV strains could be divided into three groups (SH-W-CHN-like, S-1-HUN-like and JXAT2015-like strains). All three obtained PKV genomes belong to SH-W-CHN-like strains and JSYZ1806-158 was detected as a recombinant virus. Furthermore, multiple comparisons showed that nucleotide similarities are clearly lower than amino acid similarities for PKV polyproteins. Selective pressure analysis indicated that Chinese PKV polyproteins are predominantly under negative selection. Overall, this study provided new insights into the prevalence and evolution of PKV in both diarrheic and healthy pigs in China.

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The highly infectious African swine fever virus (ASFV) is currently the only known DNA arbovirus within the Asfarviridae family which primarily infects domestic pigs and wild boars. African swine fever (ASF) leads to a mortality rate of up to 100% which has caused massive socio-economic losses worldwide. Previous research indicates that ASFV's virulence can be attributed to polyprotein pp62, which plays a crucial role in viral assembly and core maturation. This particular study utilized in silico analysis to identify highly conserved cytotoxic T-cell epitopes in pp62 that can potentially serve as key components for future ASFV vaccines. To achieve this, the researchers retrieved, clustered, and aligned the peptide sequences of pp62. Subsequently, the aligned sequences were analyzed to identify epitopes that bind promiscuously to the swine major histocompatibility complex I (MHC I) alleles and exhibiting MHC IC50 values < 500 nM. Additionally, peptide sequences with positive proteasome and TAP scores were considered. Potential cross-reactivity was assessed by comparing the peptide sequences against available proteome sequences of Sus scrofa domesticus in various databases. Furthermore, molecular docking was conducted to evaluate the binding of candidate epitopes with swine leukocyte antigen-1*0401 (SLA-1*0401). The dissociation constants, binding energies, root mean square deviation, and root mean square fluctuation values for the SLA-epitope complexes were compared with a positive reference. In the course of the study, 21 highly conserved CD8+ epitopes were identified, out of which four were further assessed for their potential immunogenicity. The results demonstrated that the highly conserved CD8+ epitopes discovered in this study are promising for integration into future ASFV vaccine formulations. As preliminary data, it is anticipated that these findings will subsequently undergo in vitro and in vivo studies in the future.

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BACKGROUND: Viral infections continue to increase morbidity and mortality severely. The flavivirus genus has fifty different species, including the dengue, Zika, and West Nile viruses that can infect 40% of individuals globally, who reside in at least a hundred different countries. Dengue, one of the oldest and most dangerous human infections, was initially documented by the Chinese Medical Encyclopedia in the Jin period. It was referred to as "water poison," connected to flying insects, i.e., Aedes aegypti and Aedes albopictus. DENV causes some medical expressions like dengue hemorrhagic fever, acute febrile illness, and dengue shock syndrome. OBJECTIVE: According to the World Health Organization report of 2012, 2500 million people are in danger of contracting dengue fever worldwide. According to a recent study, 96 million of the 390 million dengue infections yearly show some clinical or subclinical severity. There is no antiviral drug or vaccine to treat this severe infection. It can be controlled by getting enough rest, drinking plenty of water, and using painkillers. The first dengue vaccine created by Sanofi, called Dengvaxia, was previously approved by the US-FDA in 2019. All four serotypes of the DENV1-4 have shown re-infection in vaccine recipients. However, the usage of Dengvaxia has been constrained by its adverse effects. CONCLUSION: Different classes of compounds have been reported against DENV, such as nitrogen-containing heterocycles (i.e., imidazole, pyridine, triazoles quinazolines, quinoline, and indole), oxygen-containing heterocycles (i.e., coumarins), and some are mixed heterocyclic compounds of S, N (thiazole, benzothiazine, and thiazolidinediones), and N, O (i.e., oxadiazole). There have been reports of computationally designed compounds to impede the molecular functions of specific structural and non-structural proteins as potential therapeutic targets. This review summarized the current progress in developing dengue protease inhibitors.

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Positive-strand RNA viruses use long open reading frames to express large polyproteins that are processed into individual proteins by viral proteases. Polyprotein processing is highly regulated and yields intermediate species with different functions than the fully processed proteins, increasing the biochemical diversity of the compact viral genome while also presenting challenges in that proteins must remain stably folded in multiple contexts. We have used circular dichroism spectroscopy and single molecule microscopy to examine the solution structure and self-association of the poliovirus P3 region protein composed of membrane binding 3A, RNA priming 3B (VPg), 3Cpro protease, and 3Dpol RNA-dependent RNA polymerase proteins. Our data indicate that co-folding interactions within the 3ABC segment stabilize the conformational state of the 3C protease region, and this stabilization requires the full-length 3A and 3B proteins. Enzymatic activity assays show that 3ABC is also an active protease, and it cleaves peptide substrates at rates comparable to 3Cpro. The cleavage of a larger polyprotein substrate is stimulated by the addition of RNA, and 3ABCpro becomes 20-fold more active than 3Cpro in the presence of stoichiometric amounts of viral cre RNA. The data suggest that co-folding within the 3ABC region results in a protease that can be highly activated toward certain cleavage sites by localization to specific RNA elements within the viral replication center, providing a mechanism for regulating viral polyprotein processing.

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Introducing nitrogen fixation (nif  ) genes into eukaryotic genomes and targeting Nif components to mitochondria or chloroplasts is a promising strategy for engineering nitrogen-fixing plants. A prerequisite for achieving nitrogen fixation in crops is stable and stoichiometric expression of each component in organelles. Previously, we designed a polyprotein-based nitrogenase system depending on Tobacco Etch Virus protease (TEVp) to release functional Nif components from five polyproteins. Although this system satisfies the demand for specific expression ratios of Nif components in Escherichia coli, we encountered issues with TEVp cleavage of polyproteins targeted to yeast mitochondria. To overcome this obstacle, a version of the Nif polyprotein system was constructed by replacing TEVp cleavage sites with minimal peptide sequences, identified by knowledge-based engineering, that are susceptible to cleavage by the endogenous mitochondrial-processing peptidase. This replacement not only further reduces the number of genes required, but also prevents potential precleavage of polyproteins outside the target organelle. This version of the polyprotein-based nitrogenase system achieved levels of nitrogenase activity in E. coli, comparable to those observed with the TEVp-based polyprotein nitrogenase system. When applied to yeast mitochondria, stable and balanced expression of Nif components was realized. This strategy has potential advantages, not only for transferring nitrogen fixation to eukaryotic cells, but also for the engineering of other metabolic pathways that require mitochondrial compartmentalization.

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Biomolecular condensates (BMCs) play an important role in the replication of a growing number of viruses, but many important mechanistic details remain to be elucidated. Previously, we demonstrated that the pan-retroviral nucleocapsid (NC) and HIV-1 pr55Gag (Gag) proteins phase separate into condensates, and that HIV-1 protease (PR)-mediated maturation of Gag and Gag-Pol precursor proteins yields self-assembling BMCs that have HIV-1 core architecture. Using biochemical and imaging techniques, we aimed to further characterize the phase separation of HIV-1 Gag by determining which of its intrinsically disordered regions (IDRs) influence the formation of BMCs, and how the HIV-1 viral genomic RNA (gRNA) could influence BMC abundance and size. We found that mutations in the Gag matrix (MA) domain or the NC zinc finger motifs altered condensate number and size in a salt-dependent manner. Gag BMCs were also bimodally influenced by the gRNA, with a condensate-promoting regime at lower protein concentrations and a gel dissolution at higher protein concentrations. Interestingly, incubation of Gag with CD4+ T cell nuclear lysates led to the formation of larger BMCs compared to much smaller ones observed in the presence of cytoplasmic lysates. These findings suggest that the composition and properties of Gag-containing BMCs may be altered by differential association of host factors in nuclear and cytosolic compartments during virus assembly. This study significantly advances our understanding of HIV-1 Gag BMC formation and provides a foundation for future therapeutic targeting of virion assembly.

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Many elastomeric proteins, which play important roles in a wide range of biological processes, exist as parallel/antiparallelly arranged dimers or multimers to perform their mechanobiological functions. For example, in striated muscle sarcomeres, the giant muscle protein titin exists as hexameric bundles to mediate the passive elasticity of muscles. However, it has not been possible to directly probe the mechanical properties of such parallelly arranged elastomeric proteins. And it remains unknown if the knowledge obtained from single-molecule force spectroscopy studies can be directly extrapolated to such parallelly/antiparallelly arranged systems. Here, we report the development of atomic force microscopy (AFM)-based two-molecule force spectroscopy to directly probe the mechanical properties of two elastomeric proteins that are arranged in parallel. We developed a twin-molecule approach to allow two parallelly arranged elastomeric proteins to be picked up and stretched simultaneously in an AFM experiment. Our results clearly revealed the mechanical features of such parallelly arranged elastomeric proteins during force-extension measurements and allowed for the determination of mechanical unfolding forces of proteins in such an experimental setting. Our study provides a general and robust experimental strategy to closely mimic the physiological condition of such parallel elastomeric protein multimers.

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The processing of the Coronavirus polyproteins pp1a and pp1ab by the main protease Mpro to produce mature proteins is a crucial event in virus replication and a promising target for antiviral drug development. Mpro cleaves polyproteins in a defined order, but how Mpro and/or the polyproteins determine the order of cleavage remains enigmatic due to a lack of structural information about polyprotein-bound Mpro. Here, we present the cryo-EM structures of SARS-CoV-2 Mpro in an apo form and in complex with the nsp7-10 region of the pp1a polyprotein. The complex structure shows that Mpro interacts with only the recognition site residues between nsp9 and nsp10, without any association with the rest of the polyprotein. Comparison between the apo form and polyprotein-bound structures of Mpro highlights the flexible nature of the active site region of Mpro, which allows it to accommodate ten recognition sites found in the polyprotein. These observations suggest that the role of Mpro in selecting a preferred cleavage site is limited and underscores the roles of the structure, conformation, and/or dynamics of the polyproteins in determining the sequence of polyprotein cleavage by Mpro.

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