Since the emergence of SARS-CoV-2, mutations in all subunits of the RNA-dependent RNA polymerase (RdRp) of the virus have been repeatedly reported. Although RdRp represents a primary target for antiviral drugs, experimental studies exploring the phenotypic effect of these mutations have been limited. This study focuses on the phenotypic effects of substitutions in the three RdRp subunits: nsp7, nsp8, and nsp12, selected based on their occurrence rate and potential impact. We employed nano-differential scanning fluorimetry and microscale thermophoresis to examine the impact of these mutations on protein stability and RdRp complex assembly. We observed diverse impacts; notably, a single mutation in nsp8 significantly increased its stability as evidenced by a 13°C increase in melting temperature, whereas certain mutations in nsp7 and nsp8 reduced their binding affinity to nsp12 during RdRp complex formation. Using a fluorometric enzymatic assay, we assessed the overall effect on RNA polymerase activity. We found that most of the examined mutations altered the polymerase activity, often as a direct result of changes in stability or affinity to the other components of the RdRp complex. Intriguingly, a combination of nsp8 A21V and nsp12 P323L mutations resulted in a 50% increase in polymerase activity. To our knowledge, this is the first biochemical study to demonstrate the impact of amino acid mutations across all components constituting the RdRp complex in emerging SARS-CoV-2 subvariants.

• Keywords
• RNA‐dependent RNA polymerase (RdRp), SARS‐CoV‐2, mutations, phenotypic effect,
• MeSH
• COVID-19 virology   MeSH
• Coronavirus RNA-Dependent RNA Polymerase * genetics   metabolism   chemistry   MeSH
• Humans MeSH
• Mutation * MeSH
• RNA-Dependent RNA Polymerase genetics   chemistry   metabolism   MeSH
• SARS-CoV-2 * genetics   enzymology   MeSH
• Protein Stability MeSH
• Protein Binding MeSH
• Viral Nonstructural Proteins * genetics   chemistry   metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Coronavirus RNA-Dependent RNA Polymerase * MeSH
• NS8 protein, SARS-CoV-2 MeSH Browser
• NSP12 protein, SARS-CoV-2 MeSH Browser
• NSP7 protein, SARS-CoV-2 MeSH Browser
• RNA-Dependent RNA Polymerase MeSH
• Viral Nonstructural Proteins * MeSH

For most retroviruses, including HIV, association with the plasma membrane (PM) promotes the assembly of immature particles, which occurs simultaneously with budding and maturation. In these viruses, maturation is initiated by oligomerization of polyprotein precursors. In contrast, several retroviruses, such as Mason-Pfizer monkey virus (M-PMV), assemble in the cytoplasm into immature particles that are transported across the PM. Therefore, protease activation and specific cleavage must not occur until the pre-assembled particle interacts with the PM. This interaction is triggered by a bipartite signal consisting of a cluster of basic residues in the matrix (MA) domain of Gag polyprotein and a myristoyl moiety N-terminally attached to MA. Here, we provide evidence that myristoyl exposure from the MA core and its insertion into the PM occurs in M-PMV. By a combination of experimental methods, we show that this results in a structural change at the C-terminus of MA allowing efficient cleavage of MA from the downstream region of Gag. This suggests that, in addition to the known effect of the myristoyl switch of HIV-1 MA on the multimerization state of Gag and particle assembly, the myristoyl switch may have a regulatory role in initiating sequential cleavage of M-PMV Gag in immature particles.

• Keywords
• betaretrovirus, infectious disease, matrix protein, maturation, microbiology, myristoyl switch, protease, viruses,
• MeSH
• Cell Membrane MeSH
• Endopeptidases MeSH
• Gene Products, gag chemistry   MeSH
• Mason-Pfizer monkey virus * chemistry   physiology   MeSH
• Proteins MeSH
• Virus Assembly MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Endopeptidases MeSH
• Gene Products, gag MeSH
• Proteins MeSH

Approximately one third of children with steroid-resistant nephrotic syndrome (SRNS) carry pathogenic variants in one of the many associated genes. The WT1 gene coding for the WT1 transcription factor is among the most frequently affected genes. Cases from the Czech national SRNS database were sequenced for exons 8 and 9 of the WT1 gene. Eight distinct exonic WT1 variants in nine children were found. Three children presented with isolated SRNS, while the other six manifested with additional features. To analyze the impact of WT1 genetic variants, wild type and mutant WT1 proteins were prepared and the DNA-binding affinity of these proteins to the target EGR1 sequence was measured by microscale thermophoresis. Three WT1 mutants showed significantly decreased DNA-binding affinity (p.Arg439Pro, p.His450Arg and p.Arg463Ter), another three mutants showed significantly increased binding affinity (p.Gln447Pro, p.Asp469Asn and p.His474Arg), and the two remaining mutants (p.Cys433Tyr and p.Arg467Trp) showed no change of DNA-binding affinity. The protein products of WT1 pathogenic variants had variable DNA-binding affinity, and no clear correlation with the clinical symptoms of the patients. Further research is needed to clarify the mechanisms of action of the distinct WT1 mutants; this could potentially lead to individualized treatment of a so far unfavourable disease.

• MeSH
• Child MeSH
• DNA therapeutic use   MeSH
• Drug Resistance MeSH
• Humans MeSH
• Mutation MeSH
• Nephrotic Syndrome * drug therapy   genetics   metabolism   MeSH
• WT1 Proteins * genetics   metabolism   MeSH
• Steroids pharmacology   MeSH
• Check Tag
• Child MeSH
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• DNA MeSH
• WT1 Proteins * MeSH
• Steroids MeSH
• WT1 protein, human MeSH Browser

Proper assembly and disassembly of both immature and mature HIV-1 hexameric lattices are critical for successful viral replication. These processes are facilitated by several host-cell factors, one of which is myo-inositol hexaphosphate (IP6). IP6 participates in the proper assembly of Gag into immature hexameric lattices and is incorporated into HIV-1 particles. Following maturation, IP6 is also likely to participate in stabilizing capsid protein-mediated mature hexameric lattices. Although a structural-functional analysis of the importance of IP6 in the HIV-1 life cycle has been reported, the effect of IP6 has not yet been quantified. Using two in vitro methods, we quantified the effect of IP6 on the assembly of immature-like HIV-1 particles, as well as its stabilizing effect during disassembly of mature-like particles connected with uncoating. We analyzed a broad range of molar ratios of protein hexamers to IP6 molecules during assembly and disassembly. The specificity of the IP6-facilitated effect on HIV-1 particle assembly and stability was verified by K290A, K359A, and R18A mutants. In addition to IP6, we also tested other polyanions as potential assembly cofactors or stabilizers of viral particles.IMPORTANCE Various host cell factors facilitate critical steps in the HIV-1 replication cycle. One of these factors is myo-inositol hexaphosphate (IP6), which contributes to assembly of HIV-1 immature particles and helps maintain the well-balanced metastability of the core in the mature infectious virus. Using a combination of two in vitro methods to monitor assembly of immature HIV-1 particles and disassembly of the mature core-like structure, we quantified the contribution of IP6 and other small polyanion molecules to these essential steps in the viral life cycle. Our data showed that IP6 contributes substantially to increasing the assembly of HIV-1 immature particles. Additionally, our analysis confirmed the important role of two HIV-1 capsid lysine residues involved in interactions with IP6. We found that myo-inositol hexasulphate also stabilized the HIV-1 mature particles in a concentration-dependent manner, indicating that targeting this group of small molecules may have therapeutic potential.

• Keywords
• HIV-1, IP6, assembly, capsid, immature, mature, polyanion,
• MeSH
• gag Gene Products, Human Immunodeficiency Virus chemistry   genetics   metabolism   MeSH
• HIV-1 chemistry   genetics   MeSH
• Mutation, Missense MeSH
• Polyelectrolytes MeSH
• Polymers chemistry   MeSH
• Virus Assembly * MeSH
• Amino Acid Substitution MeSH
• Structure-Activity Relationship MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• gag Gene Products, Human Immunodeficiency Virus MeSH
• polyanions MeSH Browser
• Polyelectrolytes MeSH
• Polymers MeSH

Shortly after entering the cell, HIV-1 copies its genomic RNA into double-stranded DNA in a process known as reverse transcription. This process starts inside a core consisting of an enclosed lattice of capsid proteins that protect the viral RNA from cytosolic sensors and degradation pathways. To accomplish reverse transcription and integrate cDNA into the host cell genome, the capsid shell needs to be disassembled, or uncoated. Premature or delayed uncoating attenuates reverse transcription and blocks HIV-1 infectivity. Small molecules that bind to the capsid lattice of the HIV-1 core and either destabilize or stabilize its structure could thus function as effective HIV-1 inhibitors. To screen for such compounds, we modified our recently developed FAITH assay to allow direct assessment of the stability of in vitro preassembled HIV-1 capsid-nucleocapsid (CANC) tubular particles. This new assay is a high-throughput fluorescence method based on measuring the amount of nucleic acid released from CANC complexes under disassembly conditions. The amount of disassembled CANC particles and released nucleic acid is proportional to the fluorescence signal, from which the relative percentage of CANC stability can be calculated. We consider our assay a potentially powerful tool for in vitro screening for compounds that alter HIV disassembly.

• MeSH
• HIV Infections drug therapy   MeSH
• HIV-1 drug effects   physiology   MeSH
• Anti-HIV Agents chemistry   isolation & purification   pharmacology   MeSH
• Humans MeSH
• Nucleocapsid analysis   drug effects   MeSH
• Viral Core Proteins chemistry   genetics   metabolism   MeSH
• RNA, Viral genetics   MeSH
• High-Throughput Screening Assays MeSH
• Amino Acid Sequence MeSH
• Base Sequence MeSH
• Virus Uncoating drug effects   genetics   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Anti-HIV Agents MeSH
• Viral Core Proteins MeSH
• RNA, Viral MeSH

Retrovirus assembly is driven mostly by Gag polyprotein oligomerization, which is mediated by inter and intra protein-protein interactions among its capsid (CA) domains. Mason-Pfizer monkey virus (M-PMV) CA contains three cysteines (C82, C193 and C213), where the latter two are highly conserved among most retroviruses. To determine the importance of these cysteines, we introduced mutations of these residues in both bacterial and proviral vectors and studied their impact on the M-PMV life cycle. These studies revealed that the presence of both conserved cysteines of M-PMV CA is necessary for both proper assembly and virus infectivity. Our findings suggest a crucial role of these cysteines in the formation of infectious mature particles.

• Keywords
• Cysteine mutagenesis, M-PMV capsid, M-PMV infectivity, Retrovirus assembly, Virus core stability,
• MeSH
• Cell Line MeSH
• Cysteine genetics   MeSH
• Genetic Vectors MeSH
• HEK293 Cells MeSH
• Humans MeSH
• Mason-Pfizer monkey virus genetics   physiology   MeSH
• Mutation MeSH
• Proviruses genetics   MeSH
• Virus Assembly * MeSH
• Virion physiology   MeSH
• Capsid Proteins chemistry   genetics   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Extramural MeSH
• Names of Substances
• Cysteine MeSH
• Capsid Proteins MeSH

As we have shown previously, the Cu and Ag concentrations in the sporocarps of Ag-hyperaccumulating Amanita strobiliformis are correlated, and both metals share the same uptake system and are sequestered by the same metallothioneins intracellularly. To further improve our knowledge of the Cu and Ag handling in A. strobiliformis cells, we searched its transcriptome for the P1B-1-ATPases, recognizing Cu+ and Ag+ for transport. We identified transcripts encoding 1097-amino acid (AA) AsCRD1 and 978-AA AsCCC2, which were further subjected to functional studies in metal sensitive Saccharomyces cerevisiae. The expression of AsCRD1 conferred highly increased Cu and Ag tolerance to metal sensitive yeasts in which the functional AsCRD1:GFP (green fluorescent protein) fusion localized exclusively to the tonoplast, indicating that the AsCRD1-mediated Cu and Ag tolerance was a result of vacuolar sequestration of the metals. Increased accumulation of AsCRD1 transcripts observed in A. strobiliformis mycelium upon the treatments with Cu and Ag (8.7- and 4.5-fold in the presence of 5 μM metal, respectively) supported the notion that AsCRD1 can be involved in protection of the A. strobiliformis cells against the toxicity of both metals. Neither Cu nor Ag affected the levels of AsCCC2 transcripts. Heterologous expression of AsCCC2 in mutant yeasts did not contribute to Cu tolerance, but complemented the mutant genotype of the S. cerevisiae ccc2Δ strain. Consistent with the role of the yeast Ccc2 in the trafficking of Cu from cytoplasm to nascent proteins via post-Golgi, the GFP fluorescence in AsCCC2-expressing ccc2Δ yeasts localized among Golgi-like punctate foci within the cells. The AsCRD1- and AsCCC2-associated phenotypes were lost in yeasts expressing mutant transporter variants in which a conserved phosphorylation/dephosphorylation site was altered. Altogether, the data support the roles of AsCRD1 and AsCCC2 as genuine P1B-1-ATPases, and indicate their important functions in the removal of toxic excess of Cu and Ag from the cytoplasm and charging the endomembrane system with Cu, respectively.

• Keywords
• Amanita strobiliformis, P1-type ATPase, copper transporter, ectomycorrhizal fungi, metal homeostasis, silver transporter,
• Publication type
• Journal Article MeSH