The Rous sarcoma virus Gag Polyprotein Forms Biomolecular Condensates Driven by Intrinsically-disordered Regions.

Biomolecular condensates (BMCs) play important roles incellular structures includingtranscription factories, splicing speckles, and nucleoli. BMCs bring together proteins and other macromolecules, selectively concentrating them so that specific reactions can occur without interference from the surrounding environment. BMCs are often made up of proteins that contain intrinsically disordered regions (IDRs), form phase-separated spherical puncta, form liquid-like droplets that undergo fusion and fission, contain molecules that are mobile, and are disrupted with phase-dissolving drugs such as 1,6-hexanediol. In addition to cellular proteins, many viruses, including influenza A, SARS-CoV-2, and human immunodeficiency virus type 1 (HIV-1) encode proteins that undergo phase separation and rely on BMC formation for replication. In prior studies of the retrovirus Rous sarcoma virus (RSV), we observed that the Gag protein forms discrete spherical puncta in the nucleus, cytoplasm, and at the plasma membrane that co-localize with viral RNA and host factors, raising the possibility that RSV Gag forms BMCs that participate in the intracellular phase of the virion assembly pathway. In our current studies, we found that Gag contains IDRs in the N-terminal (MAp2p10) and C-terminal (NC) regions of the protein and fulfills many criteria of BMCs. Although the role of BMC formation in RSV assembly requires further study, our results suggest the biophysical properties of condensates are required for the formation of Gag complexes in the nucleus and the cohesion of these complexes as they traffic through the nuclear pore, into the cytoplasm, and to the plasma membrane, where the final assembly and release of virus particles occurs.

The Rous sarcoma virus Gag polyprotein forms biomolecular condensates driven by intrinsically-disordered regions.

Biomolecular condensates (BMCs) play important roles in cellular structures including transcription factories, splicing speckles, and nucleoli. BMCs bring together proteins and other macromolecules, selectively concentrating them so that specific reactions can occur without interference from the surrounding environment. BMCs are often made up of proteins that contain intrinsically disordered regions (IDRs), form phase-separated spherical puncta, form liquid-like droplets that undergo fusion and fission, contain molecules that are mobile, and are disrupted with phase-dissolving drugs such as 1,6-hexanediol. In addition to cellular proteins, many viruses, including influenza A, SARS-CoV-2, and human immunodeficiency virus type 1 (HIV-1) encode proteins that undergo phase separation and rely on BMC formation for replication. In prior studies of the retrovirus Rous sarcoma virus (RSV), we observed that the Gag protein forms discrete spherical puncta in the nucleus, cytoplasm, and at the plasma membrane that co-localize with viral RNA and host factors, raising the possibility that RSV Gag forms BMCs that participate in the virion intracellular assembly pathway. In our current studies, we found that Gag contains IDRs in the N-terminal (MAp2p10) and C-terminal (NC) regions of the protein and fulfills many criteria of BMCs. Although the role of BMC formation in RSV assembly requires further study, our results suggest the biophysical properties of condensates are required for the formation of Gag complexes in the nucleus and the cohesion of these complexes as they traffic through the nuclear pore, into the cytoplasm, and to the plasma membrane, where the final assembly and release of virus particles occurs.

Monoclonal Antibodies to S and N SARS-CoV-2 Proteins as Probes to Assess Structural and Antigenic Properties of Coronaviruses.

Antibodies targeting the spike (S) and nucleocapsid (N) proteins of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are essential tools. In addition to important roles in the treatment and diagnosis of infection, the availability of high-quality specific antibodies for the S and N proteins is essential to facilitate basic research of virus replication and in the characterization of mutations responsible for variants of concern. We have developed panels of mouse and rabbit monoclonal antibodies (mAbs) to the SARS-CoV-2 spike receptor-binding domain (S-RBD) and N protein for functional and antigenic analyses. The mAbs to the S-RBD were tested for neutralization of native SARS-CoV-2, with several exhibiting neutralizing activity. The panels of mAbs to the N protein were assessed for cross-reactivity with the SARS-CoV and Middle East respiratory syndrome (MERS)-CoV N proteins and could be subdivided into sets that showed unique specificity for SARS-CoV-2 N protein, cross-reactivity between SARS-CoV-2 and SARS-CoV N proteins only, or cross-reactivity to all three coronavirus N proteins tested. Partial mapping of N-reactive mAbs were conducted using truncated fragments of the SARS-CoV-2 N protein and revealed near complete coverage of the N protein. Collectively, these sets of mouse and rabbit monoclonal antibodies can be used to examine structure/function studies for N proteins and to define the surface location of virus neutralizing epitopes on the RBD of the S protein.

Systematic review of adjunct therapies to improve outcomes following botulinum toxin injection for treatment of limb spasticity.

OBJECTIVE: To determine the quality of evidence from randomized controlled trials on the efficacy of adjunct therapies following botulinum toxin injections for limb spasticity. DATA SOURCES: MEDLINE, EMBASE, CINAHL, and Cochrane Central Register of Controlled Trials electronic databases were searched for English language human studies from 1980 to 21 May 2015. STUDY SELECTION: Randomized controlled trials assessing adjunct therapies postbotulinum toxin injection for treatment of spasticity were included. Of the 268 studies screened, 17 met selection criteria. DATA EXTRACTION: Two reviewers independently assessed risk of bias using the Physiotherapy Evidence Database (PEDro) scale and graded according to Sackett's levels of evidence. DATA SYNTHESIS: Ten adjunct therapies were identified. Evidence suggests that adjunct use of electrical stimulation, modified constraint-induced movement therapy, physiotherapy (all Level 1), casting and dynamic splinting (both Level 2) result in improved Modified Ashworth Scale scores by at least 1 grade. There is Level 1 and 2 evidence that adjunct taping, segmental muscle vibration, cyclic functional electrical stimulation, and motorized arm ergometer may not improve outcomes compared with botulinum toxin injections alone. There is Level 1 evidence that casting is better than taping, taping is better than electrical stimulation and stretching, and extracorporeal shock wave therapy is better than electrical stimulation for outcomes including the Modified Ashworth Scale, range of motion and gait. All results are based on single studies. CONCLUSION: There is high level evidence to suggest that adjunct therapies may improve outcomes following botulinum toxin injection. No results have been confirmed by independent replication. All interventions would benefit from further study.

Alternative nucleophilic substrates for the endonuclease activities of human immunodeficiency virus type 1 integrase.

Retroviral integrase can use water or some small alcohols as the attacking nucleophile to nick DNA. To characterize the range of compounds that human immunodeficiency virus type 1 integrase can accommodate for its endonuclease activities, we tested 45 potential electron donors (having varied size and number or spacing of nucleophilic groups) as substrates during site-specific nicking at viral DNA ends and during nonspecific nicking reactions. We found that integrase used 22 of the 45 compounds to nick DNA, but not all active compounds were used for both activities. In particular, 13 compounds were used for site-specific and nonspecific nicking, 5 only for site-specific nicking, and 4 only for nonspecific nicking; 23 other compounds were not used for either activity. Thus, integrase can accommodate a large number of nucleophilic substrates but has selective requirements for its different activities, underscoring its dynamic properties and providing new information for modeling and understanding integrase.

Evaluation of a system to screen for stimulators of non-specific DNA nicking by HIV-1 integrase: application to a library of 50,000 compounds.

BACKGROUND: In addition to activities needed to catalyse integration, retroviral integrases exhibit non-specific endonuclease activity that is enhanced by certain small compounds, suggesting that integrase could be stimulated to damage viral DNA before integration occurs. METHODS: A non-radioactive, plate-based, solution phase, fluorescence assay was used to screen a library of 50,080 drug-like chemicals for stimulation of non-specific DNA nicking by HIV-1 integrase. RESULTS: A semi-automated workflow was established and primary hits were readily identified from a graphic output. Overall, 0.6% of the chemicals caused a large increase in fluorescence (the primary hit rate) without also having visible colour that could have artifactually caused this result. None of the potential stimulators from this moderate-size library, however, passed a secondary test that included an inactive integrase mutant that assessed whether the increased fluorescence depended on the endonuclease activity of integrase. CONCLUSIONS: This first attempt at identifying integrase stimulator compounds establishes the necessary logistics and workflow required. The results from this study should encourage larger scale high-throughput screening to advance the novel antiviral strategy of stimulating integrase to damage retroviral DNA.

A nonradioactive plate-based assay for stimulators of nonspecific DNA nicking by HIV-1 integrase and other nucleases.

Retroviral integrase enzymes have a nonspecific endonuclease activity that is stimulated by certain compounds, suggesting that integrase could be manipulated to damage viral DNA. To identify integrase stimulator (IS) compounds as potential antiviral agents, we have developed a nonradioactive assay that is suitable for high-throughput screening. The assay uses a 49-mer oligonucleotide that is 5'-labeled with a fluorophore, 3'-tagged with a quencher, and designed to form a hairpin that mimics radioactive double-stranded substrates in gel-based nicking assays. Reactions in 384-well plates are analyzed on a real-time PCR machine after a single heat denaturation and subsequent cooling to a point between the melting temperatures of unnicked substrate and nicked products (no cycling is required). Under these conditions, unnicked DNA reforms the hairpin and quenches fluorescence, whereas completely nicked DNA yields a large signal. The assay was linear with time, stimulator concentration, and amount of integrase, and 20% concentrations of the solvent used for many chemical libraries did not interfere with the assay. The assay had an excellent Z' factor, and it reliably detected known IS compounds. This assay, which is adaptable to other nonspecific nucleases, will be useful for identifying additional IS compounds to develop the novel antiviral strategy of stimulating integrase to destroy retroviral DNA.

Identifying amino acid residues that contribute to the cellular-DNA binding site on retroviral integrase.

Although retroviral integrase specifically trims the ends of viral DNA and inserts these ends into any sequence in cellular DNA, little information is available to explain how integrase distinguishes between its two DNA substrates. We recently described novel integrase mutants that were improved for specific nicking of viral DNA but impaired at joining these ends into nonviral DNA. An acidic or bulky substitution at one particular residue was critical for this activity profile, and the prototypic protein--Rous sarcoma virus integrase with an S124D substitution--was defective at nonspecifically binding DNA. We have now characterized 19 (including 16 new) mutants that contain one or more aspartic acid substitutions at residues that extend over the surface of the protein and might participate with residue 124 in binding cellular DNA. In particular, every mutant with an aspartate substitution at residue 98 or 128, similar to the original S124D protein, showed improved specific nicking of viral DNA but disturbed nonspecific nicking of nonviral DNA. These data describe a probable cellular-DNA binding platform that involves at least 5 amino acids, in the following order of importance: 124>128>(98, 125)>123. These experimental data are vital for new models of integrase and will contribute to identifying targets for the next generation of integrase inhibitors.

Effects of varying the spacing within the D,D-35-E motif in the catalytic region of retroviral integrase.

The catalytic domain of all retroviral integrases contains an Asp,Asp-35-Glu (D,D-35-E) motif with precisely 35 amino acids between the second aspartate and the glutamate. We have now made several mutations designed to alter the length or flexibility of a mobile loop within this 35-amino-acid spacer region in full-length Rous sarcoma virus integrase. Surprisingly, most of the mutants had enzymatic activity, including ones that shortened or lengthened the loop by up to 6 amino acids. Several size mutants exhibited the two biologically relevant activities of integrase in reactions with Mn(2+), although they were inactive with Mg(2+). No viruses containing integrase with an altered length, however, replicated in cell culture, and these viruses were blocked at the integration step. Thus, the conserved 35-amino-acid spacing is not absolutely required for enzymatic activity, but the correlation between infectivity and Mg(2+)-dependent activity supports magnesium as the metal cofactor used by integrase in vivo.

Retroviral integrases that are improved for processing but impaired for joining.

Retroviral integrase specifically trims (or processes) the ends of retroviral DNA, then inserts (or joins) these ends into cellular DNA nonspecifically. We previously showed that Rous sarcoma virus integrase with a serine-to-aspartate substitution at amino acid 124 was markedly improved for processing but dramatically impaired for joining, making it the first mutant to separate the activities of integrase in this way. We now show that placing glutamic acid at this residue has the same effect, whereas asparagine or glutamine, which resemble aspartate and glutamate but without the negatively charged acid group, improved processing and impaired joining to a lesser extent. Placing aspartic acid at either of the adjacent residues 123 or 125 also had an intermediate effect. Thus, the charge, structure, and position of the substitution all contribute to the properties of the S124D protein. Infectivity of virions containing these mutations paralleled the in vitro findings, with substitutions having the greatest effect on joining completely blocking replication. Additional studies indicated the replication-defective viruses were blocked at integration and that the S124D protein is impaired at binding nonviral DNA. These functional, biochemical, and genetic data implicate this particular integrase residue as a key part of the binding site for cellular DNA.

A substitution in rous sarcoma virus integrase that separates its two biologically relevant enzymatic activities.

Retroviral integrase prepares viral DNA for integration by removing 2 nucleotides from each end of unintegrated DNA in a reaction referred to as processing. However, it has been known since the processing assay was first described that avian integrases frequently nick 3 nucleotides, as well as 2 nucleotides, from viral DNA ends when reaction mixtures contain Mn2+. We now report that specificity for the biologically relevant "-2" site is enhanced when the serine at amino acid 124 of Rous sarcoma virus (RSV) integrase is replaced by alanine, valine, glycine, lysine, or aspartate. The protein with a serine-to-aspartate substitution exhibited especially high fidelity for the correct site, as evidenced by a ratio of -2 nicks to -3 nicks that was more than 40-fold greater than that for the wild-type enzyme in reactions with Mn2+. Even with Mg2+, the substituted proteins exhibited greater specificity than the wild type, especially the S124D protein. Moreover, this protein was more efficient than the wild type at processing viral DNA ends. Unexpectedly, however, the S124D protein was significantly impaired at catalyzing the insertion of viral DNA ends in reactions with Mn2+ and joining was undetectable in reactions with Mg2+. Thus, the S124D protein has separated the processing and joining activities of integrase. Similar results were found for human immunodeficiency virus integrase with the analogous substitution. No proteins with comparable properties have been described. Moreover, RSV virions containing integrase with the S124D mutation were unable to replicate in cell cultures. Together, these data suggest that integrase has evolved to have submaximal processing activity so that it can also catalyze DNA joining.

An amino acid in the central catalytic domain of three retroviral integrases that affects target site selection in nonviral DNA.

Integrase can insert retroviral DNA into almost any site in cellular DNA; however, target site preferences are noted in vitro and in vivo. We recently demonstrated that amino acid 119, in the alpha2 helix of the central domain of the human immunodeficiency virus type 1 integrase, affected the choice of nonviral target DNA sites. We have now extended these findings to the integrases of a nonprimate lentivirus and a more distantly related alpharetrovirus. We found that substitutions at the analogous positions in visna virus integrase and Rous sarcoma virus integrase changed the target site preferences in five assays that monitor insertion into nonviral DNA. Thus, the importance of this protein residue in the selection of nonviral target DNA sites is likely to be a general property of retroviral integrases. Moreover, this amino acid might be part of the cellular DNA binding site on integrase proteins.