IC-Tagging and Protein Relocation to ARV muNS Inclusions: A Method to Study Protein-Protein Interactions in the Cytoplasm or Nucleus of Living Cells

Background: Characterization of protein-protein interactions is essential for understanding cellular functions. Although there are many published methods to analyze protein-protein interactions, most of them present serious limitations. In a different study we have characterized a novel avian reovirus muNS-based protein tagging and inclusion targeting method, and demonstrated its validity to purify free an immobilized protein. Methodology/Principal Findings: Here we present a method to identify protein-protein interactions inside living eukaryotic cells (tested in primate and avian cells). When p53 was tagged with Intercoil (IC; muNS residues 477–542), it not only got integrated into muNS cytoplasmic inclusions, but also attracted its known ligand SV40 large T antigen (TAg) to these structures. We have also adapted this system to work within the cell nucleus, by creating muNS-related protein chimeras that form nuclear inclusions. We show that nuclear muNS-derived inclusions are as efficient as cytoplasmic ones in capturing IC-tagged proteins, and that the proteins targeted to nuclear inclusions are able to interact with their known ligands. Conclusions/Significance: Our protein redistribution method does not present the architectural requirement of reconstructing a transcription factor as any of the two-hybrid systems do. The method is simple and requires only cell transfection and a fluorescence microscope. Our tagging method can be used either in the cytoplasm or the nucleus o

A Versatile Molecular Tagging Method for Targeting Proteins to Avian Reovirus muNS Inclusions. Use in Protein Immobilization and Purification

Background: Avian reoviruses replicate in viral factories, which are dense cytoplasmic compartments estabilished by protein-protein interactions. The non-structural protein muNS forms the factory scaffold that attracts other viral components in a controlled fashion. To create such a three-dimensional network, muNS uses several different selfinteracting domains. Methodology/Principal Findings: In this study we have devised a strategy to identify muNS regions containing selfinteracting domains, based on the capacity of muNS-derived inclusions to recruit muNS fragments. The results revealed that the muNS region consisting of residues 477–542 was recruited with the best efficiency, and this raised the idea of using this fragment as a molecular tag for delivering foreign proteins to muNS inclusions. By combining such tagging system with our previously established method for purifying muNS inclusions from baculovirus-infected insect cells, we have developed a novel protein purification protocol. Conclusions/Significance: We show that our tagging and inclusion-targeting system can be a simple, versatile and efficient method for immobilizing and purifying active proteins expressed in baculovirus-infected cells. We also demonstrate that muNS inclusions can simultaneously recruit several tagged proteins, a finding which may be used to generate protei

The Retroviral Restriction Ability of SAMHD1, but Not Its Deoxynucleotide Triphosphohydrolase Activity, Is Regulated by Phosphorylation

SummarySAMHD1 is a cellular enzyme that depletes intracellular deoxynucleoside triphosphates (dNTPs) and inhibits the ability of retroviruses, notably HIV-1, to infect myeloid cells. Although SAMHD1 is expressed in both cycling and noncycling cells, the antiviral activity of SAMHD1 is limited to noncycling cells. We determined that SAMHD1 is phosphorylated on residue T592 in cycling cells but that this phosphorylation is lost when cells are in a noncycling state. Reverse genetic experiments revealed that SAMHD1 phosphorylated on residue T592 is unable to block retroviral infection, but this modification does not affect the ability of SAMHD1 to decrease cellular dNTP levels. SAMHD1 contains a target motif for cyclin-dependent kinase 1 (cdk1) (592TPQK595), and cdk1 activity is required for SAMHD1 phosphorylation. Collectively, these findings indicate that phosphorylation modulates the ability of SAMHD1 to block retroviral infection without affecting its ability to decrease cellular dNTP levels

Pressurized DNA state inside herpes capsids-A novel antiviral target

Drug resistance in viruses represents one of the major challenges of healthcare. As part of an effort to provide a treatment that avoids the possibility of drug resistance, we discovered a novel mechanism of action (MOA) and specific compounds to treat all nine human herpesviruses and animal herpesviruses. The novel MOA targets the pressurized genome state in a viral capsid, "turns off" capsid pressure, and blocks viral genome ejection into a cell nucleus, preventing viral replication. This work serves as a proof-of-concept to demonstrate the feasibility of a new antiviral target-suppressing pressure-driven viral genome ejection-that is likely impervious to developing drug resistance. This pivotal finding presents a platform for discovery of a new class of broad-spectrum treatments for herpesviruses and other viral infections with genome-pressure-dependent replication. A biophysical approach to antiviral treatment such as this is also a vital strategy to prevent the spread of emerging viruses where vaccine development is challenged by high mutation rates or other evasion mechanisms

Role of SAMHD1 nuclear localization in restriction of HIV-1 and SIVmac

International audienceBackground : SAMHD1 is a nuclear protein that blocks lentiviral infection before reverse transcription inmacrophages and dendritic cells. The viral accessory protein Vpx overcomes the SAMHD1-mediated lentiviral blockby inducing its proteasomal degradation.Results : Here, we identified the nuclear localization signal (NLS) of SAMHD1, and studied its contribution torestriction of HIV-1 and SIVmac. By studying the cellular distribution of different SAMHD1 variants, we mapped thenuclear localization of SAMHD1 to residues11KRPR14. Mutagenesis of these residues changed the cellulardistribution of SAMHD1 from the nucleus to the cytoplasm. SAMHD1 mutants that lost nuclear localizationrestricted HIV-1 and SIV as potently as the wild type protein. Interestingly, SAMHD1 mutants that localized to thecytoplasm were not degraded by nuclear Vpx alleles. Therefore, nuclear Vpx alleles require nuclear localization ofSAMHD1 in order to induce its degradation. In agreement, SIVmac viruses encoding Vpx did not overcome therestriction imposed by the cytoplasmic variants of SAMHD1.Conclusions : We mapped the NLS of SAMHD1 to residues11KRPR14and studied the contribution of SAMHD1nuclear localization to restriction of HIV-1 and SIV. These experiments demonstrate that cytoplasmic variants ofSAMHD1 potently block lentiviral infection and are resistant to Vpx-mediated degradation. The nuclear Vpx allelesstudied here are only capable of degrading a nuclearly localized SAMHD1 suggesting that Vpx-mediateddegradation of SAMHD1 is initiated in the nucleus

Pressure-driven release of viral genome into a host nucleus is a mechanism leading to herpes infection

Many viruses previously have been shown to have pressurized genomes inside their viral protein shell, termed the capsid. This pressure results from the tight confinement of negatively charged viral nucleic acids inside the capsid. However, the relevance of capsid pressure to viral infection has not been demonstrated. In this work, we show that the internal DNA pressure of tens of atmospheres inside a herpesvirus capsid powers ejection of the viral genome into a host cell nucleus. To our knowledge, this provides the first demonstration of a pressure-dependent mechanism of viral genome penetration into a host nucleus, leading to infection of eukaryotic cells

Avian Reovirus μNS Protein Forms Homo-Oligomeric Inclusions in a Microtubule-Independent Fashion, Which Involves Specific Regions of Its C-Terminal Domain▿

Members of the genus Orthoreovirus replicate in cytoplasmic inclusions termed viral factories. Compelling evidence suggests that the nonstructural protein μNS forms the matrix of the factories and recruits specific viral proteins to these structures. In the first part of this study, we analyzed the properties of avian reovirus factories and μNS-derived inclusions and found that they are nonaggresome cytoplasmic globular structures not associated with the cytoskeleton which do not require an intact microtubule network for formation and maturation. We next investigated the capacity of avian reovirus μNS to form inclusions in transfected and baculovirus-infected cells. Our results showed that μNS is the main component of the inclusions formed by recombinant baculovirus expression. This, and the fact that μNS is able to self-associate inside the cell, suggests that μNS monomers contain all the interacting domains required for inclusion formation. Examination of the inclusion-forming capacities of truncated μNS versions allowed us to identify the region spanning residues 448 to 635 of μNS as the smallest that was inclusion competent, although residues within the region 140 to 380 seem to be involved in inclusion maturation. Finally, we investigated the roles that four different motifs present in μNS(448-635) play in inclusion formation, and the results suggest that the C-terminal tail domain is a key determinant in dictating the initial orientation of monomer-to-monomer contacts to form basal oligomers that control inclusion shape and inclusion-forming efficiency. Our results contribute to an understanding of the generation of structured protein aggregates that escape the cellular mechanisms of protein recycling

Incorporation of HA-tagged muNS regions into muNS or muNS-Mi-derived inclusions in transfected cells.

<p><b>A</b>. muNS inclusions. Full-length muNS is schematically indicated by a horizontal black bar comprising residues 1–635 and regions 1 to 5 are also indicated. Horizontal black bars represent each single muNS fragment generated, with the HA epitope indicated as a small red box. The positions of two previously described coiled-coil elements predicted in the muNS sequence are indicated by two grey boxes and by vertical grey bars. Each construct was expressed alone (−muNS) or co-expressed with muNS (+muNS), and representative immunofluorescence images of transfected CEF cells are shown at the right side of the Figure. The HA epitope was detected by immunofluorescence (red) and nuclei were stained blue with DAPI. <b>B</b>. muNS-Mi inclusions. As in A, but the indicated constructs were expressed alone (−muNS-Mi) or co-expressed with muNS-Mi (+muNS-Mi). In the 1+2 image, the inset is an enlargement of the boxed area.</p