Characterization of nsp1 Binding to the Viral RNA Leader Sequence of Severe Acute Respiratory Syndrome Coronavirus.

Nonstructural protein 1 (nsp1) of the severe acute respiratory syndrome coronavirus (SCOV1 and SCOV2) acts as a host shutoff protein by blocking the translation of host mRNAs and triggering their decay. Surprisingly, viral RNA, which resembles host mRNAs containing a 5'-cap and a 3'-poly(A) tail, escapes significant translation inhibition and RNA decay, aiding viral propagation. Current literature proposes that, in SCOV2, nsp1 binds the viral RNA leader sequence, and the interaction may serve to distinguish viral RNA from host mRNA. However, a direct binding between SCOV1 nsp1 and the corresponding RNA leader sequence has not been established yet. Here, we show that SCOV1 nsp1 binds to the SCOV1 RNA leader sequence but forms multiple complexes at a high concentration of nsp1. These complexes are marginally different from complexes formed with SCOV2 nsp1. Finally, mutations of the RNA stem-loop did not completely abolish RNA binding by nsp1, suggesting that an RNA secondary structure is more important for binding than the sequence itself. Understanding the nature of binding of nsp1 to viral RNA will allow us to understand how this viral protein selectively suppresses host gene expression.

Proximity-dependent biotinylation detects associations between SARS coronavirus nonstructural protein 1 and stress granule-associated proteins.

The nonstructural protein 1 (nsp1) of severe acute respiratory syndrome coronavirus and severe acute respiratory syndrome coronavirus 2 is a critical viral protein that suppresses host gene expression by blocking the assembly of the ribosome on host mRNAs. To understand the mechanism of inhibition of host gene expression, we sought to identify cellular proteins that interact with nsp1. Using proximity-dependent biotinylation followed by proteomic analyses of biotinylated proteins, here we captured multiple dynamic interactions of nsp1 with host cell proteins. In addition to ribosomal proteins, we identified several pre-mRNA processing proteins that interact with nsp1, including splicing factors and transcription termination proteins, as well as exosome, and stress granule (SG)-associated proteins. We found that the interactions with transcription termination factors are primarily governed by the C-terminal region of nsp1 and are disrupted by the mutation of amino acids K164 and H165 that are essential for its host shutoff function. We further show that nsp1 interacts with Ras GTPase-activating protein SH3 domain-binding protein 1 (G3BP1) and colocalizes with G3BP1 in SGs under sodium arsenite-induced stress. Finally, we observe that the presence of nsp1 disrupts the maturation of SGs over a long period. Isolation of SG core at different times shows a gradual loss of G3BP1 in the presence of nsp1.

SARS coronavirus protein nsp1 disrupts localization of Nup93 from the nuclear pore complex.

Severe acute respiratory syndrome coronavirus nonstructural protein 1 (nsp1) is a key factor in virus-induced down-regulation of host gene expression. In infected cells, nsp1 engages in a multipronged mechanism to inhibit host gene expression by binding to the 40S ribosome to block the assembly of translationally competent ribosome, and then inducing endonucleolytic cleavage and the degradation of host mRNAs. Here, we report a previously undetected mechanism by which nsp1 exploits the nuclear pore complex and disrupts the nuclear-cytoplasmic transport of biomolecules. We identified members of the nuclear pore complex from the nsp1-associated protein assembly and found that the expression of nsp1 in HEK cells disrupts Nup93 localization around the nuclear envelope without triggering proteolytic degradation, while the nuclear lamina remains unperturbed. Consistent with its role in host shutoff, nsp1 alters the nuclear-cytoplasmic distribution of an RNA binding protein, nucleolin. Our results suggest that nsp1, alone, can regulate multiple steps of gene expression including nuclear-cytoplasmic transport.

Functional evaluation of synthetic flavonoids and chalcones for potential antiviral and anticancer properties.

Flavonoids, stilbenes, and chalcones are plant secondary metabolites that often possess diverse biological activities including anti-inflammatory, anti-cancer, and anti-viral activities. The wide range of bioactivities poses a challenge to identify their targets. Here, we studied a set of synthetically generated flavonoids and chalcones to evaluate for their biological activity, and compared similarly substituted flavonoids and chalcones. Substituted chalcones, but not flavonoids, showed inhibition of viral translation without significantly affecting viral replication in cells infected with hepatitis C virus (HCV). We suggest that the chalcones used in this study inhibit mammalian target of rapamycin (mTOR) pathway by ablating phosphorylation of ribosomal protein 6 (rps6), and also the kinase necessary for phosphorylating rps6 in Huh7.5 cells (pS6K1). In addition, selected chalcones showed inhibition of growth in Ishikawa, MCF7, and MDA-MB-231 cells resulting an IC50 of 1-6µg/mL. When similarly substituted flavonoids were used against the same set of cancer cells, we did not observe any inhibitory effect. Together, we report that chalcones show potential for anti-viral and anti-cancer activities compared to similarly substituted flavonoids.

Prolonged α-amanitin treatment of cells for studying mutated polymerases causes degradation of DSIF160 and other proteins.

A useful method for studying the function of the mammalian RNA polymerase II takes advantage of the extreme sensitivity of its largest subunit, Rpb1, to α-amanitin. Mutations of interest are introduced into an α-amanitin-resistant version of Rpb1, which is then expressed ectopically in cells. The phenotypes of these cells are then examined after inhibiting the endogenous wild-type polymerase with α-amanitin. Here, we show that cells that are enabled to grow in α-amanitin by expression of an α-amanitin-resistant Rpb1 exhibit changes in cell physiology that can lead to misleading experimental outcomes. The changes we have characterized include the accelerated degradation of some proteins, such as DSIF160, and the reduced rate of synthesis of others. In one series of experiments, we examined an α-amanitin-resistant construct, with a mutant C-terminal domain (CTD), that was unable to direct poly(A)-dependent transcription termination in cells growing in α-amanitin. The potential interpretation that the termination defect in this construct is due to the mutation in the CTD was rejected when the construct was found to be termination-competent in cells grown in the absence of α-amanitin. Instead, it appears that certain termination factors become limiting when the cells are grown in α-amanitin, presumably due to the α-amanitin-induced degradation we have characterized and/or to the inadequate transcription of certain genes by the α-amanitin-resistant Rpb1-containing polymerase.

Suppression of viral RNA binding and the assembly of infectious hepatitis C virus particles in vitro by cyclophilin inhibitors.

Nonstructural protein 5A (NS5A) of hepatitis C virus (HCV) is an indispensable component of the HCV replication and assembly machineries. Although its precise mechanism of action is not yet clear, current evidence indicates that its structure and function are regulated by the cellular peptidylprolyl isomerase cyclophilin A (CyPA). CyPA binds to proline residues in the C-terminal half of NS5A, in a distributed fashion, and modulates the structure of the disordered domains II and III. Cyclophilin inhibitors (CPIs), including cyclosporine (CsA) and its nonimmunosuppressive derivatives, inhibit HCV infection of diverse genotypes, both in vitro and in vivo. Here we report a mechanism by which CPIs inhibit HCV infection and demonstrate that CPIs can suppress HCV assembly in addition to their well-documented inhibitory effect on RNA replication. Although the interaction between NS5A and other viral proteins is not affected by CPIs, RNA binding by NS5A in cell culture-based HCV (HCVcc)-infected cells is significantly inhibited by CPI treatment, and sensitivity of RNA binding is correlated with previously characterized CyPA dependence or CsA sensitivity of HCV mutants. Furthermore, the difference in CyPA dependence between a subgenomic and a full-length replicon of JFH-1 was due, at least in part, to an additional role that CyPA plays in HCV assembly, a conclusion that is supported by experiments with the clinical CPI alisporivir. The host-directed nature and the ability to interfere with more than one step in the HCV life cycle may result in a higher genetic barrier to resistance for this class of HCV inhibitors.

A major determinant of cyclophilin dependence and cyclosporine susceptibility of hepatitis C virus identified by a genetic approach.

Since the advent of genome-wide small interfering RNA screening, large numbers of cellular cofactors important for viral infection have been discovered at a rapid pace, but the viral targets and the mechanism of action for many of these cofactors remain undefined. One such cofactor is cyclophilin A (CyPA), upon which hepatitis C virus (HCV) replication critically depends. Here we report a new genetic selection scheme that identified a major viral determinant of HCV's dependence on CyPA and susceptibility to cyclosporine A. We selected mutant viruses that were able to infect CyPA-knockdown cells which were refractory to infection by wild-type HCV produced in cell culture. Five independent selections revealed related mutations in a single dipeptide motif (D316 and Y317) located in a proline-rich region of NS5A domain II, which has been implicated in CyPA binding. Engineering the mutations into wild-type HCV fully recapitulated the CyPA-independent and CsA-resistant phenotype and four putative proline substrates of CyPA were mapped to the vicinity of the DY motif. Circular dichroism analysis of wild-type and mutant NS5A peptides indicated that the D316E/Y317N mutations (DEYN) induced a conformational change at a major CyPA-binding site. Furthermore, nuclear magnetic resonance experiments suggested that NS5A with DEYN mutations adopts a more extended, functional conformation in the putative CyPA substrate site in domain II. Finally, the importance of this major CsA-sensitivity determinant was confirmed in additional genotypes (GT) other than GT 2a. This study describes a new genetic approach to identifying viral targets of cellular cofactors and identifies a major regulator of HCV's susceptibility to CsA and its derivatives that are currently in clinical trials.

The poly(A)-dependent transcriptional pause is mediated by CPSF acting on the body of the polymerase.

Eukaryotic poly(A) signals direct mRNA 3'-end processing and also pausing and termination of transcription. We show that pausing and termination require the processing factor CPSF, which binds the AAUAAA hexamer of the mammalian poly(A) signal. Pausing does not require the RNA polymerase II C-terminal domain (CTD) or the cleavage stimulation factor, CstF, that binds the CTD. Pull-down experiments show that CPSF binds, principally through its 30-kDa subunit, to the body of the polymerase. CPSF can also bind CstF, but this seems to be mutually exclusive with polymerase binding. We suggest that CPSF, while binding the body of the polymerase, scans for hexamers in the extruding RNA. Any encounter with a hexamer triggers pausing. If the hexamer is part of a functional poly(A) signal, CstF is recruited and binds CPSF, causing it to release the polymerase body and move (with CstF) to the CTD.

Tri-snRNP-associated proteins interact with subunits of the TRAMP and nuclear exosome complexes, linking RNA decay and pre-mRNA splicing.

Nuclear RNA decay factors are involved in many different pathways including rRNA processing, snRNA and snoRNA biogenesis, pre-mRNA processing, and the rapid decay of cryptic intergenic transcripts. In contrast to its yeast counterpart, the mammalian nuclear decay machinery is largely uncharacterized. Here we report interactions of several putative components of the human nuclear RNA decay machinery, including the TRAMP complex protein Mtr4 and the nuclear exosome constituents PM/Scl-100 and PM/Scl-75, with components of the U4/U6.U5 tri-snRNP complex required for pre-mRNA splicing. The tri-snRNP component Prp31 interacts indirectly with Mtr4 and PM/Scl-100 in a manner that is dependent on the phosphorylation sites in the middle of the protein, while Prp3 and Prp4 interact with the nuclear decay complex independent of Prp31. Together our results suggest recruitment of the nuclear decay machinery to the spliceosome to ensure production of properly spliced mRNA.

A Tale of Two RNAs during Viral Infection: How Viruses Antagonize mRNAs and Small Non-Coding RNAs in The Host Cell.

Viral infection initiates an array of changes in host gene expression. Many viruses dampen host protein expression and attempt to evade the host anti-viral defense machinery. Host gene expression is suppressed at several stages of host messenger RNA (mRNA) formation including selective degradation of translationally competent messenger RNAs. Besides mRNAs, host cells also express a variety of noncoding RNAs, including small RNAs, that may also be subject to inhibition upon viral infection. In this review we focused on different ways viruses antagonize coding and noncoding RNAs in the host cell to its advantage.

The conserved AAUAAA hexamer of the poly(A) signal can act alone to trigger a stable decrease in RNA polymerase II transcription velocity.

In vivo the poly(A) signal not only directs 3'-end processing but also controls the rate and extent of transcription. Thus, upon crossing the poly(A) signal RNA polymerase II first pauses and then terminates. We show that the G/U-rich region of the poly(A) signal, although required for termination in vivo, is not required for poly(A)-dependent pausing either in vivo or in vitro. Consistent with this, neither CstF, which recognizes the G/U-rich element, nor the polymerase CTD, which binds CstF, is required for pausing. The only part of the poly(A) signal required to direct the polymerase to pause is the AAUAAA hexamer. The effect of the hexamer on the polymerase is long lasting--in many situations polymerases over 1 kb downstream of the hexamer continue to exhibit delayed progress down the template in vivo. The hexamer is the first part of the poly(A) signal to emerge from the polymerase and may play a role independent of the rest of the poly(A) signal in paving the way for subsequent events such as 3'-end processing and termination of transcription.

The RNA tether from the poly(A) signal to the polymerase mediates coupling of transcription to cleavage and polyadenylation.

We have investigated the mechanism by which transcription accelerates cleavage and polyadenylation in vitro. By using a coupled transcription-processing system, we show that rapid and efficient 3' end processing occurs in the absence of crowding agents like polyvinyl alcohol. The continuity of the RNA from the poly(A) signal down to the polymerase is critical to this processing. If this tether is cut with DNA oligonucleotides and RNaseH during transcription, the efficiency of processing is drastically reduced. The polymerase is known to be an integral part of the cleavage and polyadenylation apparatus. RNA polymerase II pull-down and immobilized template experiments suggest that the role of the tether is to hold the poly(A) signal close to the polymerase during the early stages of processing complex assembly until the complex is sufficiently mature to remain stably associated with the polymerase on its own.