Ibrahim M. Moustafa

Nucleic acid polymerases use a general acid for nucleotidyl transfer.

Nucleic acid polymerases catalyze the formation of DNA or RNA from nucleoside-triphosphate precursors. Amino acid residues in the active site of polymerases are thought to contribute only indirectly to catalysis by serving as ligands for the two divalent cations that are required for activity or substrate binding. Two proton-transfer reactions are necessary for polymerase-catalyzed nucleotidyl transfer: deprotonation of the 3′-hydroxyl nucleophile and protonation of the pyrophosphate leaving group. Using model enzymes representing all four classes of nucleic acid polymerases, we show that the proton donor to pyrophosphate is an active-site amino acid residue. The use of general acid catalysis by polymerases extends the mechanism of nucleotidyl transfer beyond that of the well-established two-metal-ion mechanism. The existence of an active-site residue that regulates polymerase catalysis may permit manipulation of viral polymerase replication speed and/or fidelity for virus attenuation and vaccine development.

Hepatitis C virus non-structural protein 3 (HCV NS3): a multifunctional antiviral target

Hepatitis C virus non-structural protein 3 contains a serine protease and an RNA helicase. Protease cleaves the genome-encoded polyprotein and inactivates cellular proteins required for innate immunity. Protease has emerged as an important target for the development of antiviral therapeutics, but drug resistance has turned out to be an obstacle in the clinic. Helicase is required for both genome replication and virus assembly. Mechanistic and structural studies of helicase have hurled this enzyme into a prominent position in the field of helicase enzymology. Nevertheless, studies of helicase as an antiviral target remain in their infancy.

Probing the Sialic Acid Binding Site of the Hemagglutinin-Neuraminidase of Newcastle Disease Virus: Identification of Key Amino Acids Involved in Cell Binding, Catalysis, and Fusion

ABSTRACT We recently reported the first crystal structure of a paramyxovirus hemagglutinin-neuraminidase (HN) from Newcastle disease virus. This multifunctional protein is responsible for binding to cellular sialyl-glycoconjugate receptors, promotion of fusion through interaction with the second viral surface fusion (F) glycoprotein, and processing progeny virions by removal of sialic acid from newly synthesized viral coat proteins. Our structural studies suggest that HN possesses a single sialic acid recognition site that can be switched between being a binding site and a catalytic site. Here we examine the effect of mutation of several conserved amino acids around the binding site on the hemagglutination, neuraminidase, and fusion functions of HN. Most mutations around the binding site result in loss of neuraminidase activity, whereas the effect on receptor binding is more variable. Residues E401, R416, and Y526 appear to be key for receptor binding. The increase in fusion promotion seen in some mutants that lack receptor binding activity presents a conundrum. We propose that in these cases HN may be switched into a fusion-promoting state through a series of conformational changes that propagate from the sialic acid binding site through to the HN dimer interface. These results further support the single-site model and suggest certain residues to be important for the triggering of fusion.

Molecular Dynamics Simulations of Viral RNA Polymerases Link Conserved and Correlated Motions of Functional Elements to Fidelity

Abstract The viral RNA-dependent RNA polymerase (RdRp) is essential for multiplication of all RNA viruses. The sequence diversity of an RNA virus population contributes to its ability to infect the host. This diversity emanates from errors made by the RdRp during RNA synthesis. The physical basis for RdRp fidelity is unclear but is linked to conformational changes occurring during the nucleotide-addition cycle. To understand RdRp dynamics that might influence RdRp function, we have analyzed all-atom molecular dynamics simulations on the nanosecond timescale of four RdRps from the picornavirus family that exhibit 30–74% sequence identity. Principal component analysis showed that the major motions observed during the simulations derived from conserved structural motifs and regions of known function. The dynamics of residues participating in the same biochemical property, for example, RNA binding, nucleotide binding or catalysis, were correlated even when spatially distant on the RdRp structure. The conserved and correlated dynamics of functional structural elements suggest coevolution of dynamics with structure and function of the RdRp. Crystal structures of all picornavirus RdRps exhibit a template–nascent RNA duplex channel too small to fully accommodate duplex RNA. Simulations revealed opening and closing motions of the RNA and nucleoside triphosphate channels, which might be relevant to nucleoside triphosphate entry, inorganic pyrophosphate exit and translocation. A role for nanosecond timescale dynamics in RdRp fidelity is supported by the altered dynamics of the high-fidelity G64S derivative of PV RdRp relative to wild-type enzyme.

Transcription from the second heavy-strand promoter of human mtDNA is repressed by transcription factor A in vitro

Cell-based studies support the existence of two promoters on the heavy strand of mtDNA: heavy-strand promoter 1 (HSP1) and HSP2. However, transcription from HSP2 has been reported only once in a cell-free system, and never when recombinant proteins have been used. Here, we document transcription from HSP2 using an in vitro system of defined composition. An oligonucleotide template representing positions 596–685 of mtDNA was sufficient to observe transcription by the human mtRNA polymerase (POLRMT) that was absolutely dependent on mitochondrial transcription factor B2 (TFB2M). POLRMT/TFB2M-dependent transcription was inhibited by concentrations of mitochondrial transcription factor A (TFAM) stoichiometric with the transcription template, a condition that activates transcription from the light-strand promoter (LSP) in vitro. Domains of TFAM required for LSP activation were also required for HSP2 repression, whereas other mtDNA binding proteins failed to alter transcriptional output. Binding sites for TFAM were located on both sides of the start site of transcription from HSP2, suggesting that TFAM binding interferes with POLRMT and/or TFB2M binding. Consistent with a competitive binding model for TFAM repression of HSP2, the impact of TFAM concentration on HSP2 transcription was diminished by elevating the POLRMT and TFB2M concentrations. In the context of our previous studies of LSP and HSP1, it is now clear that three promoters exist in human mtDNA. Each promoter has a unique requirement for and/or response to the level of TFAM present, thus implying far greater complexity in the regulation of mammalian mitochondrial transcription than recognized to date.

Dynamics: the missing link between structure and function of the viral RNA-dependent RNA polymerase?

The structural basis for nucleotide incorporation fidelity remains an open question for all nucleic acid polymerases. Addressing this question for the viral RNA-dependent RNA polymerase (RdRp) is of particular, practical significance because it is a determinant of sensitivity to antiviral nucleosides and may be a determinant of viral virulence. All polymerases are thought to employ the same catalytic mechanism, but the rate of nucleotide incorporation can vary substantially. Here we review some of the recent work with the RdRp that leads us to suggest that structure provides only a partial understanding of RdRp function and dynamics may be the missing link.

Picornavirus genome replication. Identification of the surface of the poliovirus (PV) 3C dimer that interacts with PV 3Dpol during VPg uridylylation and construction of a structural model for the PV 3C2-3Dpol complex.

Picornaviruses have a peptide termed VPg covalently linked to the 5′-end of the genome. Attachment of VPg to the genome occurs in at least two steps. First, Tyr-3 of VPg, or some precursor thereof, is used as a primer by the viral RNA-dependent RNA polymerase, 3Dpol, to produce VPg-pUpU. Second, VPg-pUpU is used as a primer to produce full-length genomic RNA. Production of VPg-pUpU is templated by a single adenylate residue located in the loop of an RNA stem-loop structure termed oriI by using a slide-back mechanism. Recruitment of 3Dpol to and its stability on oriI have been suggested to require an interaction between the back of the thumb subdomain of 3Dpol and an undefined region of the 3C domain of viral protein 3CD. We have performed surface acidic-to-alanine-scanning mutagenesis of 3C to identify the surface of 3C with which 3Dpol interacts. This analysis identified numerous viable poliovirus mutants with reduced growth kinetics that correlated to reduced kinetics of RNA synthesis that was attributable to a change in VPg-pUpU production. Importantly, these 3C derivatives were all capable of binding to oriI as well as wild-type 3C. Synthetic lethality was observed for these mutants when placed in the context of a poliovirus mutant containing 3Dpol-R455A, a residue on the back of the thumb required for VPg uridylylation. These data were used to guide molecular docking of the structures for a poliovirus 3C dimer and 3Dpol, leading to a structural model for the 3C2-3Dpol complex that extrapolates well to all picornaviruses.

Identification of Multiple Rate-limiting Steps during the Human Mitochondrial Transcription Cycle in Vitro

We have reconstituted human mitochondrial transcription in vitro on DNA oligonucleotide templates representing the light strand and heavy strand-1 promoters using protein components (RNA polymerase and transcription factors A and B2) isolated from Escherichia coli. We show that 1 eq of each transcription factor and polymerase relative to the promoter is required to assemble a functional initiation complex. The light strand promoter is at least 2-fold more efficient than the heavy strand-1 promoter, but this difference cannot be explained solely by the differences in the interaction of the transcription machinery with the different promoters. In both cases, the rate-limiting step for production of the first phosphodiester bond is open complex formation. Open complex formation requires both transcription factors; however, steps immediately thereafter only require transcription factor B2. The concentration of nucleotide required for production of the first dinucleotide product is substantially higher than that required for subsequent cycles of nucleotide addition. In vitro, promoter-specific differences in post-initiation control of transcription exist, as well as a second rate-limiting step that controls conversion of the transcription initiation complex into a transcription elongation complex. Rate-limiting steps of the biochemical pathways are often those that are targeted for regulation. Like the more complex multisubunit transcription systems, multiple steps may exist for control of transcription in human mitochondria. The tools and mechanistic framework presented here will facilitate not only the discovery of mechanisms regulating human mitochondrial transcription but also interrogation of the structure, function, and mechanism of the complexes that are regulated during human mitochondrial transcription.

Sequence-Specific Fidelity Alterations Associated with West Nile Virus Attenuation in Mosquitoes.

High rates of error-prone replication result in the rapid accumulation of genetic diversity of RNA viruses. Recent studies suggest that mutation rates are selected for optimal viral fitness and that modest variations in replicase fidelity may be associated with viral attenuation. Arthropod-borne viruses (arboviruses) are unique in their requirement for host cycling and may necessitate substantial genetic and phenotypic plasticity. In order to more thoroughly investigate the correlates, mechanisms and consequences of arbovirus fidelity, we selected fidelity variants of West Nile virus (WNV; Flaviviridae, Flavivirus) utilizing selection in the presence of a mutagen. We identified two mutations in the WNV RNA-dependent RNA polymerase associated with increased fidelity, V793I and G806R, and a single mutation in the WNV methyltransferase, T248I, associated with decreased fidelity. Both deep-sequencing and in vitro biochemical assays confirmed strain-specific differences in both fidelity and mutational bias. WNV fidelity variants demonstrated host-specific alterations to replicative fitness in vitro, with modest attenuation in mosquito but not vertebrate cell culture. Experimental infections of colonized and field populations of Cx. quinquefaciatus demonstrated that WNV fidelity alterations are associated with a significantly impaired capacity to establish viable infections in mosquitoes. Taken together, these studies (i) demonstrate the importance of allosteric interactions in regulating mutation rates, (ii) establish that mutational spectra can be both sequence and strain-dependent, and (iii) display the profound phenotypic consequences associated with altered replication complex function of flaviviruses.

Expanding knowledge of P3 proteins in the poliovirus lifecycle

Poliovirus is the most extensively studied member of the order Picornavirales, which contains numerous medical, veterinary and agricultural pathogens. The picornavirus genome encodes a single polyprotein that is divided into three regions: P1, P2 and P3. P3 proteins are known to participate more directly in genome replication, for example by containing the viral RNA-dependent RNA polymerase (RdRp or 3Dpol), among several other proteins and enzymes. We will review recent data that provide new insight into the structure, function and mechanism of P3 proteins and their complexes, which are required for initiation of genome replication. Replication of poliovirus genomes occurs within macromolecular complexes, containing viral RNA, viral proteins and host-cell membranes, collectively referred to as replication complexes. P2 proteins clearly contribute to interactions with the host cell that are required for virus multiplication, including formation of replication complexes. We will discuss recent data that suggest a role for P3 proteins in formation of replication complexes. Among the least understood steps of the poliovirus lifecycle is encapsidation of genomic RNA. We will also describe data that suggest a role for P3 proteins in this step.