Multiple poliovirus-induced organelles suggested by comparison of spatiotemporal dynamics of membranous structures and phosphoinositides.

At the culmination of poliovirus (PV) multiplication, membranes are observed that contain phosphatidylinositol-4-phosphate (PI4P) and appear as vesicular clusters in cross section. Induction and remodeling of PI4P and membranes prior to or concurrent with genome replication has not been well studied. Here, we exploit two PV mutants, termed EG and GG, which exhibit aberrant proteolytic processing of the P3 precursor that substantially delays the onset of genome replication and/or impairs virus assembly, to illuminate the pathway of formation of PV-induced membranous structures.

Unexpected sequences and structures of mtDNA required for efficient transcription from the first heavy-strand promoter.

Human mtDNA contains three promoters, suggesting a need for differential expression of the mitochondrial genome. Studies of mitochondrial transcription have used a reductionist approach, perhaps masking differential regulation. Here we evaluate transcription from light-strand (LSP) and heavy-strand (HSP1) promoters using templates that mimic their natural context.

Kinetic analysis of human PrimPol DNA polymerase activity reveals a generally error-prone enzyme capable of accurately bypassing 7,8-dihydro-8-oxo-2'-deoxyguanosine.

Recent studies have identified human PrimPol as a new RNA/DNA primase and translesion DNA synthesis polymerase (TLS pol) that contributes to nuclear and mitochondrial DNA replication. We investigated the mechanism of PrimPol polymerase activity on both undamaged and damaged DNA substrates. With Mg²⁺ as a cofactor, PrimPol binds primer-template DNA with low affinity K(d,DNA) values (∼200-1200 nM).

Expanding the proteome of an RNA virus by phosphorylation of an intrinsically disordered viral protein.

The human proteome contains myriad intrinsically disordered proteins. Within intrinsically disordered proteins, polyproline-II motifs are often located near sites of phosphorylation. We have used an unconventional experimental paradigm to discover that phosphorylation by protein kinase A (PKA) occurs in the intrinsically disordered domain of hepatitis C virus non-structural protein 5A (NS5A) on Thr-2332 near one of its polyproline-II motifs.

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.

Core human mitochondrial transcription apparatus is a regulated two-component system in vitro.

The core human mitochondrial transcription apparatus is currently regarded as an obligate three-component system comprising the bacteriophage T7-related mitochondrial RNA polymerase, the rRNA methyltransferase-related transcription factor, h-mtTFB2, and the high mobility group box transcription/DNA-packaging factor, h-mtTFA/TFAM. Using a faithful recombinant human mitochondrial transcription system from Escherichia coli, we demonstrate that specific initiation from the mtDNA promoters, LSP and HSP1, only requires mitochondrial RNA polymerase and h-mtTFB2 in vitro. When h-mtTFA is added to these basal components, LSP exhibits a much lower threshold for activation and a larger amplitude response than HSP1.

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.

Structural and functional studies of the promoter element for dengue virus RNA replication.

The 5' untranslated region (5'UTR) of the dengue virus (DENV) genome contains two defined elements essential for viral replication. At the 5' end, a large stem-loop (SLA) structure functions as the promoter for viral polymerase activity. Next to the SLA, there is a short stem-loop that contains a cyclization sequence known as the 5' upstream AUG region (5'UAR).

Structural and functional analysis of dengue virus RNA.

Sequences and structures present at the 5' and 3' UTRs of RNA viruses play crucial roles in the initiation and regulation of translation, RNA synthesis and viral assembly. In dengue virus, as well as in other mosquito-borne flaviviruses, the presence of complementary sequences at the ends of the genome mediate long-range RNA-RNA interactions. Dengue virus RNA displays two pairs of complementary sequences (CS and UAR) required for genome circularization and viral viability.

A 5' RNA element promotes dengue virus RNA synthesis on a circular genome.

The mechanisms of RNA replication of plus-strand RNA viruses are still unclear. Here, we identified the first promoter element for RNA synthesis described in a flavivirus. Using dengue virus as a model, we found that the viral RdRp discriminates the viral RNA by specific recognition of a 5' element named SLA.

Long-range RNA-RNA interactions circularize the dengue virus genome.

Secondary and tertiary RNA structures present in viral RNA genomes play essential regulatory roles during translation, RNA replication, and assembly of new viral particles. In the case of flaviviruses, RNA-RNA interactions between the 5' and 3' ends of the genome have been proposed to be required for RNA replication. We found that two RNA elements present at the ends of the dengue virus genome interact in vitro with high affinity.