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HIV-1 reverse transcriptase (RT) possesses both DNA polymerase activity and RNase H activity that act in concert to convert single-stranded RNA of the viral genome to double-stranded DNA that is then integrated into the DNA of the infected cell. Reverse transcriptase- catalyzed reverse transcription critically relies on the proper generation of a polypurine tract (PPT) primer. However, the mechanism of PPT primer generation and the features of the PPT sequence that are critical for its recognition by HIV-1RTremain unclear. Here, we used a chemical cross-linking method together with molecular dynamics simulations and single-molecule assays to study the mechanism of PPT primer generation. We found that the PPT was specifically and properly recognized within covalently tethered HIV-1 RT-nucleic acid complexes. These findings indicated that recognition of the PPT occurs within a stable catalytic complex after its formation.Wefound that this unique recognition is based on two complementary elements that rely on the PPT sequence: RNaseHsequence preference and incompatibility of the poly(rA/dT) tract of the PPT with the nucleic acid conformation that is required for RNaseHcleavage. The latter results from rigidity of the poly(rA/dT) tract and leads to base-pair slippageof this sequenceupondeformation into a catalytically relevant geometry. In summary, our results reveal an unexpected mechanism of PPT primer generation based on specific dynamic properties of the poly(rA/dT) segment and help advance our understanding of the mechanisms in viral RNA reverse transcription. [ABSTRACT FROM AUTHOR]

A hallmark of Ebola virus (EBOV) infection is the formation of viral inclusions in the cytoplasm of infected cells. These viral inclusions contain the EBOV nucleocapsids and are sites of viral replication and nucleocapsid maturation. Although there is growing evidence that viral inclusions create a protected environment that fosters EBOV replication, little is known about their role in the host response to infection. The cellular stress response is an effective antiviral strategy that leads to stress granule (SG) formation and translational arrest mediated by the phosphorylation of a translation initiation factor, the subunit of eukaryotic initiation factor 2 (eIF2α). Here, we show that selected SG proteins are sequestered within EBOV inclusions, where they form distinct granules that colocalize with viral RNA. These inclusion-bound (IB) granules are functionally and structurally different from canonical SGs. Formation of IB granules does not indicate translational arrest in the infected cells. We further show that EBOV does not induce formation of canonical SGs or eIF2α phosphorylation at any time postinfection but is unable to fully inhibit SG formation induced by different exogenous stressors, including sodium arsenite, heat, and hippuristanol. Despite the sequestration of SG marker proteins into IB granules, canonical SGs are unable to form within inclusions, which we propose might be mediated by a novel function of VP35, which disrupts SG formation. This function is independent of VP35's RNA binding activity. Further studies aim to reveal the mechanism for SG protein sequestration and precise function within inclusions. [ABSTRACT FROM AUTHOR]

Conformational control of biomolecular activities can reveal functional insights and enable the engineering of novel activities. Here we show that conformational control through intramolecular cross-linking of a helicase monomer with undetectable unwinding activity converts it into a superhelicase that can unwind thousands of base pairs processively, even against a large opposing force. A natural partner that enhances the helicase activity is shown to achieve its stimulating role also by selectively stabilizing the active conformation. Our work provides insight into the regulation of nucleic acid unwinding activity and introduces a monomeric superhelicase without nuclease activities, which may be useful for biotechnological applications. [ABSTRACT FROM AUTHOR]

Base-pairing interactions between nucleic acids mediate target recognition in many biological processes. We developed a super-resolution imaging and modeling platform that enabled the in vivo determination of base pairing-mediated target recognition kinetics. We examined a stress-induced bacterial small RNA, SgrS, which induces the degradation of target messenger RNAs (mRNAs). SgrS binds to a primary target mRNA in a reversible and dynamic fashion, and formation of SgrS-mRNA complexes is rate-limiting, dictating the overall regulation efficiency in vivo. Examination of a secondary target indicated that differences in the target search kinetics contribute to setting the regulation priority among different target mRNAs. This super-resolution imaging and analysis approach provides a conceptual framework that can be generalized to other small RNA systems and other target search processes. [ABSTRACT FROM AUTHOR]

Besides an essential transcriptional factor for B cell development and function, cellular interferon regulatory factor 4 (c-IRF4) directly regulates expression of the c-Myc gene, which is not only associated with various B cell lymphomas but also required for herpesvirus latency and pathogenesis. Kaposi's sarcoma-associated herpesvirus (KSHV), the etiological agent of Kaposi's sarcoma and primary effusion lymphoma, has developed a unique mechanism to deregulate host antiviral innate immunity and growth control by incorporating four viral homologs (vIRF1 to -4) of cellular IRFs into its genome. Previous studies have shown that several KSHV latent proteins, including vIRF3, vFLIP, and LANA, target the expression, function, and stability of c-Myc to establish and maintain viral latency. Here we report that the KSHV vIRF4 lytic protein robustly suppresses expression of c-IRF4 and c-Myc, reshaping host gene expression profiles to facilitate viral lytic replication. Genomewide gene expression analysis revealed that KSHV vIRF4 grossly affects host gene expression by upregulating and downregulating 118 genes and 166 genes, respectively, by at least 2-fold. Remarkably, vIRF4 suppressed c-Myc expression by 11-fold, which was directed primarily by the deregulation of c-IRF4 expression. Real-time quantitative PCR (RT-qPCR), single-molecule in situ hybridization, and chromatin immunoprecipitation assays showed that vIRF4 not only reduces c-IRF4 expression but also competes with c-IRF4 for binding to the specific promoter region of the c-Myc gene, resulting in drastic suppression of c-Myc expression. Consequently, the loss of vIRF4 function in the suppression of c-IRF4 and c-Myc expression ultimately led to a reduction of KSHV lytic replication capacity. These results indicate that the KSHV vIRF4 lytic protein comprehensively targets the expression and function of c-IRF4 to downregulate c-Myc expression, generating a favorable environment for viral lytic replication. Finally, this study further reinforces the important role of the c-Myc gene in KSHV lytic replication and latency. [ABSTRACT FROM AUTHOR]

Frequent collisions between cellular DNA replication complexes (replisomes) and obstacles such as damaged DNA or frozen protein complexes make DNA replication fork progression surprisingly sporadic. These collisions can lead to the ejection of replisomes prior to completion of replication, which, if left unrepaired, results in bacterial cell death. As such, bacteria have evolved DNA replication restart mechanisms that function to reload replisomes onto abandonedDNAreplication forks. Here, we define a direct interaction between PriC, a key Escherichia coli DNA replication restart protein, and the single-stranded DNA-binding protein (SSB), a protein that is ubiquitously associated withDNAreplication forks. PriC/SSB complex formation requires evolutionarily conserved residues from both proteins, including a pair of Arg residues from PriC and the C terminus of SSB. In vitro, disruption of the PriC/SSB interface by sequence changes in either protein blocks the first step ofDNAreplication restart, reloading of the replicative DnaB helicase onto an abandoned replication fork. Consistent with the critical role of PriC/ SSB complex formation in DNA replication restart, PriC variants that cannot bind SSB are non-functional in vivo. Singlemolecule experiments demonstrate that PriC binding to SSB alters SSB/DNA complexes, exposing single-stranded DNA and creating a platform for other proteins to bind. These data lead to a model in which PriC interaction with SSB remodels SSB/DNA structures at abandoned DNA replication forks to create a DNA structure that is competent for DnaB loading. [ABSTRACT FROM AUTHOR]

The archaeal rninichromosome maintenance protein MCM forms a homohexameric complex that functions as the DNA replicative helicase and serves as a model system for its eukaryotic counterpart. Here, we applied single molecule fluorescence resonance energy transfer methods to probe the substrate specificity and binding mechanism of MCM from the hyperthermophilic Archaea Sulfolobus solfataricus on various DNA substrates. S. solfataricus MCM displays a binding preference for forked substrates relative to partial or full duplex substrates. Moreover, the nature of MCM binding to Y-shaped substrates is distinct in that MCM loads on the 3′-tail while interacting with the 5′-tail likely via the MCM surface. These results provide the first elucidation of a dynamic nature of interaction between a ring-shaped helicase interacting with an opposing single-stranded DNA tail. This interaction contributes to substrate selectivity and increases the stability of the forked DNA-MCM complex, with possible implications for the MCM unwinding mechanism. [ABSTRACT FROM AUTHOR]

Single-stranded DNA-binding proteins and their functional homologs, replication protein A, are essential components of cellular DNA replication, repair and recombination. We describe here the isolation and characterization of multiple replication protein A homologs, RPA1, RPA2, and RPA3, from the archaeon Methanosarcina acetivorans. RPA1 comprises four single-stranded DNA-binding domains, while RPA2 and RPA3 are each composed of two such domains and a zinc finger domain. Gel filtration analysis suggested that RPA1 exists as homotetramers and homodimers in solution, while RPA2 and RPA3 form only homodimers. Unlike the multiple RPA proteins found in other Archaea and eukaryotes, each of the M. acetivorans RPAs can act as a distinct single-stranded DNA-binding protein. Fluorescence resonance energy transfer and fluorescence polarization anisotropy studies revealed that the M. acetivorans RPAs bind to as few as 10 single-stranded DNA bases. However, more stable binding is achieved with single-stranded DNA of 18-23 bases, and for such substrates the estimated Kd was 3.82 ± 0.28 nM, 173.6 ± 105.17 nM, and 5.92 ± 0.23 nM, for RPA1, RPA2, and RPA3, respectively. The architectures of the M. acetivorans RPAs are different from those of hitherto reported homologs. Thus, these proteins may represent novel forms of replication protein A. Most importantly, our results show that the three RPAs and their combinations highly stimulate the primer extension capacity of M. acetivorans DNA polymerase BI. Although bacterial SSB and eukaryotic RPA have been shown to stimulate DNA synthesis by their cognate DNA polymerases, our findings provide the first in vitro biochemical evidence for the conservation of this property in an archaeon. [ABSTRACT FROM AUTHOR]

The article examines a single-molecule technique combining fluorescence microscopy and optical tweezers that allows for both being measured simultaneously. It presents measurements of the DNA repair helicase UvrD that revealed it exhibits two distinct unwinding activity types regulated by its stoichiometry. It mentions the two conformational states of UvrD correlate with movement toward or away from the fork in DNA.