Structural and biochemical characterization of cauliflower mosaic virus reverse transcriptase.

Reverse transcriptases (RTs) are enzymes with DNA polymerase and RNase H activities. They convert ssRNA into dsDNA and are key enzymes for the replication of retroviruses and retroelements. Caulimoviridae is a major family of plant-infecting viruses. Caulimoviruses have a circular dsDNA genome that is replicated by reverse transcription, but in contrast to retroviruses, they lack integrase. Caulimoviruses are related to Ty3 retroelements. Ty3 RT has been extensively studied structurally and biochemically, but corresponding information for caulimoviral RTs is unavailable. In the present study, we report the first crystal structure of cauliflower mosaic virus (CaMV) RT in complex with a duplex made of RNA and DNA strands (RNA/DNA hybrid). CaMV RT forms a monomeric complex with the hybrid, unlike Ty3 RT, which does so as a dimer. Results of the RNA-dependent DNA polymerase and DNA-dependent DNA polymerase activity assays showed that individual CaMV RT molecules are able to perform full polymerase functions. However, our analyses showed that an additional CaMV RT molecule needs to transiently associate with a polymerase-competent RT molecule to execute RNase H cuts of the RNA strand. Collectively, our results provide details into the structure and function of CaMV RT and describe how the enzyme compares to other related RTs.

Cryo-EM structure of rotavirus B NSP2 reveals its unique tertiary architecture.

Rotavirus (RV) NSP2 is a multifunctional RNA chaperone that exhibits numerous activities that are essential for replication and viral genome packaging. We performed an in silico analysis that highlighted a distant relationship of NSP2 from rotavirus B (RVB) to proteins from other human RVs. We solved a cryo-electron microscopy structure of RVB NSP2 that shows structural differences with corresponding proteins from other human RVs. Based on the structure, we identified amino acid residues that are involved in RNA interactions. Anisotropy titration experiments showed that these residues are important for nucleic acid binding. We also identified structural motifs that are conserved in all RV species. Collectively, our data complete the structural characterization of rotaviral NSP2 protein and demonstrate its structural diversity among RV species.IMPORTANCERotavirus B (RVB), also known as adult diarrhea rotavirus, has caused epidemics of severe diarrhea in China, India, and Bangladesh. Thousands of people are infected in a single RVB epidemic. However, information on this group of rotaviruses remains limited. As NSP2 is an essential protein in the viral life cycle, including its role in the formation of replication factories, it may be a target for future antiviral strategy against viruses with similar mechanisms.

Evolutionary History and Activity of RNase H1-Like Proteins in Arabidopsis thaliana.

RNase H1 is an endonuclease specific toward the RNA strand of RNA:DNA hybrids. Members of this protein family are present in most living organisms and are essential for removing RNA that base pairs with DNA. It prevents detrimental effects of RNA:DNA hybrids and is involved in several biological processes. Arabidopsis thaliana has been previously shown to contain three genes encoding RNase H1 proteins that localize to three distinct cellular compartments. We show that these genes originate from two gene duplication events. One occurred in the common ancestor of dicots and produced nuclear and organellar RNase H1 paralogs. Second duplication occurred in the common ancestor of Brassicaceae and produced mitochondrial- and plastid-localized proteins. These proteins have the canonical RNase H1 activity, which requires at least four ribonucleotides for endonucleolytic digestion. Analysis of mutants in the RNase H1 genes revealed that the nuclear RNH1A and mitochondrial RNH1B are dispensable for development under normal growth conditions. However, the presence of at least one organellar RNase H1 (RNH1B or RNH1C) is required for embryonic development. The plastid-localized RNH1C affects plastid DNA copy number and sensitivity to replicative stress. Our results present the evolutionary history of RNH1 proteins in A. thaliana, demonstrate their canonical RNase H1 activity and indicate their role in early embryonic development.

Mechanism of RecF-RecO-RecR cooperation in bacterial homologous recombination.

In bacteria, one type of homologous-recombination-based DNA-repair pathway involves RecFOR proteins that bind at the junction between single-stranded (ss) and double-stranded (ds) DNA. They facilitate the replacement of SSB protein, which initially covers ssDNA, with RecA, which mediates the search for homologous sequences. However, the molecular mechanism of RecFOR cooperation remains largely unknown. We used Thermus thermophilus proteins to study this system. Here, we present a cryo-electron microscopy structure of the RecF-dsDNA complex, and another reconstruction that shows how RecF interacts with two different regions of the tetrameric RecR ring. Lower-resolution reconstructions of the RecR-RecO subcomplex and the RecFOR-DNA assembly explain how RecO is positioned to interact with ssDNA and SSB, which is proposed to lock the complex on a ssDNA-dsDNA junction. Our results integrate the biochemical data available for the RecFOR system and provide a framework for its complete understanding.