In Planta Interaction and Transreplication of Distinct Begomoviruses and their Associated Components

The studies described here were intended to examine the transreplication and interactions abilities of a widespread ToLCNDV, and an emerging begomovirus PeLCV associated with its cognate betasatellite TbLCuB. PeLCV, a monopartite begomovirus, has been characterized from many important crops, vegetables and weeds along with its associated TbLCuB. The DNA-B of bipartite ToLCNDV genome has been successfully transreplicated by the DNA-A of different bipartite begomoviruses, albeit with low frequency. Whether PeLCV can transreplicate DNA-B of ToLCNDV is unknown. To unravel this notion, both these viruses were inoculated to the model Nicotiana benthamiana plants in all possible combinations and the in planta existence of viral components were verified by PCR and Southern blot hybridization. The results demonstrated that PeLCV transreplicated and maintained ToLCNDV DNA-B. Whereas, ToLCNDV DNA-A could not transreplicate TbLCuB. Analyses of Rep proteins structure of ToLCNDV and PeLCV revealed a structural resemblance, whereas putative iteron-binding sequences of PeLCV were compatible with the Rep-binding iterons of ToLCNDV-B. The results suggested that PeLCV and ToLCNDV DNA-B can interact synergistically and can be disastrous under field conditions. © 2021 Friends Science Publishers

Molecular detection and phylogenetic analysis of pigeon circovirus from racing pigeons in China during 2016-2019

The Pigeon circovirus (PiCV) containing a circular single stranded DNA (approximately 2 kb) genome belongs to the genus Circovirus of the family Circoviridae. PiCV infections in pigeons ( Columba livia) have been reported worldwide. Currently, pigeon racing is becoming increasingly popular and considered to be a national sport in China, and even, the greatest competitions of racing pigeons are taking place in China. However, there are still no epidemiologic data regarding PiCV infections among racing pigeons in China. To trace the prevalence, genetic variation and evolution of PiCV in sick and healthy racing pigeons, 622 samples were collected from 11 provinces or municipalities in China from 2016 to 2019. Samples were tested by polymerase chain reaction. The results showed that the positive rate of PiCV was 19.3% (120/622) at the sample level and 59.0% (23/39) at the club level, thus suggesting that the virus was prevalent in Chinese racing pigeons. A sequence analysis revealed that the cap genes of the PiCV strains identified in our study displayed a high genetic diversity and shared nucleotide homologies of 71.9%–100% and amino acid homologies of 71.7%–100%. 28 and 36 unique amino acid substitutions were observed in the Cap and Rep proteins derived from our PiCV strains, respectively. Furthermore, The ATT and GTG were highly suspected to be the start codons of the Cap protein. A cap-gene-based phylogenetic analysis showed that the strains in this study could be further divided into seven clades (A, B, C, E, G, H, and I) and some of them were closely related to worldwide strains from different types of pigeons. A large number of recombination events (31 events) were also detected in the PiCV genomes from Chinese racing pigeons. These findings indicate that PiCV strains circulating in China exhibit a high genetic diversity.

Improved Genome Packaging Efficiency of AAV Vectors Using Rep Hybrids

Recombinant Adeno-associated viruses (rAAVs) are one of the most commonly used vectors for a variety of gene therapy applications. In the last two decades research focused primarily on the characterization and isolation of new cap genes resulting in hundreds of natural and engineered AAV capsid variants while the rep gene, the other major AAV open reading frame, has been less studied. This is due to the fact that the rep gene from AAV serotype 2 (AAV2) enables the ssDNA packaging of recombinant genomes into most AAV serotype and engineered capsids. However, a major byproduct of all vector productions is empty AAV capsids, lacking the encapsidated vector genome, especially for non-AAV2 vectors. Despite the packaging process being considered the rate-limiting step for rAAV production, none of the rep genes from the other AAV serotypes have been characterized for their packaging efficiency. Thus, in this study AAV2 rep was replaced with the rep gene of a select number of AAV serotypes. However, this led to a lowering of capsid protein expression, relative to the standard AAV2-rep system. In further experiments the 3’end of the AAV2 rep gene was reintroduced to promote increased capsid expression and a series of chimeras between the different AAV Rep proteins were generated and characterized for their vector genome packaging ability. The utilization of these novel Rep hybrids increased the percentage of genome containing (full) capsids ~2-4-fold for all of the non-AAV2 serotypes tested. Thus, these Rep chimeras could revolutionize rAAV production.

The Cryo-EM structure of AAV2 Rep68 in complex with ssDNA reveals a malleable AAA+ machine that can switch between oligomeric states

The adeno-associated virus (AAV) non-structural Rep proteins catalyze all the DNA transactions required for virus viability including, DNA replication, transcription regulation, genome packaging, and during the latent phase, site-specific integration. Rep proteins contain two multifunctional domains: an Origin Binding Domain (OBD) and a SF3 helicase domain (HD). Studies have shown that Rep proteins have a dynamic oligomeric behavior where the nature of the DNA substrate molecule modulates its oligomeric state. In the presence of ssDNA, Rep68 forms a large double-octameric ring complex. To understand the mechanisms underlying AAV Rep function, we investigated the cryo-EM and X-ray structures of Rep68–ssDNA complexes. Surprisingly, Rep68 generates hybrid ring structures where the OBD forms octameric rings while the HD forms heptamers. Moreover, the binding to ATPγS promotes a large conformational change in the entire AAA+ domain that leads the HD to form both heptamer and hexamers. The HD oligomerization is driven by an interdomain linker region that acts as a latch to ‘catch’ the neighboring HD subunit and is flexible enough to permit the formation of different stoichiometric ring structures. Overall, our studies show the structural basis of AAV Rep's structural flexibility required to fulfill its multifunctional role during the AAV life cycle.

The Cryo-EM structure of AAV2 Rep68 in complex with ssDNA reveals a malleable AAA+ machine that can switch between oligomeric states

The adeno-associated virus (AAV) Rep proteins use a unique AAA+ domain to catalyze DNA replication, transcription regulation, and genome packaging. Also, they mediate site-specific integration during a latent phase. To understand the mechanisms underlying AAV Rep function, we investigated the cryo-EM and X-ray structures of Rep68-ssDNA complexes. Surprisingly, Rep68 generates hybrid ring structures where the Origin-Binding-Domain (OBD) forms octameric rings while the helicase domain (HD) forms heptamers. Moreover, the binding to ATPγS promotes a large conformational change in the entire AAA+ domain that leads the HD to form both heptamer and hexamers. The HD oligomerization is driven by an interdomain linker region that acts as a latch to ‘catch’ the neighboring HD subunit and is flexible enough to permit the formation of different stoichiometric ring structures. Overall, our studies show the structural basis of AAV Rep’s structural flexibility required to fulfill its multifunctional role during the AAV life cycle.

Porcine Circovirus Type 2 Rep Enhances IL-10 Production in Macrophages via Activation of p38-MAPK Pathway

Porcine circovirus type 2 (PCV2) is one of the major threats to pig farms worldwide. Although PCV2 has been identified to promote IL-10 production, the detailed regulatory roles of PCV2 Rep for IL-10 production remain unclear. Herein, we first found that PCV2 Rep, rather than PCV1 Rep, enhanced IL-10 expression at the later phase of PCV2 infection in porcine alveolar macrophages (PAMs). Furthermore, we found that PCV2 Rep directly activated the p38-MAPK pathway to promote transcription factors NF-κB p50 and Sp1 binding to the il10 promoter, but PCV1 Rep did not. During PCV2 infection, however, PCV2 Rep promoted the binding activities of NF-κB p50 and Sp1 with the il10 promoter only at the later phase of PCV2 infection, since Rep proteins only expressed at the later phase of the infection. Moreover, silence of the thymine DNA glycosylase (TDG), a Rep-binding protein, significantly reduced the binding activities of NF-κB p50 and Sp1 with il10 promoter, resulting in the reduction of IL-10 production in PCV2-inoculated PAMs at the later phase of infection. Taken together, our results demonstrate that Rep proteins enhance IL-10 production during PCV2 infection of PAMs via activation of p38-MAPK pathways, in which host TDG is a critical mediator.

Molecular dissection of the replication system of plasmid pIGRK encoding two in-frame Rep proteins with antagonistic functions

Background Gene overlapping is a frequent phenomenon in microbial genomes. Excluding so-called “trivial overlapping”, there are significant implications of such genetic arrangements, including regulation of gene expression and modification of protein activity. It is also postulated that, besides gene duplication, the appearance of overlapping genes (OGs) is one of the most important factors promoting a genome’s novelty and evolution. OGs coding for in-frame proteins with different functions are a particularly interesting case. In this study we identified and characterized two in-frame proteins encoded by OGs on plasmid pIGRK from Klebsiella pneumoniae, a representative of the newly distinguished pHW126 plasmid family. Results A single repR locus located within the replication system of plasmid pIGRK encodes, in the same frame, two functional polypeptides: a full-length RepR protein and a RepR’ protein (with N-terminal truncation) translated from an internal START codon. Both proteins form homodimers, and interact with diverse DNA regions within the plasmid replication origin and repR promoter operator. Interestingly, RepR and RepR’ have opposing functions – RepR is crucial for initiation of pIGRK replication, while RepR’ is a negative regulator of this process. Nevertheless, both proteins act cooperatively as negative transcriptional regulators of their own expression. Conclusions Regulation of the initiation of pIGRK replication is a complex process in which a major role is played by two in-frame proteins with antagonistic functions. In-frame encoded Rep proteins are uncommon, having been described in only a few plasmids. This is the first description of such proteins in a plasmid of the pHW126 family.

A Lineage of Begomoviruses Encode Rep and AC4 Proteins of Enigmatic Ancestry: Hints on the Evolution of Geminiviruses in the New World

The begomoviruses (BGVs) are plant pathogens that evolved in the Old World during the Cretaceous and arrived to the New World (NW) in the Cenozoic era. A subgroup of NW BGVs, the “Squash leaf curl virus (SLCV) lineage” (S-Lin), includes viruses with unique characteristics. To get clues on the evolutionary origin of this lineage, a search for divergent members was undertaken. Four novel BGVs were characterized, including one that is basal to the group. Comparative analyses led to discover a ~670 bp genome module that is nearly exclusive of this lineage, encompassing the replication origin, the AC4 gene, and 480 bp of the Rep gene. A similar DNA module was found in two curtoviruses, hence suggesting that the S-Lin ancestor acquired its distinctive genomic segment by recombination with a curtovirus. This hypothesis was definitely disproved by an in-depth sequence analysis. The search for homologs of S-Lin Rep uncover the common origin of Rep proteins encoded by diverse Geminiviridae genera and viral “fossils” integrated at plant genomes. In contrast, no homolog of S-Lin Rep was found in public databases. Consequently, it was concluded that the SLCV clade ancestor evolved by a recombination event between a primitive NW BGV and a virus from a hitherto unknown lineage.

The specificity of ParR binding determines the compatibility of conjugative plasmids inClostridium perfringens

ABSTRACTPlasmids that encode the same replication machinery are generally unable to coexist in the same bacterial cell. However,Clostridium perfringensstrains often carry multiple conjugative toxin or antibiotic resistance plasmids that are closely related and encode similar Rep proteins. In many bacteria, plasmid partitioning upon cell division involves a ParMRC system and there are ~10 different ParMRC families inC. perfringens, with differences in amino acid sequences between each ParM family (15% − 54% identity). Since plasmids encoding genes belonging to the same ParMRC family are not observed in the same strain, these families appear to represent the basis for plasmid compatibility inC. perfringens. To understand this process, we examined the key recognition steps between ParR DNA-binding proteins and theirparCbinding sites. The ParR proteins bound to sequences within aparCsite from the same ParMRC family, but could not interact with aparCsite from a different ParMRC family. These data provide evidence that compatibility of the conjugative toxin plasmids ofC. perfringensis mediated by theirparMRC-like partitioning systems. This process provides a selective advantage by enabling the host bacterium to maintain separate plasmids that encode toxins that are specific for different host targets.