High parasite diversity maintained after an alga-virus coevolutionary arms race.

Arms race dynamics are a common outcome of host-parasite coevolution. While they can theoretically be maintained indefinitely, realistic arms races are expected to be finite. Once an arms race has ended, for example due to the evolution of a generalist-resistant host, the system may transition into coevolutionary dynamics that favour long-term diversity. In microbial experiments, host-parasite arms races often transition into a stable coexistence of generalist-resistant hosts, (semi-)susceptible hosts, and parasites. While long-term host diversity is implicit in these cases, parasite diversity is usually overlooked. In this study, we examined parasite diversity after the end of an experimental arms race between a unicellular alga (Chlorella variabilis) and its lytic virus (PBCV-1). First, we isolated virus genotypes from multiple time points from two replicate microcosms. A time-shift experiment confirmed that the virus isolates had escalating host ranges, i.e., that arms races had occurred. We then examined the phenotypic and genetic diversity of virus isolates from the post-arms race phase. Post-arms race virus isolates had diverse host ranges, survival probabilities, and growth rates; they also clustered into distinct genetic groups. Importantly, host range diversity was maintained throughout the post-arms race phase, and the frequency of host range phenotypes fluctuated over time. We hypothesize that this dynamic polymorphism was maintained by a combination of fluctuating selection and demographic stochasticity. Together with previous work in prokaryotic systems, our results link experimental observations of arms races to natural observations of long-term host and parasite diversity.

Chimeric padlock and iLock probes for increased efficiency of targeted RNA detection.

Many approaches exist to detect RNA using complementary oligonucleotides. DNA ligation-based techniques can improve discrimination of subtle sequence variations, but they have been difficult to implement for direct RNA analysis due to the infidelity and inefficiency of most DNA ligases on RNA. In this report, we have systematically studied if ribonucleotide substitutions in padlock probes can provide higher catalytic efficiencies for Chlorella virus DNA ligase (PBCV-1 DNA ligase) and T4 RNA ligase 2 (T4Rnl2) on RNA. We provide broad characterization of end-joining fidelity for both enzymes in RNA-templated 3'-OH RNA/5'-pDNA chimeric probe ligation. Both ligases showed increased ligation efficiency toward chimeric substrates on RNA. However, end-joining fidelity of PBCV-1 DNA ligase remained poor, while T4Rnl2 showed a somewhat better end-joining fidelity compared to PBCV-1 DNA ligase. The recently presented invader padlock (iLock) probes overcome the poor end-joining fidelity of PBCV-1 DNA ligase by the requirement of target-dependent 5' flap removal prior to ligation. Here we show that two particular ribonucleotide substitutions greatly improve the activation and ligation rate of chimeric iLock probes on RNA. We characterized the end-joining efficiency and fidelity of PBCV-1 DNA ligase and T4Rnl2 with chimeric iLock probes on RNA and found that both enzymes exhibit high ligation fidelities for single nucleotide polymorphisms on RNA. Finally, we applied the chimeric probe concept to directly differentiate between human and mouse ACTB mRNA in situ, demonstrating chimeric padlock and iLock probes as superior to their DNA equivalents.

Structure of the chlorovirus PBCV-1 major capsid glycoprotein determined by combining crystallographic and carbohydrate molecular modeling approaches.

The glycans of the major capsid protein (Vp54) of Paramecium bursaria chlorella virus (PBCV-1) were recently described and found to be unusual. This prompted a reexamination of the previously reported Vp54 X-ray structure. A detailed description of the complete glycoprotein was achieved by combining crystallographic data with molecular modeling. The crystallographic data identified most of the monosaccharides located close to the protein backbone, but failed to detect those further from the glycosylation sites. Molecular modeling complemented this model by adding the missing monosaccharides and examined the conformational preference of the whole molecule, alone or within the crystallographic environment. Thus, combining X-ray crystallography with carbohydrate molecular modeling resulted in determining the complete glycosylated structure of a glycoprotein. In this case, it is the chlorovirus PBCV-1 major capsid protein.

The N-glycan structures of the antigenic variants of chlorovirus PBCV-1 major capsid protein help to identify the virus-encoded glycosyltransferases.

The chlorovirus Paramecium bursaria chlorella virus 1 (PBCV-1) is a large dsDNA virus that infects the microalga Chlorella variabilis NC64A. Unlike most other viruses, PBCV-1 encodes most, if not all, of the machinery required to glycosylate its major capsid protein (MCP). The structures of the four N-linked glycans from the PBCV-1 MCP consist of nonasaccharides, and similar glycans are not found elsewhere in the three domains of life. Here, we identified the roles of three virus-encoded glycosyltransferases (GTs) that have four distinct GT activities in glycan synthesis. Two of the three GTs were previously annotated as GTs, but the third GT was identified in this study. We determined the GT functions by comparing the WT glycan structures from PBCV-1 with those from a set of PBCV-1 spontaneous GT gene mutants resulting in antigenic variants having truncated glycan structures. According to our working model, the virus gene a064r encodes a GT with three domains: domain 1 has a ß-l-rhamnosyltransferase activity, domain 2 has an α-l-rhamnosyltransferase activity, and domain 3 is a methyltransferase that decorates two positions in the terminal α-l-rhamnose (Rha) unit. The a075l gene encodes a ß-xylosyltransferase that attaches the distal d-xylose (Xyl) unit to the l-fucose (Fuc) that is part of the conserved N-glycan core region. Last, gene a071r encodes a GT that is involved in the attachment of a semiconserved element, α-d-Rha, to the same l-Fuc in the core region. Our results uncover GT activities that assemble four of the nine residues of the PBCV-1 MCP N-glycans.

The Roles of Electrostatic Interactions in Capsid Assembly Mechanisms of Giant Viruses.

In the last three decades, many giant DNA viruses have been discovered. Giant viruses present a unique and essential research frontier for studies of self-assembly and regulation of supramolecular assemblies. The question on how these giant DNA viruses assemble thousands of proteins so accurately to form their protein shells, the capsids, remains largely unanswered. Revealing the mechanisms of giant virus assembly will help to discover the mysteries of many self-assembly biology problems. Paramecium bursaria Chlorella virus-1 (PBCV-1) is one of the most intensively studied giant viruses. Here, we implemented a multi-scale approach to investigate the interactions among PBCV-1 capsid building units called capsomers. Three binding modes with different strengths are found between capsomers around the relatively flat area of the virion surface at the icosahedral 2-fold axis. Furthermore, a capsomer structure manipulation package is developed to simulate the capsid assembly process. Using these tools, binding forces among capsomers were investigated and binding funnels were observed that were consistent with the final assembled capsid. In addition, total binding free energies of each binding mode were calculated. The results helped to explain previous experimental observations. Results and tools generated in this work established an initial computational approach to answer current unresolved questions regarding giant virus assembly mechanisms. Results will pave the way for studying more complicated process in other biomolecular structures.

Strong selection and high mutation supply characterize experimental Chlorovirus evolution.

Characterizing how viruses evolve expands our understanding of the underlying fundamental processes, such as mutation, selection and drift. One group of viruses whose evolution has not yet been extensively studied is the Phycodnaviridae, a globally abundant family of aquatic large double-stranded (ds)DNA (dsDNA) viruses. Here we studied the evolutionary change of Paramecium bursaria chlorella virus 1 during experimental coevolution with its algal host. We used pooled genome sequencing of six independently evolved populations to characterize genomic change over five time points. Across six experimental replicates involving either strong or weak demographic fluctuations, we found single nucleotide polymorphisms (SNPs) at sixty-seven sites. The occurrence of genetic variants was highly repeatable, with just two of the SNPs found in only a single experimental replicate. Three genes A122/123R, A140/145R and A540L showed an excess of variable sites, providing new information about potential targets of selection during Chlorella-Chlorovirus coevolution. Our data indicated that the studied populations were not mutation-limited and experienced strong positive selection. Our investigation highlighted relevant processes governing the evolution of aquatic large dsDNA viruses, which ultimately contributes to a better understanding of the functioning of natural aquatic ecosystems.

Chlorovirus PBCV-1 Multidomain Protein A111/114R Has Three Glycosyltransferase Functions Involved in the Synthesis of Atypical N-Glycans.

The structures of the four N-linked glycans from the prototype chlorovirus PBCV-1 major capsid protein do not resemble any other glycans in the three domains of life. All known chloroviruses and antigenic variants (or mutants) share a unique conserved central glycan core consisting of five sugars, except for antigenic mutant virus P1L6, which has four of the five sugars. A combination of genetic and structural analyses indicates that the protein coded by PBCV-1 gene a111/114r, conserved in all chloroviruses, is a glycosyltransferase with three putative domains of approximately 300 amino acids each. Here, in addition to in silico sequence analysis and protein modeling, we measured the hydrolytic activity of protein A111/114R. The results suggest that domain 1 is a galactosyltransferase, domain 2 is a xylosyltransferase and domain 3 is a fucosyltransferase. Thus, A111/114R is the protein likely responsible for the attachment of three of the five conserved residues of the core region of this complex glycan, and, if biochemically corroborated, it would be the second three-domain protein coded by PBCV-1 that is involved in glycan synthesis. Importantly, these findings provide additional support that the chloroviruses do not use the canonical host endoplasmic reticulum-Golgi glycosylation pathway to glycosylate their glycoproteins; instead, they perform glycosylation independent of cellular organelles using virus-encoded enzymes.

The Role of Tape Measure Protein in Nucleocytoplasmic Large DNA Virus Capsid Assembly.

Nucleocytoplasmic large DNA viruses (NCLDVs) are a group of large viruses that infect a wide range of hosts, from animals to protists. These viruses are grouped together in NCLDV based on genomic sequence analyses. They share a set of essential genes for virion morphogenesis and replication. Most NCLDVs generally have large physical sizes while their morphologies vary in different families, such as icosahedral, brick, or oval shape, raising the question of the possible regulatory factor on their morphogenesis. The capsids of icosahedral NCLDVs are assembled from small building blocks, named capsomers, which are the trimeric form of the major capsid proteins. Note that the capsids of immature poxvirus are spherical even though they are assembled from capsomers that share high structural conservation with those icosahedral NCLDVs. The recently published high resolution structure of NCLDVs, Paramecium bursaria Chlorella virus 1 and African swine fever virus, described the intensive network of minor capsid proteins that are located underneath the capsomers. Among these minor proteins is the elongated tape measure protein (TmP) that spans from one icosahedral fivefold vertex to another. In this study, we focused on the critical roles that TmP plays in the assembly of icosahedral NCLDV capsids, answering a question raised in a previously proposed spiral mechanism. Interestingly, basic local alignment search on the TmPs showed no significant hits in poxviruses, which might be the factor that differentiates poxviruses and icosahedral NCLDVs in their morphogenesis.

Identification of a Chlorovirus PBCV-1 Protein Involved in Degrading the Host Cell Wall during Virus Infection.

Chloroviruses are unusual among viruses infecting eukaryotic organisms in that they must, like bacteriophages, penetrate a rigid cell wall to initiate infection. Chlorovirus PBCV-1 infects its host, Chlorella variabilis NC64A by specifically binding to and degrading the cell wall of the host at the point of contact by a virus-packaged enzyme(s). However, PBCV-1 does not use any of the five previously characterized virus-encoded polysaccharide degrading enzymes to digest the Chlorella host cell wall during virus entry because none of the enzymes are packaged in the virion. A search for another PBCV-1-encoded and virion-associated protein identified protein A561L. The fourth domain of A561L is a 242 amino acid C-terminal domain, named A561LD4, with cell wall degrading activity. An A561LD4 homolog was present in all 52 genomically sequenced chloroviruses, infecting four different algal hosts. A561LD4 degraded the cell walls of all four chlorovirus hosts, as well as several non-host Chlorella spp. Thus, A561LD4 was not cell-type specific. Finally, we discovered that exposure of highly purified PBCV-1 virions to A561LD4 increased the specific infectivity of PBCV-1 from about 25-30% of the particles forming plaques to almost 50%. We attribute this increase to removal of residual host receptor that attached to newly replicated viruses in the cell lysates.

SMRT Sequencing of Paramecium Bursaria Chlorella Virus-1 Reveals Diverse Methylation Stability in Adenines Targeted by Restriction Modification Systems.

Chloroviruses (family Phycodnaviridae) infect eukaryotic, freshwater, unicellular green algae. A unique feature of these viruses is an abundance of DNA methyltransferases, with isolates dedicating up to 4.5% of their protein coding potential to these genes. This diversity highlights just one of the long-standing values of the chlorovirus model system; where group-wide epigenomic characterization might begin to elucidate the function(s) of DNA methylation in large dsDNA viruses. We characterized DNA modifications in the prototype chlorovirus, PBCV-1, using single-molecule real time (SMRT) sequencing (aka PacBio). Results were compared to total available sites predicted in silico based on DNA sequence alone. SMRT-software detected N6-methyl-adenine (m6A) at GATC and CATG recognition sites, motifs previously shown to be targeted by PBCV-1 DNA methyltransferases M.CviAI and M. CviAII, respectively. At the same time, PacBio analyses indicated that 10.9% of the PBCV-1 genome had large interpulse duration ratio (ipdRatio) values, the primary metric for DNA modification identification. These events represent 20.6x more sites than can be accounted for by all available adenines in GATC and CATG motifs, suggesting base or backbone modifications other than methylation might be present. To define methylation stability, we cross-compared methylation status of each GATC and CATG sequence in three biological replicates and found ∼81% of sites were stably methylated, while ∼2% consistently lack methylation. The remaining 17% of sites were stochastically methylated. When methylation status was analyzed for both strands of each target, we show that palindromes existed in completely non-methylated states, fully-methylated states, or hemi-methylated states, though GATC sites more often lack methylation than CATG sequences. Given that both sequences are targeted by not just methyltransferases, but by restriction endonucleases that are together encoded by PBCV-1 as virus-originating restriction modification (RM) systems, there is strong selective pressure to modify all target sites. The finding that most instances of non-methylation are associated with hemi-methylation is congruent with observations that hemi-methylated palindromes are resistant to cleavage by restriction endonucleases. However, sites where hemi-methylation is conserved might represent a unique regulatory function for PBCV-1. This study serves as a baseline for future investigation into the epigenomics of chloroviruses and their giant virus relatives.

Sensitive and specific microRNA detection by RNA dependent DNA ligation and rolling circle optical signal amplification.

MicroRNAs (miRNAs) have been regarded as potential biomarkers in early diagnosis of cancer. Since the high sequence similarity among miRNA family members, biosensing miRNAs with single-base resolution is still a challenge, particularly when the different base is located at the terminal of miRNA. Herein, we developed two real-time fluorescence monitoring methods for miRNA detection utilizing efficient PBCV-1 DNA ligase mediated target miRNA dependent DNA ligation, followed by rolling circle signal amplification. Compared to duplex-specific nuclease (DSN) enhanced rolling circle transcription (RCT) system, nicking endonuclease (NEase) assisted rolling circle amplification (PRCA-NESA) can provide higher amplification efficiency, and achieve a limit-of-detection of 0.5 amol for miR-17 in 10 µL sample. More importantly, benefiting from the unique characteristics of PBCV-1 DNA ligase, we designed an asymmetric PRCA-NESA method, which can greatly discriminate the single-base difference at either 5'- or 3'-terminals of miRNAs. MiR-17 from various tumor cells also can be reliably detected. In conclusion, our strategy exploited the application potential of PBCV-1 DNA ligase in biosensing, and provided a new idea to highly specific miRNA detection, thereby would possess a promising potential for further application in biomedical research and early cancer diagnosis.

Dynamic attachment of Chlorovirus PBCV-1 to Chlorella variabilis.

Chloroviruses infect their hosts by specifically binding to and degrading the cell wall of their algal hosts at the site of attachment, using an intrinsic digesting enzyme(s). Chlorovirus PBCV-1 stored as a lysate survived longer than virus alone, suggesting virus attachment to cellular debris may be reversible. Ghost cells (algal cells extracted with methanol) were used as a model to study reversibility of PBCV-1 attachment because ghost cells are as susceptible to attachment and wall digestion as are live cells. Reversibility of attachment to ghost cells was examined by releasing attached virions with a cell wall degrading enzyme extract. The majority of the released virions retained infectivity even after re-incubating the released virions with ghost cells two times. Thus the chloroviruses appear to have a dynamic attachment strategy that may be beneficial in indigenous environments where cell wall debris can act as a refuge until appropriate host cells are available.