Background Short read sequencing has been used extensively to decipher the genome diversity of human cytomegalovirus (HCMV) strains, but falls short to reveal individual genomes in mixed HCMV strain populations. Novel third-generation sequencing platforms offer an extended read length and promise to resolve how distant polymorphic sites along individual genomes are linked. In the present study, we established a long amplicon PacBio sequencing workflow to identify the absolute and relative quantities of unique HCMV haplotypes spanning over multiple hypervariable sites in mixtures. Initial validation of this approach was performed with defined HCMV DNA templates derived from cell-culture enriched viruses and was further tested for its suitability on patient samples carrying mixed HCMV infections. Results Total substitution and indel error rate of mapped reads ranged from 0.17 to 0.43% depending on the stringency of quality trimming. Artificial HCMV DNA mixtures were correctly determined down to 1% abundance of the minor DNA source when the total HCMV DNA input was 4 × 104 copies/ml. PCR products of up to 7.7 kb and a GC content < 55% were efficiently generated when DNA was directly isolated from patient samples. In a single sample, up to three distinct haplotypes were identified showing varying relative frequencies. Alignments of distinct haplotype sequences within patient samples showed uneven distribution of sequence diversity, interspersed by long identical stretches. Moreover, diversity estimation at single polymorphic regions as assessed by short amplicon sequencing may markedly underestimate the overall diversity of mixed haplotype populations. Conclusions Quantitative haplotype determination by long amplicon sequencing provides a novel approach for HCMV strain characterisation in mixed infected samples which can be scaled up to cover the majority of the genome by multi-amplicon panels. This will substantially improve our understanding of intra-host HCMV strain diversity and its dynamic behaviour.
Human cytomegalovirus (HCMV) envelope glycoprotein complexes, gH/gL/gO trimer and gH/gL/UL128-131 pentamer, are important for cell-free HCMV entry. While soluble NRP2-Fc (sNRP2-Fc) interferes with epithelial/endothelial cell entry through UL128, soluble platelet-derived growth factor receptor α-Fc (sPDGFRα-Fc) interacts with gO, thereby inhibiting infection of all cell types. Since gO is the most variable subunit, we investigated the influence of gO polymorphism on the inhibitory capacities of sPDGFRα-Fc and sNRP2-Fc. Accordingly, gO genotype 1c (GT1c) sequence was fully or partially replaced by gO GT2b, GT3, and GT5 sequences in the bacterial artificial chromosome (BAC) TB40-BAC4-luc background (where luc is luciferase). All mutants were tested for fibroblast and epithelial cell infectivity, for virion content of gB, gH, and gO, and for infection inhibition by sPDGFRα-Fc and sNRP2-Fc. Full-length and partial gO GT swapping may increase epithelial-to-fibroblast ratios due to subtle alterations in fibroblast and/or epithelial infectivity but without substantial changes in gB and gH levels in mutant virions. All gO GT mutants except recombinant gO GT1c/3 displayed a nearly complete inhibition at 1.25 μg/ml sPDGFRα-Fc on epithelial cells (98% versus 91%), and all experienced complete inhibition on fibroblasts (≥99%). While gO GT replacement did not influence sNRP2-Fc inhibition at 1.25 μg/ml on epithelial cells (97% to 99%), it rendered recombinant mutant GT1c/3 moderately accessible to fibroblast inhibition (40%). In contrast to the steep sPDGFRα-Fc inhibition curves (slope of >1.0), sNRP2-Fc dose-response curves on epithelial cells displayed slopes of ∼1.0, suggesting functional differences between these entry inhibitors. Our findings demonstrate that artificially generated gO recombinants rather than the major gO genotypic forms may affect the inhibitory capacities of sPDGFRα and sNRP2 in a cell type-dependent manner. IMPORTANCE Human cytomegalovirus (HCMV) is known for its broad cell tropism, as reflected by the different organs and tissues affected by HCMV infection. Hence, inhibition of HCMV entry into distinct cell types could be considered a promising therapeutic option to limit cell-free HCMV infection. Soluble forms of cellular entry receptor PDGFRα rather than those of entry receptor neuropilin-2 inhibit infection of multiple cell types. sPDGFRα specifically interacts with gO of the trimeric gH/gL/gO envelope glycoprotein complex. HCMV strains may differ with respect to the amounts of trimer in virions and the highly polymorphic gO sequence. In this study, we show that the major gO genotypes of HCMV that are also found in vivo are similarly well inhibited by sPDGFRα. Novel gO genotypic forms potentially emerging through recombination, however, may evade sPDGFRα inhibition on epithelial cells. These findings provide useful additional information for the future development of anti-HCMV therapeutic compounds based on sPDGFRα.
Ice nucleation (IN) active bacteria such as Pseudomonas syringae promote the growth of ice crystals more effectively than any material known. Using the specialized ice nucleation protein (INP) InaZ, P. syringae-the well studied epiphytic plant pathogen-attacks plants by frost damage and, likewise fascinating, drives ice nucleation within clouds when airborne in the atmosphere by linkage to the Earth's water cycle. While ice nucleation proteins play a tremendous role for life on the planet, the molecular details of their activity on the bacterial membrane surface are largely unknown. Bacterial ghosts (BGs) derived from Escherichia coli can be used as simplified model systems to study the mode of action of InaZ. In this work, the authors used BGs to study the role of InaZ localization on the luminal side of the bacterial inner membrane. Naturally, P. syringae INPs are displayed on the surface of the outer membrane; so in contrast, the authors engineered an N-terminal truncated form of inaZ lacking the transport sequence for anchoring of InaZ on the outer membrane. This construct was fused to N-and C-terminal inner membrane anchors and expressed in Escherichia coli C41. The IN activity of the corresponding living recombinant E. coli catalyzing interfacial ice formation of supercooled water at high subzero temperatures was tested by a droplet-freezing assay and surface spectroscopy. The median freezing temperature (T 50 ) of the parental living E. coli C41 cells without INP was detected at −20.1 °C and with inner membrane anchored INPs at a T 50 value between −7 and −9 °C, demonstrating that the induction of IN from the inside of the bacterium by inner membrane anchored INPs facing the luminal inner membrane side is very similar to IN induced by bacterial INPs located at the outer membrane. Bacterial ghosts derived from these different constructs showed first droplet freezing values between −6 and −8 °C, whereas E. coli C41 BGs alone without carrying inner membrane anchored INPs exhibit a T 50 of −18.9 °C. Sum frequency generation spectroscopy showed structural ordered water at the BG/water interface, which increased close to the water melting point. Together, this indicates that the more efficient IN of INP-BGs compared to their living parental strains can be explained by the free access of inner membrane anchored INP constructs to ultrapure water filling the inner space of the BGs.
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