The flaviviruses dengue virus (DENV) and Zika virus (ZIKV) are severe health threats with rapidly expanding ranges. To identify the host cell dependencies of DENV and ZIKV, we completed orthologous ...functional genomic screens using RNAi and CRISPR/Cas9 approaches. The screens recovered the ZIKV entry factor AXL as well as multiple host factors involved in endocytosis (RAB5C and RABGEF), heparin sulfation (NDST1 and EXT1), and transmembrane protein processing and maturation, including the endoplasmic reticulum membrane complex (EMC). We find that both flaviviruses require the EMC for their early stages of infection. Together, these studies generate a high-confidence, systems-wide view of human-flavivirus interactions and provide insights into the role of the EMC in flavivirus replication.
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•RNAi and CRISPR/Cas9 screens were used to find flavivirus dependencies•The screens recovered host factors involved in endocytosis and heparin sulfation•The EMC is required by DENV and ZIKV in the early stages of replication•These studies give a systems-wide view of human-flavivirus interactions
Savidis et al. identify DENV and ZIKV dependencies using orthologous RNAi and CRISPR/Cas9 approaches. Multiple host factors involved in endocytosis and transmembrane protein processing, including the endoplasmic reticulum membrane complex (EMC), are important for flaviviral replication. Together, their studies generate a systems-wide view of human-flavivirus interactions.
Research has shown that RNA virus populations are highly variable, most likely due to low fidelity replication of RNA genomes. It is generally assumed that populations of DNA viruses will be less ...complex and show reduced variability when compared to RNA viruses. Here, we describe the use of high throughput sequencing for a genome wide study of viral populations from urine samples of neonates with congenital human cytomegalovirus (HCMV) infections. We show that HCMV intrahost genomic variability, both at the nucleotide and amino acid level, is comparable to many RNA viruses, including HIV. Within intrahost populations, we find evidence of selective sweeps that may have resulted from immune-mediated mechanisms. Similarly, genome wide, population genetic analyses suggest that positive selection has contributed to the divergence of the HCMV species from its most recent ancestor. These data provide evidence that HCMV, a virus with a large dsDNA genome, exists as a complex mixture of genome types in humans and offer insights into the evolution of the virus.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Human cytomegalovirus (HCMV) is the leading cause of birth defects associated with infections and a leading cause of transplantation failure. This study reveals the patterns and limits of HCMV ...genomic diversity by performing a large-scale analysis of HCMV sequences sampled from human hosts, identifying the hot and cold spots of variability. We find that the diversity is unevenly distributed across three host compartments and show that HCMV populations of vascular compartments are genetically constrained while enriched for polymorphisms of glycoproteins and regulatory proteins. This work significantly advances our understanding of the genomic diversity of HCMV in humans and has clear implications for the development of therapeutics against HCMV.
Human cytomegalovirus (HCMV) exhibits surprisingly high genomic diversity during natural infection although little is known about the limits or patterns of HCMV diversity among humans. To address this deficiency, we analyzed genomic diversity among congenitally infected infants. We show that there is an upper limit to HCMV genomic diversity in these patient samples, with â¼25% of the genome being devoid of polymorphisms. These low diversity regions were distributed across 26 loci that were preferentially located in DNA-processing genes. Furthermore, by developing, to our knowledge, the first genome-wide mutation and recombination rate maps for HCMV, we show that genomic diversity is positively correlated with these two rates. In contrast, median levels of viral genomic diversity did not vary between putatively single or mixed strain infections. We also provide evidence that HCMV populations isolated from vascular compartments of hosts from different continents are genetically similar and that polymorphisms in glycoproteins and regulatory proteins are enriched in these viral populations. This analysis provides the most highly detailed map of HCMV genomic diversity in human hosts to date and informs our understanding of the distribution of HCMV genomic diversity within human hosts.
Populations of human cytomegalovirus (HCMV), a large DNA virus, are highly polymorphic in patient samples, which may allow for rapid evolution within human hosts. To understand HCMV evolution, ...longitudinally sampled genomic populations from the urine and plasma of 5 infants with symptomatic congenital HCMV infection were analyzed. Temporal and compartmental variability of viral populations were quantified using high throughput sequencing and population genetics approaches. HCMV populations were generally stable over time, with ~88% of SNPs displaying similar frequencies. However, samples collected from plasma and urine of the same patient at the same time were highly differentiated with approximately 1700 consensus sequence SNPs (1.2% of the genome) identified between compartments. This inter-compartment differentiation was comparable to the differentiation observed in unrelated hosts. Models of demography (i.e., changes in population size and structure) and positive selection were evaluated to explain the observed patterns of variation. Evidence for strong bottlenecks (>90% reduction in viral population size) was consistent among all patients. From the timing of the bottlenecks, we conclude that fetal infection occurred between 13-18 weeks gestational age in patients analyzed, while colonization of the urine compartment followed roughly 2 months later. The timing of these bottlenecks is consistent with the clinical histories of congenital HCMV infections. We next inferred that positive selection plays a small but measurable role in viral evolution within a single compartment. However, positive selection appears to be a strong and pervasive driver of evolution associated with compartmentalization, affecting ≥ 34 of the 167 open reading frames (~20%) of the genome. This work offers the most detailed map of HCMV in vivo evolution to date and provides evidence that viral populations can be stable or rapidly differentiate, depending on host environment. The application of population genetic methods to these data provides clinically useful information, such as the timing of infection and compartment colonization.
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Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The challenge of distinguishing genetic drift from selection remains a central focus of population genetics. Time-sampled data may provide a powerful tool for distinguishing these processes, and we ...here propose approximate Bayesian, maximum likelihood, and analytical methods for the inference of demography and selection from time course data. Utilizing these novel statistical and computational tools, we evaluate whole-genome datasets of an influenza A H1N1 strain in the presence and absence of oseltamivir (an inhibitor of neuraminidase) collected at thirteen time points. Results reveal a striking consistency amongst the three estimation procedures developed, showing strongly increased selection pressure in the presence of drug treatment. Importantly, these approaches re-identify the known oseltamivir resistance site, successfully validating the approaches used. Enticingly, a number of previously unknown variants have also been identified as being positively selected. Results are interpreted in the light of Fisher's Geometric Model, allowing for a quantification of the increased distance to optimum exerted by the presence of drug, and theoretical predictions regarding the distribution of beneficial fitness effects of contending mutations are empirically tested. Further, given the fit to expectations of the Geometric Model, results suggest the ability to predict certain aspects of viral evolution in response to changing host environments and novel selective pressures.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Despite tremendous progress in our understanding of human immunodeficiency virus (HIV) natural history and advances in HIV treatment, there is neither an approved vaccine nor a cure for infection. ...Here, we describe the development and characterization of a novel replicating vaccine vector utilizing Cytomegalovirus (CMV) and a TLR5 adjuvant. After partial truncation of the central, immunodominant hypervariable domain, flagellin (fliC) from Salmonella was cloned downstream of a codon optimized gag gene from simian immunodeficiency virus (SIV) and transiently expressed in telomerized rhesus fibroblast (TeloRF) cells in culture. Lysates generated from these transfected cells induced the tumor necrosis factor alpha (TNF-α), in a mouse macrophage cell line, in a TLR5-dependent manner. The Gag/FliC expression construct was cloned into a bacterial artificial chromosome encoding the rhesus CMV (RhCMV) genome, and infectious RhCMV was generated following transfection of TeloRF cells. This virus stably expressed an SIV Gag/FliC fusion protein through four serial passages. Lysates generated from infected cells induced TNF-α in a TLR5-dependent manner. Western blot analysis of infected cell lysates verified expression of a Gag/FliC fusion protein using a SIV p27 capsid monoclonal antibody. Lastly, rhesus macaques inoculated with this novel RhCMV virus demonstrated increased inflammatory responses at the site of inoculation seven days post-infection when compared to the parental RhCMV. These results demonstrate that an artificially constructed replicating RhCMV expressing an SIV Gag/FliC fusion protein is capable of activating TLR5 in a macrophage cell line in vitro and induction of an altered inflammatory response in vivo. Ongoing animals studies are aimed at determining vaccine efficacy, including subsequent challenge with pathogenic SIV.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
RecA is essential for recombination, DNA repair and SOS induction in Escherichia coli. ATP hydrolysis is known to be important for RecA's roles in recombination and DNA repair. In vitro reactions ...modelling SOS induction minimally require ssDNA and non-hydrolyzable ATP analogues. This predicts that ATP hydrolysis will not be required for SOS induction in vivo. The requirement of ATP binding and hydrolysis for SOS induction in vivo is tested here through the study of recA4159 (K72A) and recA2201 (K72R). RecA4159 is thought to have reduced affinity for ATP. RecA2201 binds, but does not hydrolyse ATP. Neither mutant was able to induce SOS expression after UV irradiation. RecA2201, unlike RecA4159, could form filaments on DNA and storage structures as measured with RecA-GFP. RecA2201 was able to form hybrid filaments and storage structures and was either recessive or dominant to RecA⁺, depending on the ratio of the two proteins. RecA4159 was unable to enter RecA⁺ filaments on DNA or storage structures and was recessive to RecA⁺. It is concluded that ATP hydrolysis is essential for SOS induction. It is proposed that ATP binding is essential for storage structure formation and ability to interact with other RecA proteins in a filament.
Summary
RecA is important in recombination, DNA repair and repair of replication forks. It functions through the production of a protein–DNA filament. To study the localization of RecA in live ...Escherichia coli cells, the RecA protein was fused to the green fluorescence protein (GFP). Strains with this gene have recombination/DNA repair activities three‐ to tenfold below wild type (or about 1000‐fold above that of a recA null mutant). RecA–GFP cells have a background of green fluorescence punctuated with up to five foci per cell. Two types of foci have been defined: 4,6‐diamidino‐2‐phenylindole (DAPI)‐sensitive foci that are bound to DNA and DAPI‐insensitive foci that are DNA‐less aggregates/storage structures. In log phase cells, foci were not localized to any particular region. After UV irradiation, the number of foci increased and they localized to the cell centre. This suggested colocalization with the DNA replication factory. recA, recB and recF strains showed phenotypes and distributions of foci consistent with the predicted effects of these mutations.
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