Olfactory entry promotes herpesvirus recombination

2021 ◽  
Author(s):  
Wanxiaojie Xie ◽  
Kimberley Bruce ◽  
Helen E. Farrell ◽  
Philip G. Stevenson
Keyword(s):  
Point Mutations ◽  
Murine Cytomegalovirus ◽  
Entry Site ◽  
Recombinant Viruses ◽  
Viral Loads ◽  
Show Evidence ◽  
Olfactory Cells ◽  
Herpesvirus 4 ◽  
Respiratory Epithelial ◽  
Human Herpesviruses

Herpesvirus genomes show abundant evidence of past recombination. Its functional importance is unknown. A key question is whether recombinant viruses can outpace the immunity induced by their parents to reach higher loads. We tested this by co-infecting mice with attenuated mutants of Murid Herpesvirus-4 (MuHV-4). Infection by the natural olfactory route routinely allowed mutant viruses to reconstitute wild-type genotypes and reach normal viral loads. Lung co-infections rescued much less well. Attenuated murine cytomegalovirus mutants similarly showed recombinational rescue via the nose but not the lungs. These infections spread similarly, so route-specific rescue implied that recombination occurred close to the olfactory entry site. Rescue of replication-deficient MuHV-4 confirmed this, showing that coinfection occurred in the first encountered olfactory cells. This worked even with asynchronous inoculation, implying that a defective virus can wait here for later rescue. Virions entering the nose get caught on respiratory mucus, which the respiratory epithelial cilia push back towards the olfactory surface. Early infection was correspondingly focussed on the anterior olfactory edge. Thus, by concentrating incoming infection into a small area, olfactory entry seems to promote functionally significant recombination. Importance All organisms depend on genetic diversity to cope with environmental change. Small viruses rely on frequent point mutations. This is harder for herpesviruses because they have larger genomes. Recombination provides another means of genetic optimization. Human herpesviruses often co-infect, and they show evidence of past recombination, but whether this is rare and incidental or functionally important is unknown. We showed that herpesviruses entering mice via the natural olfactory route meet reliably enough for recombination routinely to repair crippling mutations and restore normal viral loads. It appeared to occur in the first encountered olfactory cells and reflected a concentration of infection at the anterior olfactory edge. Thus, natural host entry incorporates a significant capacity for herpesvirus recombination.

scholarly journals Olfactory host entry supports herpesvirus recombination

2020 ◽  
Author(s):  
Wanxiaojie Xie ◽  
Kimberley Bruce ◽  
Helen E. Farrell ◽  
Philip G. Stevenson
Keyword(s):  
Salivary Gland ◽  
Lung Infection ◽  
Latent Virus ◽  
Murine Cytomegalovirus ◽  
Normal Host ◽  
New Hosts ◽  
Olfactory Cells ◽  
Lung Infections ◽  
Herpesvirus 4 ◽  
Virus Strains

AbstractHerpesvirus genomes record abundant recombination. Its impact on infection remains ill-defined. When co-infecting mice by the natural olfactory route, individually incapacitated Murid Herpesvirus-4 (MuHV-4) mutants routinely recombined to restore normal host colonization. Lung infection rescued much less well. Murine cytomegalovirus mutants deficient in salivary gland colonization also showed rescue via the nose but not the lungs. As nose and lung infections show similar spread, efficient recombination seemed specific to olfactory entry. Rescue of replication-deficient MuHV-4 implied co-infection of the first encountered cells, and this worked also with asynchronous inoculation, suggesting that latent virus could lie in wait for later reactivation. Inhaled MuHV-4 is commonly caught on respiratory mucus, which epithelial cilia push back towards the olfactory surface, and infection was correspondingly frequent at the anterior olfactory edge. Thus olfactory entry provides a general means for herpesviruses to meet.Author summaryInter-strain recombination allows viruses to optimise infection in diverse hosts. Many herpesviruses show past recombination. Yet they are ancient pathogens, so this past may be remote and recombination rare. Diverse herpesviruses enter new hosts via olfactory cells. We show that such entry routinely allows recombination between co-infecting virus strains, even when one strain cannot spread. Recombination was contrastingly rare after lung infection. Thus, entry via olfactory cells specifically supports frequent herpesvirus recombination.

scholarly journals Scans of the MYC mRNA reveal multiple stable secondary structures—including a 3′ UTR motif, conserved across vertebrates, that can affect gene expression

2019 ◽  
Author(s):  
Collin A. O’Leary ◽  
Ryan J. Andrews ◽  
Van S. Tompkins ◽  
Jonathan L. Chen ◽  
Jessica L. Childs-Disney ◽  
...  
Keyword(s):  
Internal Ribosomal Entry Site ◽  
Folding Energy ◽  
Entry Site ◽  
Rna Targets ◽  
High Propensity ◽  
Transcriptional Regulatory ◽  
Show Evidence ◽  
Analysis Window ◽  
Affect Gene Expression ◽  
Human Transcription Factor

AbstractThe MYC gene encodes a human transcription factor and proto-oncogene that is dysregulated in over half of all known cancers. To better understand potential post-transcriptional regulatory features affecting MYC expression, we analyzed secondary structure in the MYC mRNA using a program that is optimized for finding small locally-folded motifs with a high propensity for function. This was accomplished by calculating folding metrics across the MYC sequence using a sliding analysis window and generating unique consensus base pairing models weighted by their lower-than-random predicted folding energy. A series of 30 motifs were identified, primarily in the 5’ and 3’ untranslated regions, which show evidence of structural conservation and compensating mutations across vertebrate MYC homologs. This analysis was able to recapitulate known elements found within an internal ribosomal entry site, as well as discover a novel element in the 3’ UTR that is unusually stable and conserved. This novel motif was shown to affect MYC expression: likely via modulation of miRNA target accessibility. In addition to providing basic insights into mechanisms that regulate MYC expression, this study provides numerous, potentially druggable RNA targets for the MYC gene, which is considered “undruggable” at the protein level.

The transmission rate of MCMV in house mice in pens: implications for virally vectored immunocontraception

2009 ◽  
Vol 36 (5) ◽  
pp. 386 ◽  
Author(s):  
A. D. Arthur ◽  
C. J. Krebs ◽  
R. P. Pech ◽  
L. N. Farroway ◽  
G. R. Singleton
Keyword(s):  
Transmission Rate ◽  
Wild Mouse ◽  
Fertility Control ◽  
Murine Cytomegalovirus ◽  
House Mice ◽  
Recombinant Viruses ◽  
Economic Thresholds ◽  
Rapid Transmission ◽  
Future Work ◽  
Free Ranging

Pest mammals have severe economic, environmental and social impacts throughout the world. Fertility control could reduce these impacts. Murine cytomegalovirus (MCMV) is being considered as an immunocontraceptive vector to control outbreaks of house mice (Mus domesticus) in Australian grain-growing regions. For successful control, a modified MCMV must transmit at a sufficient rate to keep populations of house mice below acceptable economic thresholds. We used disease models developed previously by using observations of free-ranging wild-mouse populations to assess the transmission rate of two laboratory strains of MCMV (N1 and G4) collected in a previous experiment. Mice contained in pens were deliberately infected with the N1 strain only, or with the N1 strain followed by the G4 strain. If we assume density-dependent transmission, which is the more likely mode of transmission, we found the N1 strain of MCMV transmitted at a rate ~1/300 of the rate of field strains, and hence too slowly for successful virally vectored immunocontraception (VVIC). If transmission was frequency-dependent, the rate of transmission was ~1/3 of the rate of field strains, and hence may allow successful VVIC. The G4 strain transmitted at least as slowly as the N1 strain, and possibly much more slowly; however, we could not determine whether this was an inherent property of the G4 strain or whether it was caused by competition with the N1 strain. Given the reliance of successful VVIC on rapid transmission, we recommend that future work in any VVIC system explicitly quantifies the transmission rate of recombinant viruses relative to field strains, both in the presence and absence of competing strains.

scholarly journals An acidic residue buried in the dimer interface of isocitrate dehydrogenase 1 (IDH1) helps regulate catalysis and pH sensitivity

2020 ◽  
Author(s):  
Lucas A. Luna ◽  
Zachary Lesecq ◽  
Katharine A. White ◽  
An Hoang ◽  
David A. Scott ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Isocitrate Dehydrogenase ◽  
Point Mutations ◽  
Catalytic Efficiency ◽  
Ph Sensitivity ◽  
Isocitrate Dehydrogenase 1 ◽  
Dimer Interface ◽  
Molecular Determinants ◽  
Show Evidence ◽  
Ionizable Residues

ABSTRACTIsocitrate dehydrogenase 1 (IDH1) catalyzes the reversible NADP+-dependent conversion of isocitrate to α-ketoglutarate (α-KG) to provide critical cytosolic substrates and drive NADPH-dependent reactions like lipid biosynthesis and glutathione regeneration. In biochemical studies, the forward reaction is studied at neutral pH, while the reverse reaction is typically characterized in more acidic buffers. This led us to question whether IDH1 catalysis is pH-regulated, which would have functional implications under conditions that alter cellular pH, like apoptosis, hypoxia, cancer, and neurodegenerative diseases. Here, we show evidence of catalytic regulation of IDH1 by pH, identifying a trend of increasing kcat values for α-KG production upon increasing pH in the buffers we tested. To understand the molecular determinants of IDH1 pH sensitivity, we used the pHinder algorithm to identify buried ionizable residues predicted to have shifted pKa values. Such residues can serve as pH sensors, with changes in protonation states leading to conformational changes that regulate catalysis. We identified an acidic residue buried at the IDH1 dimer interface, D273, with a predicted pKa value upshifted into the physiological range. D273 point mutations had decreased catalytic efficiency and, importantly, loss of pH-regulated catalysis. Based on these findings, we conclude that IDH1 activity is regulated, at least in part, by pH. We show this regulation is mediated by at least one buried acidic residue ∼12 Å from the IDH1 active site. By establishing mechanisms of regulation of this well-conserved enzyme, we highlight catalytic features that may be susceptible to pH changes caused by cell stress and disease.

scholarly journals Docosahexaenoic Acid Supplementation Represses the Early Immune Response Against Murine Cytomegalovirus but Enhances NK Cell Effector Function

2021 ◽  
Author(s):  
Shuting Wu ◽  
Lili Wang ◽  
Hongyan Peng ◽  
Shuju Zhang ◽  
Qinglan Yang ◽  
...  
Keyword(s):  
Docosahexaenoic Acid ◽  
Nk Cells ◽  
Nk Cell ◽  
Cell Subset ◽  
Early Response ◽  
Murine Cytomegalovirus ◽  
Mitochondrial Activity ◽  
Cmv Infection ◽  
Viral Loads ◽  
Cell Effector

Abstract Background Docosahexaenoic acid (DHA) supplementation is beneficial for several chronic diseases; however, its effect on immune regulation is still debated. Given the prevalence of cytomegalovirus (CMV) infection and because natural killer (NK) cells are the component of innate immunity critical for controlling CMV infection, the current study explored the effect of a DHA-enriched diet on susceptibility and the NK cell effector response to murine (M) CMV infection. Results Male C57BL/6 mice fed a control or DHA-enriched diet for 3 weeks were infected with MCMV and sacrificed at the indicated time points postinfection. Compared with control mice, DHA-fed mice had higher liver and spleen viral loads on day 7 postinfection, but final MCMV clearance was not affected. The ratio and total numbers of NK cells and their terminal mature cell subset (KLRG1+ and Ly49H+ NK cells) were reduced compared with those in control mice on day 7 but not day 21 postinfection. DHA feeding resulted in higher IFN-γ and granzyme B expression in splenic NK cells on day 7 postinfection. A mechanistic analysis showed that the splenic NK cells of DHA-fed mice showed enhanced glucose uptake, increased CD71 and CD98 expression, and higher mitochondrial mass and activity than those of control mice. In addition, DHA-fed mice showed reductions in the total numbers and activation of CD4+ and CD8+ T cells. Conclusions These results suggest that DHA supplementation represses the early response to CMV infection but preserves NK cell effector functions by improving mitochondrial activity, which may play critical roles in subsequent MCMV clearance.

scholarly journals Murine cytomegalovirus disseminates independently of CX3CR1, CCL2 or its m131/m129 chemokine homologue

2019 ◽  
Vol 100 (12) ◽  
pp. 1695-1700 ◽  
Author(s):  
Helen E. Farrell ◽  
Kimberley Bruce ◽  
Alec J. Redwood ◽  
Philip G. Stevenson
Keyword(s):  
Salivary Gland ◽  
Murine Cytomegalovirus ◽  
Entry Site ◽  
Fractalkine Receptor ◽  
Systemic Spread ◽  
Infected Cells ◽  
Initial Seeding ◽  
Receptor Cx3cr1 ◽  
Patrolling Monocytes

Cytomegaloviruses (CMVs) use myeloid cells to move within their hosts. Murine CMV (MCMV) colonizes the salivary glands for long-term shedding, and reaches them via CD11c+ infected cells. A need to recruit patrolling monocytes for systemic spread has been proposed, based on poor salivary gland infection in fractalkine receptor (CX3CR1)-deficient mice. We found no significant CX3CR1 dependence of salivary gland infection. CCL2 and the viral m131/m129 chemokine homologue were also redundant for acute MCMV spread, arguing against a need for inflammation or infection to recruit additional monocytes to the entry site. M131/m129 promoted salivary gland infection, but only after the initial seeding of infected cells to this site. Our data support the idea that MCMV disseminates by infecting and mobilizing tissue-resident dendritic cells.

scholarly journals A Deleted Deletion Site in a New Vector Strain and Exceptional Genomic Stability of Plaque-Purified Modified Vaccinia Ankara (MVA)

2019 ◽  
Vol 35 (2) ◽  
pp. 212-226 ◽  
Author(s):  
Ingo Jordan ◽  
Deborah Horn ◽  
Kristin Thiele ◽  
Lars Haag ◽  
Katharina Fiddeke ◽  
...  
Keyword(s):  
Regulatory Gene ◽  
Extracellular Volume ◽  
Point Mutations ◽  
Cost Effective ◽  
Defined Medium ◽  
The Novel ◽  
Continuous Cell Lines ◽  
Recombinant Viruses ◽  
The Right ◽  
Chicken Embryo Fibroblasts

AbstractVectored vaccines based on highly attenuated modified vaccinia Ankara (MVA) are reported to be immunogenic, tolerant to pre-existing immunity, and able to accommodate and stably maintain very large transgenes. MVA is usually produced on primary chicken embryo fibroblasts, but production processes based on continuous cell lines emerge as increasingly robust and cost-effective alternatives. An isolate of a hitherto undescribed genotype was recovered by passage of a non-plaque-purified preparation of MVA in a continuous anatine suspension cell line (CR.pIX) in chemically defined medium. The novel isolate (MVA-CR19) replicated to higher infectious titers in the extracellular volume of suspension cultures and induced fewer syncytia in adherent cultures. We now extend previous studies with the investigation of the point mutations in structural genes of MVA-CR19 and describe an additional point mutation in a regulatory gene. We furthermore map and discuss an extensive rearrangement of the left telomer of MVA-CR19 that appears to have occurred by duplication of the right telomer. This event caused deletions and duplications of genes that may modulate immunologic properties of MVA-CR19 as a vaccine vector. Our characterizations also highlight the exceptional genetic stability of plaque-purified MVA: although the phenotype of MVA-CR19 appears to be advantageous for replication, we found that all genetic markers that differentiate wildtype and MVA-CR19 are stably maintained in passages of recombinant viruses based on either wildtype or MVA-CR.

scholarly journals Polarized glycoprotein targeting affects the spread of measles virus in vitro and in vivo

2004 ◽  
Vol 85 (4) ◽  
pp. 1019-1027 ◽  
Author(s):  
Markus Moll ◽  
Joanna Pfeuffer ◽  
Hans-Dieter Klenk ◽  
Stefan Niewiesk ◽  
Andrea Maisner
Keyword(s):  
Measles Virus ◽  
Single Point ◽  
Point Mutations ◽  
Recombinant Viruses ◽  
Systemic Spread ◽  
Spread Of Infection ◽  
Syncytia Formation ◽  
Basolateral Targeting

We have shown previously that basolateral targeting of plasmid-encoded measles virus (MV) F and H protein is dependent on single tyrosine residues in the cytoplasmic tails of the glycoproteins and is essential for fusion activity in polarized epithelial cells. Here, we present data on the functional importance of polarized glycoprotein expression for the cytopathic properties of infectious MV in culture and for pathogenesis in vivo. By the introduction of single point mutations, we generated recombinant viruses in which the basolateral targeting signal of either one or both glycoproteins was destroyed (tyrosine mutants). As a consequence, the mutated glycoproteins were predominantly expressed on the apical membrane of polarized Madin–Darby canine kidney cells. In contrast to parental MV, none of these virus mutants was able to spread by syncytia formation in polarized cells showing that the presence of both MV glycoproteins at the basolateral cell surface is required for cell-to-cell fusion in vitro. Using cotton rats as an animal model that allows MV replication in the respiratory tract, we showed that basolateral glycoprotein targeting is also of importance for the spread of infection in vivo. Whereas parental MV was able to spread laterally within the respiratory epithelium and from there to cells in the underlying tissue, tyrosine mutants infected only single epithelial and very few subepithelial cells. These data strongly suggest that basolateral targeting of MV glycoproteins helps to overcome the epithelial barrier and thereby facilitates the systemic spread of MV infection in vivo.

scholarly journals Lymph Node Macrophages Restrict Murine Cytomegalovirus Dissemination

2015 ◽  
Vol 89 (14) ◽  
pp. 7147-7158 ◽  
Author(s):  
Helen E. Farrell ◽  
Nick Davis-Poynter ◽  
Kimberley Bruce ◽  
Clara Lawler ◽  
Lars Dolken ◽  
...  
Keyword(s):  
Lymph Nodes ◽  
Vascular Endothelial Cells ◽  
Virus Production ◽  
Systemic Infection ◽  
Lytic Replication ◽  
Murine Cytomegalovirus ◽  
Chronic Infections ◽  
Entry Site ◽  
Mcmv Infection ◽  
Human Cmv

ABSTRACTCytomegaloviruses (CMVs) establish chronic infections that spread from a primary entry site to secondary vascular sites, such as the spleen, and then to tertiary shedding sites, such as the salivary glands. Human CMV (HCMV) is difficult to analyze, because its spread precedes clinical presentation. Murine CMV (MCMV) offers a tractable model. It is hypothesized to spread from peripheral sites via vascular endothelial cells and associated monocytes. However, viral luciferase imaging showed footpad-inoculated MCMV first reaching the popliteal lymph nodes (PLN). PLN colonization was rapid and further spread was slow, implying that LN infection can be a significant bottleneck. Most acutely infected PLN cells were CD169+subcapsular sinus macrophages (SSM). Replication-deficient MCMV also reached them, indicating direct infection. Many SSM expressed viral reporter genes, but few expressed lytic genes. SSM expressed CD11c, and MCMV with a cre-sensitive fluorochrome switch showed switched infected cells in PLN of CD11c-cre mice but yielded little switched virus. SSM depletion with liposomal clodronate or via a CD169-diphtheria toxin receptor transgene shifted infection to ER-TR7+stromal cells, increased virus production, and accelerated its spread to the spleen. Therefore, MCMV disseminated via LN, and SSM slowed this spread by shielding permissive fibroblasts and poorly supporting viral lytic replication.IMPORTANCEHCMV chronically infects most people, and it can cause congenital disability and harm the immunocompromised. A major goal of vaccination is to prevent systemic infection. How this is established is unclear. Restriction to humans makes HCMV difficult to analyze. We show that peripheral MCMV infection spreads via lymph nodes. Here, MCMV infected filtering macrophages, which supported virus replication poorly. When these macrophages were depleted, MCMV infected susceptible fibroblasts and spread faster. The capacity of filtering macrophages to limit MCMV spread argued that their infection is an important bottleneck in host colonization and might be a good vaccine target.

scholarly journals Genetic Determinants of Cell Type-Specific Poliovirus Propagation in HEK 293 Cells

2005 ◽  
Vol 79 (10) ◽  
pp. 6281-6290 ◽  
Author(s):  
Stephanie A. Campbell ◽  
Jennifer Lin ◽  
Elena Y. Dobrikova ◽  
Matthias Gromeier
Keyword(s):  
Point Mutations ◽  
Structural Features ◽  
Genetically Engineered ◽  
Nucleotide Substitutions ◽  
Entry Site ◽  
Stem Loop ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
293 Cells ◽  
Domain V

ABSTRACT The ability of poliovirus to propagate in neuronal cells can be reduced by introducing appropriate nucleotide substitutions into the viral genome. Specific mutations scattered throughout the poliovirus genome yielded the live attenuated vaccine strains of poliovirus. Neuron-specific propagation deficits of the Sabin strains are partially encrypted within a confined region of the internal ribosomal entry site (IRES), which carries attenuating point mutations in all three serotypes. Recently, high levels of neurovirulence attenuation were achieved with genetically engineered polioviruses containing heterologous IRES elements. This is exemplified with poliovirus recombinants replicating under control of a human rhinovirus type 2 (HRV2) IRES element. We have carried out experiments delineating the genetic basis for neuronal IRES function. Neuronal dysfunction of the HRV2 IRES is determined mainly by IRES stem-loop domain V, the locus for attenuating point mutations within the Sabin strains. Neuronal incompetence associated with HRV2 IRES domain V is substantially more pronounced than that observed with the attenuating IRES point mutation of the Sabin serotype 1 vaccine strain. Mix-and-match recombination of polio and HRV2 IRES domain V suggests that the attenuation phenotype correlates with overall structural features rather than primary sequence. Our experiments have identified HEK 293 cells as a novel system for the study of neuron-specific replication phenotypes of poliovirus. This cell line, originally derived from embryonic human kidney, has recently been described to display neuronal characteristics. We report propagation properties in HEK 293 cells for poliovirus recombinants with attenuated neurovirulence in experimental animals that corroborate this observation.

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