scholarly journals Drug Susceptibility and Replicative Capacity of Multidrug-Resistant Recombinant Human Cytomegalovirus Harboring Mutations in UL56 and UL54 Genes

2017 ◽  
Vol 61 (11) ◽  
Author(s):  
Jocelyne Piret ◽  
Nathalie Goyette ◽  
Guy Boivin
Keyword(s):  
Human Cytomegalovirus ◽  
Double Mutant ◽  
Antiviral Agent ◽  
Drug Susceptibility ◽  
Multidrug Resistant ◽  
Wild Type Virus ◽  
Replicative Capacity ◽  
Wild Type ◽  
The Impact

ABSTRACT Letermovir is an investigational antiviral agent with a novel mechanism of action involving the viral terminase (pUL56). We evaluated the impact of the V236M mutation in the UL56 gene alone and in combination with the E756K mutation in the UL54 gene on drug susceptibility and viral replicative capacity of recombinant human cytomegalovirus. The double mutant exhibited at least borderline resistance to all antivirals tested (ganciclovir, foscarnet, cidofovir, brincidofovir, and letermovir) and replicated less efficiently than the wild-type virus in vitro.

scholarly journals Production of Noncapped Genomic RNAs Is Critical to Sindbis Virus Disease and Pathogenicity

mBio ◽  
2020 ◽  
Vol 11 (6) ◽  
Author(s):  
Autumn T. LaPointe ◽  
V Douglas Landers ◽  
Claire E. Westcott ◽  
Kevin J. Sokoloski
Keyword(s):  
Sindbis Virus ◽  
Virus Disease ◽  
Severe Disease ◽  
Wild Type Virus ◽  
Wild Type ◽  
Genomic Rnas ◽  
Mortality And Morbidity ◽  
The Impact

ABSTRACT Alphaviruses are positive-sense RNA viruses that utilize a 5′ cap structure to facilitate translation of viral proteins and to protect the viral RNA genome. Nonetheless, significant quantities of viral genomic RNAs that lack a canonical 5′ cap structure are produced during alphaviral replication and packaged into viral particles. However, the role/impact of the noncapped genomic RNA (ncgRNA) during alphaviral infection in vivo has yet to be characterized. To determine the importance of the ncgRNA in vivo, the previously described D355A and N376A nsP1 mutations, which increase or decrease nsP1 capping activity, respectively, were incorporated into the neurovirulent AR86 strain of Sindbis virus to enable characterization of the impact of altered capping efficiency in a murine model of infection. Mice infected with the N376A nsP1 mutant exhibited slightly decreased rates of mortality and delayed weight loss and neurological symptoms, although levels of inflammation in the brain were similar to those of wild-type infection. Although the D355A mutation resulted in decreased antiviral gene expression and increased resistance to interferon in vitro, mice infected with the D355A mutant showed significantly reduced mortality and morbidity compared to mice infected with wild-type virus. Interestingly, expression of proinflammatory cytokines was found to be significantly decreased in mice infected with the D355A mutant, suggesting that capping efficiency and the production of ncgRNA are vital to eliciting pathogenic levels of inflammation. Collectively, these data indicate that the ncgRNA have important roles during alphaviral infection and suggest a novel mechanism by which noncapped viral RNAs aid in viral pathogenesis. IMPORTANCE Mosquito-transmitted alphaviruses have been the cause of widespread outbreaks of disease that can range from mild illness to lethal encephalitis or severe polyarthritis. There are currently no safe and effective vaccines or therapeutics with which to prevent or treat alphaviral disease, highlighting the need to better understand alphaviral pathogenesis to develop novel antiviral strategies. This report reveals production of noncapped genomic RNAs (ncgRNAs) to be a novel determinant of alphaviral virulence and offers insight into the importance of inflammation to pathogenesis. Taken together, the findings reported here suggest that the ncgRNAs contribute to alphaviral pathogenesis through the sensing of the ncgRNAs during alphaviral infection and are necessary for the development of severe disease.

scholarly journals Human Cytomegalovirus Resistance to Deoxyribosylindole Nucleosides Maps to a Transversion Mutation in the Terminase Subunit-Encoding GeneUL89

2014 ◽  
Vol 59 (1) ◽  
pp. 226-232 ◽  
Author(s):  
Brian G. Gentry ◽  
Quang Phan ◽  
Ellie D. Hall ◽  
Julie M. Breitenbach ◽  
Katherine Z. Borysko ◽  
...  
Keyword(s):  
Human Cytomegalovirus ◽  
Structural Similarity ◽  
Open Reading Frames ◽  
Wild Type Virus ◽  
Wild Type ◽  
Term Administration ◽  
Transversion Mutation ◽  
Exon 1

ABSTRACTHuman cytomegalovirus (HCMV) infection can cause severe illnesses, including encephalopathy and mental retardation, in immunocompromised and immunologically immature patients. Current pharmacotherapies for treating systemic HCMV infections include ganciclovir, cidofovir, and foscarnet. However, long-term administration of these agents can result in serious adverse effects (myelosuppression and/or nephrotoxicity) and the development of viral strains with reduced susceptibility to drugs. The deoxyribosylindole (indole) nucleosides demonstrate a 20-fold greater activityin vitro(the drug concentration at which 50% of the number of plaques was reduced with the presence of drug compared to the number in the absence of drug [EC50] = 0.34 μM) than ganciclovir (EC50= 7.4 μM) without any observed increase in cytotoxicity. Based on structural similarity to the benzimidazole nucleosides, we hypothesize that the indole nucleosides target the HCMV terminase, an enzyme responsible for packaging viral DNA into capsids and cleaving the DNA into genome-length units. To test this hypothesis, an indole nucleoside-resistant HCMV strain was isolated, the open reading frames of the genes that encode the viral terminase were sequenced, and a G766C mutation in exon 1 ofUL89was identified; this mutation resulted in an E256Q change in the amino acid sequence of the corresponding protein. An HCMV wild-type strain, engineered with this mutation to confirm resistance, demonstrated an 18-fold decrease in susceptibility to the indole nucleosides (EC50= 3.1 ± 0.7 μM) compared to that of wild-type virus (EC50= 0.17 ± 0.04 μM). Interestingly, this mutation did not confer resistance to the benzimidazole nucleosides (EC50for wild-type HCMV = 0.25 ± 0.04 μM, EC50for HCMV pUL89 E256Q = 0.23 ± 0.04 μM). We conclude, therefore, that the G766C mutation that results in the E256Q substitution is unique for indole nucleoside resistance and distinct from previously discovered substitutions that confer both indole and benzimidazole nucleoside resistance (D344E and A355T).

scholarly journals TMC125 Displays a High Genetic Barrier to the Development of Resistance: Evidence from In Vitro Selection Experiments

2005 ◽  
Vol 79 (20) ◽  
pp. 12773-12782 ◽  
Author(s):  
Johan Vingerhoets ◽  
Hilde Azijn ◽  
Els Fransen ◽  
Inky De Baere ◽  
Liesbet Smeulders ◽  
...  
Keyword(s):  
Wild Type Virus ◽  
Cross Resistance ◽  
Resistant Virus ◽  
Wild Type ◽  
Genetic Barrier ◽  
Development Of Resistance ◽  
Selection Experiments ◽  
The Impact ◽  
Hiv 1

ABSTRACT TMC125 is a potent new investigational nonnucleoside reverse transcriptase inhibitor (NNRTI) that is active against human immunodeficiency virus type 1 (HIV-1) with resistance to currently licensed NNRTIs. Sequential passage experiments with both wild-type virus and NNRTI-resistant virus were performed to identify mutations selected by TMC125 in vitro. In addition to “classic” selection experiments at a low multiplicity of infection (MOI) with increasing concentrations of inhibitors, experiments at a high MOI with fixed concentrations of inhibitors were performed to ensure a standardized comparison between TMC125 and current NNRTIs. Both low- and high-MOI experiments demonstrated that the development of resistance to TMC125 required multiple mutations which frequently conferred cross-resistance to efavirenz and nevirapine. In high-MOI experiments, 1 μM TMC125 completely inhibited the breakthrough of resistant virus from wild-type and NNRTI-resistant HIV-1, in contrast to efavirenz and nevirapine. Furthermore, breakthrough of virus from site-directed mutant (SDM) SDM-K103N/Y181C occurred at the same time or later with TMC125 as breakthrough from wild-type HIV-1 with efavirenz or nevirapine. The selection experiments identified mutations selected by TMC125 that included known NNRTI-associated mutations L100I, Y181C, G190E, M230L, and Y318F and the novel mutations V179I and V179F. Testing the antiviral activity of TMC125 against a panel of SDMs indicated that the impact of these individual mutations on resistance was highly dependent upon the presence and identity of coexisting mutations. These results demonstrate that TMC125 has a unique profile of activity against NNRTI-resistant virus and possesses a high genetic barrier to the development of resistance in vitro.

scholarly journals Minimising severity of dengue serotype 1 infection by transmissible interfering particles

2020 ◽  
Author(s):  
A. Shausan ◽  
J. Aaskov ◽  
C. Drovandi ◽  
K. Mengersen
Keyword(s):  
Therapeutic Agent ◽  
Antiviral Agent ◽  
Host Cells ◽  
Wild Type Virus ◽  
Parameter Estimates ◽  
Type Virus ◽  
Wild Type ◽  
Significant Difference ◽  
Serotype 1

AbstractTransmissible interfering dengue particles (DENV–TIPs) are engineered dengue virus mutants which are defective and can replicate only with the help of dengue wild–type virus (DENV). In vitro studies have found that when DENV–TIPs and DENV coinfect a cell, they compete for viral genomes and cell proteins for replication and packaging, and DENV–TIPs outperform DENV in this process. Thus, it is hypothesised that DENV–TIPs may be used as a novel therapeutic agent. However, the effectiveness of DENV–TIPs as an antiviral agent is yet to be explored at an epidemiological scale. We present a mathematical model for the replication of DENV and DENV–TIPs as they interact with human host cells, accounting for the effectiveness of DENV–TIPs in blocking DENV from coinfected cells. We fit the model to sequentially measured plasma viral titre data from primary and secondary dengue serotype 1 infected patients in Vietnam. We show that variation in initial DENV load is sufficient to recreate the observed variation between patients. Parameter estimates, differing in primary and secondary infections, do not confirm a significant difference between these two types of infection. We use our model to investigate the potential impact of DENV–TIPs as an antiviral agent. We conclude that, when the effectiveness of DENV–TIPs in inhibiting DENV from coinfected cells is at least 80%, a dose as high as 1012 copies per millilitre of blood is required to reduce duration of infection and peak DENV serotype 1 infection level at any time point of infection. This work provides a quantitative understanding of the relationship between DENV–TIPs levels and their efficiency in clearing dengue viral infection. It will guide future development of mechanistic models of how DENV–TIPs might contribute as an antiviral agent in limiting natural dengue infection.Author summaryInhibition of dengue wild–type virus (DENV) by transmissible interfering dengue particles (DENV–TIPs) is seen in some in vitro studies, and it is hypothesised that DENV–TIPs may be used as a therapeutic agent. However, the efficiency of DENV–TIPs in limiting DENV infection in patients is yet to be explored at an epidemiological scale. Using data collected from dengue serotype 1 infected patients, we model how DENV replicates in an infected patient and how effective DENV–TIPs are in controlling that replication. Our results are of use in the evaluation of DENV–TIPs as a potential antiviral agent.

scholarly journals The Human Cytomegalovirus UL44 Protein Is a Substrate for the UL97 Protein Kinase

2003 ◽  
Vol 77 (14) ◽  
pp. 7720-7727 ◽  
Author(s):  
Paula M. Krosky ◽  
Moon-Chang Baek ◽  
Wan Jin Jahng ◽  
Imma Barrera ◽  
Robert J. Harvey ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Human Cytomegalovirus ◽  
Nucleoside Analogs ◽  
Wild Type Virus ◽  
Null Mutant ◽  
Natural Substrate ◽  
Wild Type ◽  
Two Dimensional Gel Electrophoresis ◽  
Infected Cells

ABSTRACT The human cytomegalovirus UL97 protein is an unusual protein kinase that is able to autophosphorylate and to phosphorylate certain exogenous substrates, including nucleoside analogs such as ganciclovir. However, no natural substrate of UL97 in infected cells has been identified. We report here that recombinant UL44 protein became radiolabeled when incubated with recombinant UL97 and [32P]ATP and that both proteins could be coimmunoprecipitated by an antibody that recognizes either protein. Subsequent studies showed that highly purified, recombinant UL97 phosphorylated purified, recombinant UL44. This phosphorylation occurred on serine and threonine residues and was sensitive to inhibition by maribavir and to a mutation that inactivates UL97 catalytic activity. Two-dimensional gel electrophoresis revealed the absence of specific phosphorylated forms of UL44 in immunoprecipitates from lysates of cells infected with a UL97 null mutant virus or with wild-type virus in the presence of maribavir. The results indicate that UL97 is sufficient to phosphorylate UL44 in vitro and is necessary for the normal phosphorylation of UL44 in infected cells. This strongly suggests that UL44 is a natural substrate of UL97.

The URNA Genes of Herpesvirus Saimiri (Strain C488) Are Dispensable for Transformation of Human T Cells In Vitro

1999 ◽  
Vol 73 (12) ◽  
pp. 10551-10555 ◽  
Author(s):  
Armin Ensser ◽  
André Pfinder ◽  
Ingrid Müller-Fleckenstein ◽  
Bernhard Fleckenstein
Keyword(s):  
Cell Culture ◽  
Herpesvirus Saimiri ◽  
Wild Type Virus ◽  
Type Virus ◽  
Wild Type ◽  
Human T Cell ◽  
Human T Cells ◽  
Small Nuclear Rnas ◽  
T Cell Lines

ABSTRACT The herpesvirus saimiri strain C488 genome contains five genes for small nuclear RNAs, termed herpesvirus saimiri URNAs (or HSURs). Using a cosmid-based approach, all HSURs were precisely deleted from the genome. The mutant virus replicated at levels that were similar to those of wild-type viruses in OMK cells. Although the HSURs are expressed in wild-type virus-transformed human T-cell lines, the deletion does not affect viral transformation in cell culture.

scholarly journals Vaccinia Virus Envelope H3L Protein Binds to Cell Surface Heparan Sulfate and Is Important for Intracellular Mature Virion Morphogenesis and Virus Infection In Vitro and In Vivo

2000 ◽  
Vol 74 (7) ◽  
pp. 3353-3365 ◽  
Author(s):  
Chi-Long Lin ◽  
Che-Sheng Chung ◽  
Hans G. Heine ◽  
Wen Chang
Keyword(s):  
Cell Surface ◽  
Vaccinia Virus ◽  
Heparan Sulfate ◽  
Mutant Virus ◽  
Wild Type Virus ◽  
Type Virus ◽  
Wild Type ◽  
Virion Morphogenesis

ABSTRACT An immunodominant antigen, p35, is expressed on the envelope of intracellular mature virions (IMV) of vaccinia virus. p35 is encoded by the viral late gene H3L, but its role in the virus life cycle is not known. This report demonstrates that soluble H3L protein binds to heparan sulfate on the cell surface and competes with the binding of vaccinia virus, indicating a role for H3L protein in IMV adsorption to mammalian cells. A mutant virus defective in expression of H3L (H3L−) was constructed; the mutant virus has a small plaque phenotype and 10-fold lower IMV and extracellular enveloped virion titers than the wild-type virus. Virion morphogenesis is severely blocked and intermediate viral structures such as viral factories and crescents accumulate in cells infected with the H3L− mutant virus. IMV from the H3L− mutant virus are somewhat altered and less infectious than wild-type virions. However, cells infected by the mutant virus form multinucleated syncytia after low pH treatment, suggesting that H3L protein is not required for cell fusion. Mice inoculated intranasally with wild-type virus show high mortality and severe weight loss, whereas mice infected with H3L− mutant virus survive and recover faster, indicating that inactivation of the H3L gene attenuates virus virulence in vivo. In summary, these data indicate that H3L protein mediates vaccinia virus adsorption to cell surface heparan sulfate and is important for vaccinia virus infection in vitro and in vivo. In addition, H3L protein plays a role in virion assembly.

scholarly journals Involvement of the N-Terminal Deubiquitinating Protease Domain of Human Cytomegalovirus UL48 Tegument Protein in Autoubiquitination, Virion Stability, and Virus Entry

2016 ◽  
Vol 90 (6) ◽  
pp. 3229-3242 ◽  
Author(s):  
Young-Eui Kim ◽  
Se Eun Oh ◽  
Ki Mun Kwon ◽  
Chan Hee Lee ◽  
Jin-Hyun Ahn
Keyword(s):  
Human Cytomegalovirus ◽  
Virus Entry ◽  
Terminal Region ◽  
Mutant Virus ◽  
Wild Type Virus ◽  
Type Virus ◽  
Wild Type ◽  
Viral Growth ◽  
Internal Region ◽  
Deubiquitinating Protease

ABSTRACTHuman cytomegalovirus (HCMV) protein pUL48 is closely associated with the capsid and has a deubiquitinating protease (DUB) activity in its N-terminal region. Although this DUB activity moderately increases virus replication in cultured fibroblast cells, the requirements of the N-terminal region of pUL48 in the viral replication cycle are not fully understood. In this study, we characterized the recombinant viruses encoding UL48(ΔDUB/NLS), which lacks the DUB domain and the adjacent nuclear localization signal (NLS), UL48(ΔDUB), which lacks only the DUB, and UL48(Δ360–1200), which lacks the internal region (amino acids 360 to 1200) downstream of the DUB/NLS. While ΔDUB/NLS and Δ360–1200 mutant viruses did not grow in fibroblasts, the ΔDUB virus replicated to titers 100-fold lower than those for wild-type virus and showed substantially reduced viral gene expression at low multiplicities of infection. The DUB domain contained ubiquitination sites, and DUB activity reduced its own proteasomal degradation intrans. Deletion of the DUB domain did not affect the nuclear and cytoplasmic localization of pUL48, whereas the internal region (360–1200) was necessary for cytoplasmic distribution. In coimmunoprecipitation assays, pUL48 interacted with three tegument proteins (pUL47, pUL45, and pUL88) and two capsid proteins (pUL77 and pUL85) but the DUB domain contributed to only pUL85 binding. Furthermore, we found that the ΔDUB virus showed reduced virion stability and less efficiently delivered its genome into the cell than the wild-type virus. Collectively, our results demonstrate that the N-terminal DUB domain of pUL48 contributes to efficient viral growth by regulating its own stability and promoting virion stabilization and virus entry.IMPORTANCEHCMV pUL48 and its herpesvirus homologs play key roles in virus entry, regulation of immune signaling pathways, and virion assembly. The N terminus of pUL48 contains the DUB domain, which is well conserved among all herpesviruses. Although studies using the active-site mutant viruses revealed that the DUB activity promotes viral growth, the exact role of this region in the viral life cycle is not fully understood. In this study, using the mutant virus lacking the entire DUB domain, we demonstrate that the DUB domain of pUL48 contributes to viral growth by regulating its own stability via autodeubiquitination and promoting virion stability and virus entry. This report is the first to demonstrate the characteristics of the mutant virus with the entire DUB domain deleted, which, along with information on the functions of this region, is useful in dissecting the functions associated with pUL48.

scholarly journals Prediction of Minocycline Activity in the Gut From a Pig Preclinical Model Using a Pharmacokinetic -Pharmacodynamic Approach

2021 ◽  
Vol 12 ◽  
Author(s):  
Quentin Vallé ◽  
Béatrice B. Roques ◽  
Alain Bousquet-Mélou ◽  
David Dahlhaus ◽  
Felipe Ramon-Portugal ◽  
...  
Keyword(s):  
Intestinal Content ◽  
Multidrug Resistant ◽  
Preclinical Model ◽  
Intestinal Contents ◽  
Potential Activity ◽  
Potential Impact ◽  
The Impact ◽  
Pharmacodynamic Approach

The increase of multidrug-resistant (MDR) bacteria has renewed interest in old antibiotics, such as minocycline, that can be active against various MDR Gram-negative pathogens. The elimination of minocycline by both kidneys and liver makes it suitable for impaired renal function patients. However, the drawback is the possible elimination of a high amount of drug in the intestines, with potential impact on the digestive microbiota during treatment. This study aimed to predict the potential activity of minocycline against Enterobacterales in the gut after parenteral administration, by combining in vivo and in vitro studies. Total minocycline concentrations were determined by UPLC-UV in the plasma and intestinal content of piglets following intravenous administration. In parallel, the in vitro activity of minocycline was assessed against two Escherichia coli strains in sterilized intestinal contents, and compared to activity in a standard broth. We found that minocycline concentrations were 6–39 times higher in intestinal contents than plasma. Furthermore, minocycline was 5- to 245-fold less active in large intestine content than in a standard broth. Using this PK-PD approach, we propose a preclinical pig model describing the link between systemic and gut exposure to minocycline, and exploring its activity against intestinal Enterobacterales by taking into account the impact of intestinal contents.

scholarly journals In VitroDrug Combination Studies of Letermovir (AIC246, MK-8228) with Approved Anti-Human Cytomegalovirus (HCMV) and Anti-HIV Compounds in Inhibition of HCMV and HIV Replication

2015 ◽  
Vol 59 (6) ◽  
pp. 3140-3148 ◽  
Author(s):  
Steffen Wildum ◽  
Holger Zimmermann ◽  
Peter Lischka
Keyword(s):  
Human Cytomegalovirus ◽  
Treatment Strategies ◽  
Opportunistic Pathogen ◽  
Multidrug Resistant ◽  
Severe Toxicity ◽  
Transplant Recipients ◽  
Hiv Drugs ◽  
Anti Hiv

ABSTRACTDespite modern prevention and treatment strategies, human cytomegalovirus (HCMV) remains a common opportunistic pathogen associated with serious morbidity and mortality in immunocompromised individuals, such as transplant recipients and AIDS patients. All drugs currently licensed for the treatment of HCMV infection target the viral DNA polymerase and are associated with severe toxicity issues and the emergence of drug resistance. Letermovir (AIC246, MK-8228) is a new anti-HCMV agent in clinical development that acts via a novel mode of action and has demonstrated anti-HCMV activityin vitroandin vivo. For the future, drug combination therapies, including letermovir, might be indicated under special medical conditions, such as the emergence of multidrug-resistant virus strains in transplant recipients or in HCMV-HIV-coinfected patients. Accordingly, knowledge of the compatibility of letermovir with other HCMV or HIV antivirals is of medical importance. Here, we evaluated the inhibition of HCMV replication by letermovir in combination with all currently approved HCMV antivirals using cell culture checkerboard assays. In addition, the effects of letermovir on the antiviral activities of selected HIV drugs, and vice versa, were analyzed. Using two different mathematical techniques to analyze the experimental data, (i) additive effects were observed for the combination of letermovir with anti-HCMV drugs and (ii) no interaction was found between letermovir and anti-HIV drugs. Since none of the tested drug combinations significantly antagonized letermovir efficacy (or vice versa), our findings suggest that letermovir may offer the potential for combination therapy with the tested HCMV and HIV drugs.

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