scholarly journals New Insights into Human Cytomegalovirus pUL52 Structure

Viruses ◽  
2021 ◽  
Vol 13 (8) ◽  
pp. 1638
Author(s):  
Clotilde Muller ◽  
Sophie Alain ◽  
Claire Gourin ◽  
Thomas F. Baumert ◽  
Gaëtan Ligat ◽  
...  
Keyword(s):  
Viral Replication ◽  
Human Cytomegalovirus ◽  
New Drugs ◽  
Prolonged Treatment ◽  
Human Organism ◽  
Polymorphism Analysis ◽  
Resistance Mutations ◽  
Recombinant Viruses ◽  
Conserved Regions ◽  
Zinc Finger Motifs

Human cytomegalovirus (HCMV) can cause serious diseases in immunocompromised patients. Current antiviral inhibitors all target the viral DNA polymerase. They have adverse effects, and prolonged treatment can select for drug resistance mutations. Thus, new drugs targeting other stages of replication are an urgent need. The terminase complex (pUL56–pUL89–pUL51) is highly specific, has no counterpart in the human organism, and thus represents a target of choice for new antivirals development. This complex is required for DNA processing and packaging. pUL52 was shown to be essential for the cleavage of concatemeric HCMV DNA and crucial for viral replication, but its functional domains are not yet identified. Polymorphism analysis was performed by sequencing UL52 from 61 HCMV naive strains and from 14 HCMV strains from patients treated with letermovir. Using sequence alignment and homology modeling, we identified conserved regions and potential functional motifs within the pUL52 sequence. Recombinant viruses were generated with specific serine or alanine substitutions in these putative patterns. Within conserved regions, we identified residues essential for viral replication probably involved in CXXC-like or zinc finger motifs. These results suggest that they are essential for pUL52 structure/function. Thus, these patterns represent potential targets for the development of new antivirals.

scholarly journals Two Sp1/Sp3 Binding Sites in the Major Immediate-Early Proximal Enhancer of Human Cytomegalovirus Have a Significant Role in Viral Replication

2005 ◽  
Vol 79 (15) ◽  
pp. 9597-9607 ◽  
Author(s):  
Hiroki Isomura ◽  
Mark F. Stinski ◽  
Ayumi Kudoh ◽  
Tohru Daikoku ◽  
Noriko Shirata ◽  
...  
Keyword(s):  
Viral Replication ◽  
Human Cytomegalovirus ◽  
Significant Role ◽  
Binding Sites ◽  
Human Fibroblast ◽  
Immediate Early ◽  
Fibroblast Cells ◽  
Recombinant Viruses ◽  
Human Fibroblast Cells ◽  
Hcmv Replication

ABSTRACT We previously demonstrated that the major immediate early (MIE) proximal enhancer containing one GC box and the TATA box containing promoter are minimal elements required for transcription and viral replication in human fibroblast cells (H. Isomura, T. Tsurumi, M. F. Stinski, J. Virol. 78:12788-12799, 2004). After infection, the level of Sp1 increased while Sp3 remained constant. Here we report that either Sp1 or Sp3 transcription factors bind to the GC boxes located at approximately positions −55 and −75 relative to the transcription start site (+1). Both the Sp1 and Sp3 binding sites have a positive and synergistic effect on the human cytomegalovirus (HCMV) major immediate-early (MIE) promoter. There was little to no change in MIE transcription or viral replication for recombinant viruses with one or the other Sp1 or Sp3 binding site mutated. In contrast, mutation of both the Sp1 and Sp3 binding sites caused inefficient MIE transcription and viral replication. These data indicate that the Sp1 and Sp3 binding sites have a significant role in HCMV replication in human fibroblast cells.

scholarly journals A Recombinant Human Cytomegalovirus with a Large Deletion in UL97 Has a Severe Replication Deficiency

1999 ◽  
Vol 73 (7) ◽  
pp. 5663-5670 ◽  
Author(s):  
Mark N. Prichard ◽  
Ning Gao ◽  
Sanju Jairath ◽  
Gilbert Mulamba ◽  
Paula Krosky ◽  
...  
Keyword(s):  
Viral Replication ◽  
Human Cytomegalovirus ◽  
Large Deletion ◽  
Primary Cells ◽  
Parent Virus ◽  
Recombinant Viruses ◽  
Large Deletions ◽  
Good Target ◽  
Primary Fibroblasts ◽  
Antiviral Chemotherapy

ABSTRACT Human cytomegalovirus encodes a protein kinase (UL97) that confers sensitivity to ganciclovir by phosphorylating it to the monophosphate. The function of this unusual kinase in viral replication is unknown. We constructed two independent isolates of a recombinant virus, RCΔ97, that contain large deletions in this gene and carry a 4.8-kb insertion containing a selectable genetic marker. These mutant viruses were isolated by using a population of primary cells (HEL97) that express this gene from integrated copies of a defective retroviral vector. The recombinant viruses were severely impaired in their ability to replicate in primary fibroblasts, attaining virus titers that were 2 to 3 orders of magnitude lower than those produced by the parent virus. Despite the severe replication deficit, both of these viruses retained the ability to form small, slowly growing plaques in primary fibroblasts, demonstrating that UL97 is not absolutely essential for replication in cell culture. The replication deficit was relieved when UL97 was provided in trans in the complementing cell line, showing that the phenotype was due to a deficiency in UL97. Thus, theUL97 gene product plays a very important role in viral replication in tissue culture and may be a good target for antiviral chemotherapy.

scholarly journals A cis Repression Sequence Adjacent to the Transcription Start Site of the Human Cytomegalovirus US3 Gene Is Required To Down Regulate Gene Expression at Early and Late Times after Infection

1998 ◽  
Vol 72 (12) ◽  
pp. 9575-9584 ◽  
Author(s):  
Philip E. Lashmit ◽  
Mark F. Stinski ◽  
Eain A. Murphy ◽  
Grant C. Bullock
Keyword(s):  
Gene Expression ◽  
Viral Replication ◽  
Transcription Start Site ◽  
Human Cytomegalovirus ◽  
Maximum Level ◽  
Start Site ◽  
Wild Type ◽  
Transcription Start ◽  
Recombinant Viruses

ABSTRACT Human cytomegalovirus has two enhancer-containing immediate-early (IE) promoters with a cis repression sequence (CRS) positioned immediately upstream of the transcription start site, designated the major IE (MIE) promoter and the US3 promoter. The role of the CRS upstream of the US3 transcription start site in the context of the viral genome was determined by comparing the levels of transcription from these two enhancer-containing promoters in recombinant viruses with a wild-type or mutant CRS. Upstream of the CRS of the US3 promoter was either the endogenous enhancer (R2) or silencer (R1). The downstream US3 gene was replaced with the indicator gene chloramphenicol acetyltransferase (CAT). Infected permissive human fibroblast cells or nonpermissive, undifferentiated monocytic THP-1 cells were analyzed for expression from the US3 promoter containing either the wild-type or mutant CRS. With the wild-type CRS, the maximum level of transcription in permissive cells was detected within 4 to 6 h after infection and then declined. With the mutant CRS and the R2 enhancer upstream, expression from the US3 promoter continued to increase throughout the viral replication cycle to levels 20- to 40-fold higher than for the wild type. In nonpermissive or permissive monocytic THP-1 cells, expression from the US3 promoter was also significantly higher when the CRS was mutated. Less expression was obtained when only the R1 element was present, but expression was higher when the CRS was mutated. Thus, the CRS in the enhancer-containing US3 promoter appears to allow for a short burst of US3 gene expression followed by repression at early and late times after infection. Overexpression of US3 may be detrimental to viral replication, and its level of expression must be stringently controlled. The role of the CRS and the viral IE86 protein in controlling enhancer-containing promoters is discussed.

scholarly journals A Multi-Pronged Computational Pipeline for Prioritizing Drug Target Strategies for Latent Tuberculosis

2020 ◽  
Vol 8 ◽  
Author(s):  
Ushashi Banerjee ◽  
Santhosh Sankar ◽  
Amit Singh ◽  
Nagasuma Chandra
Keyword(s):  
Drug Targets ◽  
Metabolic Flux ◽  
Latent Tuberculosis ◽  
Interaction Network ◽  
New Drugs ◽  
Prolonged Treatment ◽  
Flux Analysis ◽  
Protein Protein Interaction ◽  
Candidate Drug ◽  
Global Population

Tuberculosis is one of the deadliest infectious diseases worldwide and the prevalence of latent tuberculosis acts as a huge roadblock in the global effort to eradicate tuberculosis. Most of the currently available anti-tubercular drugs act against the actively replicating form of Mycobacterium tuberculosis (Mtb), and are not effective against the non-replicating dormant form present in latent tuberculosis. With about 30% of the global population harboring latent tuberculosis and the requirement for prolonged treatment duration with the available drugs in such cases, the rate of adherence and successful completion of therapy is low. This necessitates the discovery of new drugs effective against latent tuberculosis. In this work, we have employed a combination of bioinformatics and chemoinformatics approaches to identify potential targets and lead candidates against latent tuberculosis. Our pipeline adopts transcriptome-integrated metabolic flux analysis combined with an analysis of a transcriptome-integrated protein-protein interaction network to identify perturbations in dormant Mtb which leads to a shortlist of 6 potential drug targets. We perform a further selection of the candidate targets and identify potential leads for 3 targets using a range of bioinformatics methods including structural modeling, binding site association and ligand fingerprint similarities. Put together, we identify potential new strategies for targeting latent tuberculosis, new candidate drug targets as well as important lead clues for drug design.

scholarly journals Prolonged activation of NF-κB by human cytomegalovirus promotes efficient viral replication and late gene expression

Virology ◽  
2006 ◽  
Vol 346 (1) ◽  
pp. 15-31 ◽  
Author(s):  
Ian B. DeMeritt ◽  
Jagat P. Podduturi ◽  
A. Michael Tilley ◽  
Maciej T. Nogalski ◽  
Andrew D. Yurochko
Keyword(s):  
Gene Expression ◽  
Viral Replication ◽  
Human Cytomegalovirus ◽  
Late Gene ◽  
Late Gene Expression ◽  
Efficient Viral Replication

Viral replication in HeLa/fibroblast hybrid cells infected with human cytomegalovirus

1987 ◽  
Vol 95 (1-2) ◽  
pp. 29-40 ◽  
Author(s):  
Y. Tsutsui ◽  
S. Sonta ◽  
A. Kashiwai ◽  
T. Nogami ◽  
T. Furukawa
Keyword(s):  
Viral Replication ◽  
Human Cytomegalovirus ◽  
Hybrid Cells

Human Cytomegalovirus Forms Phase-Separated Compartments at Viral Genomes to Facilitate Viral Replication

2021 ◽  
Author(s):  
Enrico Caragliano ◽  
Stefano Bonazza ◽  
Giada Frascaroli ◽  
Jiajia Tang ◽  
Timothy K. Soh ◽  
...  
Keyword(s):  
Viral Replication ◽  
Human Cytomegalovirus ◽  
Viral Genomes

scholarly journals MiDRMpol: A High-Throughput Multiplexed Amplicon Sequencing Workflow to Quantify HIV-1 Drug Resistance Mutations against Protease, Reverse Transcriptase, and Integrase Inhibitors

Viruses ◽  
2019 ◽  
Vol 11 (9) ◽  
pp. 806
Author(s):  
Shambhu G. Aralaguppe ◽  
Anoop T. Ambikan ◽  
Manickam Ashokkumar ◽  
Milner M. Kumar ◽  
Luke Elizabeth Hanna ◽  
...  
Keyword(s):  
Drug Resistance ◽  
High Throughput ◽  
Large Scale ◽  
High Throughput Sequencing ◽  
Polymorphism Analysis ◽  
Resistance Mutations ◽  
Subtype C ◽  
Bioinformatics Pipeline ◽  
Drug Resistance Mutations ◽  
Hiv 1

The detection of drug resistance mutations (DRMs) in minor viral populations is of potential clinical importance. However, sophisticated computational infrastructure and competence for analysis of high-throughput sequencing (HTS) data lack at most diagnostic laboratories. Thus, we have proposed a new pipeline, MiDRMpol, to quantify DRM from the HIV-1 pol region. The gag-vpu region of 87 plasma samples from HIV-infected individuals from three cohorts was amplified and sequenced by Illumina HiSeq2500. The sequence reads were adapter-trimmed, followed by analysis using in-house scripts. Samples from Swedish and Ethiopian cohorts were also sequenced by Sanger sequencing. The pipeline was validated against the online tool PASeq (Polymorphism Analysis by Sequencing). Based on an error rate of <1%, a value of >1% was set as reliable to consider a minor variant. Both pipelines detected the mutations in the dominant viral populations, while discrepancies were observed in minor viral populations. In five HIV-1 subtype C samples, minor mutations were detected at the <5% level by MiDRMpol but not by PASeq. MiDRMpol is a computationally as well as labor efficient bioinformatics pipeline for the detection of DRM from HTS data. It identifies minor viral populations (<20%) of DRMs. Our method can be incorporated into large-scale surveillance of HIV-1 DRM.

scholarly journals Direct infection of CD34+ progenitor cells by human cytomegalovirus: evidence for inhibition of hematopoiesis and viral replication

Blood ◽  
1996 ◽  
Vol 88 (4) ◽  
pp. 1277-1283 ◽  
Author(s):  
M Movassagh ◽  
J Gozlan ◽  
B Senechal ◽  
C Baillou ◽  
JC Petit ◽  
...  
Keyword(s):  
Viral Replication ◽  
Human Cytomegalovirus ◽  
Laboratory Strain ◽  
Inhibitory Effect ◽  
Rt Pcr ◽  
Reverse Transcription Pcr ◽  
Differentiated Cells ◽  
Cd34 Antigen ◽  
Liquid Suspension ◽  
Infected Cells

We successfully infected fluorescence-activated cell-sorted CD34+ cells from normal cord blood by the human cytomegalovirus (HCMV) laboratory strain Towne. An inhibitory effect of HCMV on clonogenic myeloid progenitors was observed in primary methylcellulose cultures. After an initial 7-day liquid culture of CD34(+)-infected cells, this inhibition was further amplified in secondary methylcellulose cultures, then involving both the myeloid and erythroid lineages. Under these conditions, viral DNA was detected both in erythroid and myeloid colonies using the polymerase chain reaction (PCR), but reverse transcription PCR (RT-PCR) failed to detect viral RNA. In contrast, when CD34(+)-infected cells were maintained in liquid suspension, both immediate, early, and late transcripts were detected as soon as day 3. In addition, viral production was demonstrated in the culture supernatants, thus confirming that a complete viral cycle occurred under liquid conditions. Furthermore, by resorting cells into CD34+ and CD34- fractions, we showed by RT-PCR that viral replication took place in cells still expressing CD34 antigen, whereas no RNA was found in more differentiated cells that had subsequently lost their CD34 antigen. These findings suggest that HCMV replication can occur at the early steps of progenitor differentiation and may be involved in the viral-induced myelosuppression.

High-level resistance to bictegravir and cabotegravir in subtype A- and D-infected HIV-1 patients failing raltegravir with multiple resistance mutations

2021 ◽  
Author(s):  
Emmanuel Ndashimye ◽  
Yue Li ◽  
Paul S Reyes ◽  
Mariano Avino ◽  
Abayomi S Olabode ◽  
...  
Keyword(s):  
Cross Resistance ◽  
Strand Transfer ◽  
Resistance Mutations ◽  
Middle Income ◽  
Recombinant Viruses ◽  
Third Line ◽  
Long Acting ◽  
High Level ◽  
Subtype A ◽  
Hiv 1

Abstract Objectives The second-generation integrase strand transfer inhibitor (INSTI) bictegravir is becoming accessible in low- and middle-income countries (LMICs), and another INSTI, cabotegravir, has recently been approved as a long-acting injectable. Data on bictegravir and cabotegravir susceptibility in raltegravir-experienced HIV-1 subtype A- and D-infected patients carrying drug resistance mutations (DRMs) remain very scarce in LMICs. Patients and methods HIV-1 integrase (IN)-recombinant viruses from eight patients failing raltegravir-based third-line therapy in Uganda were genotypically and phenotypically tested for susceptibility to bictegravir and cabotegravir. Ability of these viruses to integrate into human genomes was assessed in MT-4 cells. Results HIV-1 IN-recombinant viruses harbouring single primary mutations (N155H or Y143R/S) or in combination with secondary INSTI mutations (T97A, M50I, L74IM, E157Q, G163R or V151I) were susceptible to both bictegravir and cabotegravir. However, combinations of primary INSTI-resistance mutations such as E138A/G140A/G163R/Q148R or E138K/G140A/S147G/Q148K led to decreased susceptibility to both cabotegravir (fold change in EC50 values from 429 to 1000×) and bictegravir (60 to 100×), exhibiting a high degree of cross-resistance. However, these same IN-recombinant viruses showed impaired integration capacity (14% to 48%) relative to the WT HIV-1 NL4-3 strain in the absence of drug. Conclusions Though not currently widely accessible in most LMICs, bictegravir and cabotegravir offer a valid alternative to HIV-infected individuals harbouring subtype A and D HIV-1 variants with reduced susceptibility to first-generation INSTIs but previous exposure to raltegravir may reduce efficacy, more so with cabotegravir.

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