Molecular Requirements for Self-Interaction of the Respiratory Syncytial Virus Matrix Protein in Living Mammalian Cells

Trevisan, Marta; Antonio, Veronica Di; Radeghieri, Annalisa; Palù, Giorgio; Ghildyal, Reena; Alvisi, Gualtiero

doi:10.3390/v10030109

Cited by 14 publications

(20 citation statements)

References 46 publications

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“…M protein also forms dimers and has been crystalized in its dimeric form. Recently, we have shown that RSV M protein can self-interact when expressed in living mammalian cells, and that this requires both the N- and C-terminal domains of the protein, consistent with the reported head to tail dimeric structure of M, whereby the N-terminal domain of one subunit interacts with the C-terminal domain of the other subunit [ 7 , 37 ] . Together with the data reported in this study, this suggests that M’s interaction with polymerized actin may require formation of the homodimer; however, this remains to be elucidated.…”

Section: Discussionsupporting

confidence: 81%

Respiratory Syncytial Virus Matrix (M) Protein Interacts with Actin In Vitro and in Cell Culture

Shahriari

Wei

Ghildyal

2018

Viruses

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The virus–host protein interactions that underlie respiratory syncytial virus (RSV) assembly are still not completely defined, despite almost 60 years of research. RSV buds from the apical surface of infected cells, once virion components have been transported to the budding sites. Association of RSV matrix (M) protein with the actin cytoskeleton may play a role in facilitating this transport. We have investigated the interaction of M with actin in vitro and cell culture. Purified wildtype RSV M protein was found to bind directly to polymerized actin in vitro. Vero cells were transfected to express full-length M (1–256) as a green fluorescent protein-(GFP) tagged protein, followed by treatment with the microfilament destabilizer, cytochalasin D. Destabilization of the microfilament network resulted in mislocalization of full-length M, from mostly cytoplasmic to diffused across both cytoplasm and nucleus, suggesting that M interacts with microfilaments in this system. Importantly, treatment of RSV-infected cells with cytochalasin D results in lower infectious virus titers, as well as mislocalization of M to the nucleus. Finally, using deletion mutants of M in a transfected cell system, we show that both the N- and C-terminus of the protein are required for the interaction. Together, our data suggest a possible role for M–actin interaction in transporting virion components in the infected cell.

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Section: Discussionsupporting

confidence: 81%

Respiratory Syncytial Virus Matrix (M) Protein Interacts with Actin In Vitro and in Cell Culture

Shahriari

Wei

Ghildyal

2018

Viruses

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“…In vitro , the affinity of UL44 self-interaction has been reported to be similar to that of the UL44-UL54 interaction, although using different methods and thus not being directly comparable (16, 22). We therefore decided to compare the affinity of such interactions using a bioluminescent resonant energy transfer (BRET) assay that we recently developed to study respiratory syncytial virus (RSV) matrix protein self-association in living cells (55). HEK 293T cells were transfected to express either RLuc-UL44 or RLuc-UL44(405-433) either in the absence or in the presence of YFP-UL44, and 48 h later cells were processed for BRET analysis.…”

Section: Resultsmentioning

confidence: 99%

“…BRET experiments were performed essentially as described previously using a reader compatible with BRET measurements (VICTOR X2 Multilabel Plate Reader, Perkin Elmer) (55). BRET saturation curves were generated using the GraphPad Prism software (Graphpad Software Inc.) by plotting each individual BRET ratio value to the YFPnet/RLuc signal, and interpolating such values using the one-site binding hyperbola function of GraphPad Prism (Graphpad Software Inc.) to calculate BRETmax (Bmax) and BRET 50 (B 50 ) values, indicative of maximum energy transfer and relative affinity of each BRET pair tested (56).…”

Section: Methodsmentioning

confidence: 99%

Divide et Impera: Identification of Small-Molecule Inhibitors of HCMV Replication Interfering with Dimerization of DNA Polymerase Processivity Factor UL44

Ghassabian

Falchi

Antonio

et al. 2020

Preprint

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16Human cytomegalovirus (HCMV) is a leading cause of severe diseases in immunocompromised 17 individuals, including AIDS and transplanted patients, and in congenitally infected newborns. Despite 18 the availability of several antiviral drugs, their utility is limited by poor bioavailability, toxicity, and 19 resistant strains emergence. Therefore, it is crucial to identify new targets of therapeutic intervention. 20 The dimerization of HCMV DNA polymerase processivity factor UL44 plays an essential role in the 21 viral life cycle being required for oriLyt-dependent DNA replication. We validated the existence of 22 UL44 homodimers both in vitro and in living cells by a variety of approaches, including GST 23 pulldown, thermal shift, FRET and BRET assays. Dimerization occurred with an affinity comparable 24 to that of the UL54/UL44 interaction, and was impaired by amino acid substitutions at the 25 dimerization interface. Subsequently, we performed an in-silico screening to select 18 small 26 molecules (SMs) potentially interfering with UL44 homodimerization. Antiviral assays using 27 recombinant HCMV TB4-UL83-YFP in the presence of the 18 selected SMs led to the identification 28 of four active SMs. The most active one also inhibited AD169 in plaque reduction assays, and 29 impaired replication of an AD169-GFP reporter virus and its ganciclovir-resistant counterpart to a 30 similar extent. As assessed by Western blotting experiments, treatment of infected cells specifically 31 reduced viral gene expression starting from 48 h post infection, consistent with activity on viral DNA 32 synthesis. Therefore, SMs inhibitors of UL44 dimerization could represent a new class of HCMV 33 inhibitors, alternative to those targeting the DNA polymerase catalytic subunit or the viral terminase 34 complex. 35 IMPORTANCE 36 HCMV is a ubiquitous infectious agent causing life-lasting infections in humans. HCMV primary 37 infections and reactivation in non-immunocompetent individuals often result in life-threatening 38 conditions. Antiviral therapy mainly targets the DNA polymerase catalytic subunit UL54 and is often 39 limited by toxicity and selection of drug-resistant viral strains, making the identification of new 40 targets of therapeutic intervention crucial for a successful management of HCMV infections. The 41 49The b-Herpesvirinae member human cytomegalovirus (HCMV) is a major human pathogen, causing 50 severe and life-threatening infections in immunocompromised patients (1) and in congenitally 51 infected newborns (2). Herpesviruses are opportunistic double-stranded DNA viruses, whose genome 52 transcription, replication, and packaging occur in the host cell nucleus (3). The molecular mechanisms 53 involved in herpesvirus DNA replication and its regulation have been widely studied as they provide 54 important models for the study of eukaryotic DNA replication and because viral enzymes involved 55 in the process represent targets for antiviral therapy. HCMV DNA polymerase holoenzyme is a multi-56 functional enzyme that play...

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“…A schematic representation of ppUL44 derivatives tested in this study is shown in Figure 1 . Entry clones were used to generate C-terminal fusion mammalian expression vectors, following LR recombination reactions with the pDESTnYFP, pDESTnCFP, pDESTnRLuc Gateway compatible vectors [ 31 ], as described in [ 32 ]. Plasmid pWPI-puro-YFP-UL44 was generated by ligation a NheI-XbaI fragment from pDESTnYFP-UL44 in vector pWPI-puro.…”

Section: Methodsmentioning

confidence: 99%

Live-Cell Analysis of Human Cytomegalovirus DNA Polymerase Holoenzyme Assembly by Resonance Energy Transfer Methods

2021

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Human cytomegalovirus (HCMV) genome replication is a complex and still not completely understood process mediated by the highly coordinated interaction of host and viral products. Among the latter, six different proteins form the viral replication complex: a single-stranded DNA binding protein, a trimeric primase/helicase complex and a two subunit DNA polymerase holoenzyme, which in turn contains a catalytic subunit, pUL54, and a dimeric processivity factor ppUL44. Being absolutely required for viral replication and representing potential therapeutic targets, both the ppUL44–pUL54 interaction and ppUL44 homodimerization have been largely characterized from structural, functional and biochemical points of view. We applied fluorescence and bioluminescence resonance energy transfer (FRET and BRET) assays to investigate such processes in living cells. Both interactions occur with similar affinities and can take place both in the nucleus and in the cytoplasm. Importantly, single amino acid substitutions in different ppUL44 domains selectively affect its dimerization or ability to interact with pUL54. Intriguingly, substitutions preventing DNA binding of ppUL44 influence the BRETmax of protein–protein interactions, implying that binding to dsDNA induces conformational changes both in the ppUL44 homodimer and in the DNA polymerase holoenzyme. We also compared transiently and stably ppUL44-expressing cells in BRET inhibition assays. Transient expression of the BRET donor allowed inhibition of both ppUL44 dimerization and formation of the DNA polymerase holoenzyme, upon overexpression of FLAG-tagged ppUL44 as a competitor. Our approach could be useful both to monitor the dynamics of assembly of the HCMV DNA polymerase holoenzyme and for antiviral drug discovery.

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Molecular Requirements for Self-Interaction of the Respiratory Syncytial Virus Matrix Protein in Living Mammalian Cells

Cited by 14 publications

References 46 publications

Respiratory Syncytial Virus Matrix (M) Protein Interacts with Actin In Vitro and in Cell Culture

Respiratory Syncytial Virus Matrix (M) Protein Interacts with Actin In Vitro and in Cell Culture

Divide et Impera: Identification of Small-Molecule Inhibitors of HCMV Replication Interfering with Dimerization of DNA Polymerase Processivity Factor UL44

Live-Cell Analysis of Human Cytomegalovirus DNA Polymerase Holoenzyme Assembly by Resonance Energy Transfer Methods

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