Rapid Titration of Measles and Other Viruses: Optimization with Determination of Replication Cycle Length

Vesicular stomatitis virus (VSV; the prototype rhabdovirus) fusion is triggered at low pH and mediated by glycoprotein G, which undergoes a low-pH-induced structural transition. A unique feature of rhabdovirus G is that its conformational change is reversible. This allows G to recover its native prefusion state at the viral surface after its transport through the acidic Golgi compartments. The crystal structures of G pre-and postfusion states have been elucidated, leading to the identification of several acidic amino acid residues, clustered in the postfusion trimer, as potential pH-sensitive switches controlling the transition back toward the prefusion state. We mutated these residues and produced a panel of single and double mutants whose fusion properties, conformational change characteristics, and ability to pseudotype a virus lacking the glycoprotein gene were assayed. Some of these mutations were also introduced in the genome of recombinant viruses which were further characterized. We show that D268, located in the segment consisting of residues 264 to 273, which refolds into postfusion helix F during G structural transition, is the major pH sensor while D274, D395, and D393 have additional contributions. Furthermore, a single passage of recombinant virus bearing the mutation D268L (which was demonstrated to stabilize the G postfusion state) resulted in a pseudorevertant with a compensatory second mutation, L271P. This revealed that the propensity of the segment of residues 264 to 273 to refold into helix F has to be finely tuned since either an increase (mutation D268L alone) or a decrease (mutation L271P alone) of this propensity is detrimental to the virus. IMPORTANCE Vesicular stomatitis virus enters cells via endocytosis. Endosome acidification induces a structural transition of its unique glycoprotein (G), which mediates fusion between viral and endosomal membranes. G conformational change is reversible upon increases in pH. This allows G to recover its native prefusion state at the viral surface after its transport through the acidic Golgi compartments. We mutated five acidic residues, proposed to be pH-sensitive switches controlling the structural transition back toward the prefusion state. Our results indicate that residue D268 is the major pH sensor, while other acidic residues have additional contributions, and reveal that the propensity of the segment consisting of residues 264 to 273 to adopt a helical conformation is finely regulated. This segment might be a good target for antiviral compounds.

Section: Resultsmentioning

confidence: 99%

Characterization of pH-Sensitive Molecular Switches That Trigger the Structural Transition of Vesicular Stomatitis Virus Glycoprotein from the Postfusion State toward the Prefusion State

Ferlin

Raux

Baquero

et al. 2014

“…BSR-T7-Slam cells are BSR-T7 cells transduced by a LentiVector to stably express the human Slam receptor. HeLa cells (28), Vero/hSLAM cells (29), or BSR-T7-hSlam and brain slices from 9-day-old SLAM ϫ IFNAR ko mice (30) were infected with the MeV Schwarz vaccine strain (31), recombinant MeV Mor-Flag/L expressing an L protein tagged with a Flag peptide at its N terminus and built according to reference 26, MeV-IC323-GFPm [1], MeV Mor-Flag/L[Gaussia], NiV-enhanced green fluorescent protein (EGFP) (32), or VSV-GFP (33). MeV-IC323-GFPm [1] was built to express GFP in an additional transcription unit located in the first position before the N gene.…”

Section: Methodsmentioning

confidence: 99%

“…Recombinant MeVs were rescued as previously described (26,34). Viruses were titrated by the 50% tissue culture infective dose (TCID 50 ) titration method (31). Cells were treated as indicated by MG132 (Peptide International, Inc.), 17-dimethylaminoethylamino-17-demethoxygeldanamycin (17-DMAG), NVP-AUY922 (InvivoGen), cycloheximide, or 3-methyladenine (3-MA; Sigma).…”

Section: Methodsmentioning

confidence: 99%

HSP90 Chaperoning in Addition to Phosphoprotein Required for Folding but Not for Supporting Enzymatic Activities of Measles and Nipah Virus L Polymerases

Bloyet

Welsch

Enchéry

et al. 2016

Self Cite

Nonsegmented negative-stranded RNA viruses, or members of the order Mononegavirales, share a conserved gene order and the use of elaborate transcription and replication machinery made up of at least four molecular partners. These partners have coevolved with the acquisition of the permanent encapsidation of the entire genome by the nucleoprotein (N) and the use of this N-RNA complex as a template for the viral polymerase composed of the phosphoprotein (P) and the large enzymatic protein (L). Not only is P required for polymerase function, but it also stabilizes the L protein through an unknown underlying molecular mechanism. By using NVP-AUY922 and/or 17-dimethylaminoethylamino-17-demethoxygeldanamycin as specific inhibitors of cellular heat shock protein 90 (HSP90), we found that efficient chaperoning of L by HSP90 requires P in the measles, Nipah, and vesicular stomatitis viruses. While the production of P remains unchanged in the presence of HSP90 inhibitors, the production of soluble and functional L requires both P and HSP90 activity. Measles virus P can bind the N terminus of L in the absence of HSP90 activity. Both HSP90 and P are required for the folding of L, as evidenced by a luciferase reporter insert fused within measles virus L. HSP90 acts as a true chaperon; its activity is transient and dispensable for the activity of measles and Nipah virus polymerases of virion origin. That the cellular chaperoning of a viral polymerase into a soluble functional enzyme requires the assistance of another viral protein constitutes a new paradigm that seems to be conserved within the Mononegavirales order. IMPORTANCEViruses are obligate intracellular parasites that require a cellular environment for their replication. Some viruses particularly depend on the cellular chaperoning apparatus. We report here that for measles virus, successful chaperoning of the viral L polymerase mediated by heat shock protein 90 (HSP90) requires the presence of the viral phosphoprotein (P). Indeed, while P protein binds to the N terminus of L independently of HSP90 activity, both HSP90 and P are required to produce stable, soluble, folded, and functional L proteins. Once formed, the mature P؉L complex no longer requires HSP90 to exert its polymerase functions. Such a new paradigm for the maturation of a viral polymerase appears to be conserved in several members of the Mononegavirales order, including the Nipah and vesicular stomatitis viruses. Viruses with a nonsegmented negative-stranded RNA genome, or members of the order Mononegavirales, share a common and highly conserved genomic organization and replication machinery that are unique in the living world. Indeed, the L protein, or polymerase, which is endowed with all of the enzymatic activities required for the synthesis of RNA and the capping and polyadenylation of viral transcripts, cannot use naked viral genomic RNA in a processive way (1). Instead, L associates with the phosphoprotein (P) to dynamically anchor the polymerase complex to the nucleocapsid and/or to enable...

“…Briefly, after being blocked and permeabilized in phosphate-buffered saline (PBS)-4% FBS-0.3% Triton X-100, sections were sequentially incubated with a primary Ab overnight at 4°C and with a secondary Ab for 2 h at room temperature (RT). The primary Abs used were anti-glial fibrillary acidic protein (GFAP) rabbit polyclonal serum (Z0334; Dako), anti-microtubule-associated protein 2 (MAP-2) rabbit polyclonal IgG (H300/sc-20172; Santa Cruz), and an anti-MV F-specific monoclonal Ab (clone Y503) (39).…”

Section: Plasmids and Reagentsmentioning

confidence: 99%

Fatal Measles Virus Infection Prevented by Brain-Penetrant Fusion Inhibitors

Welsch

Talekar

Mathieu

et al. 2013

106

e Measles virus (MV) infection causes an acute childhood disease that can include infection of the central nervous system and can rarely progress to severe neurological disease for which there is no specific treatment. We generated potent antiviral peptide inhibitors of MV entry and spreading and MV-induced cell fusion. Dimers of MV-specific peptides derived from the C-terminal heptad repeat region of the MV fusion protein, conjugated to cholesterol, efficiently protect SLAM transgenic mice from fatal MV infection. Fusion inhibitors hold promise for the prophylaxis of MV infection in unvaccinated and immunocompromised people, as well as potential for the treatment of grave neurological complications of measles.