La Crosse virus (LACV) Gc fusion peptide mutants have impaired growth and fusion phenotypes, but remain neurotoxic

Abstract

La Crosse virus is a leading cause of pediatric encephalitis in the Midwestern United States and an emerging pathogen in the American South. The LACV glycoprotein Gc plays a critical role in entry as the virus attachment protein. A 22 amino acid hydrophobic region within Gc (1066-1087) was recently identified as the LACV fusion peptide. To further define the role of Gc (1066-1087) in virus entry, fusion, and neuropathogenesis, a panel of recombinant LACV (rLACV) fusion peptide mutant viruses was generated. Replication of mutant rLACVs was significantly reduced. In addition, the fusion peptide mutants demonstrated decreased fusion phenotypes relative to LACV-WT. Interestingly, these viruses maintained their ability to cause neuronal loss in culture, suggesting that the fusion peptide of LACV Gc is a determinant of properties associated with neuroinvasion (growth to high titer in muscle cells and a robust fusion phenotype), but not necessarily of neurovirulence.

Mouse Embryonic Stem Cells Are Deficient in Type I Interferon Expression in Response to Viral Infections and Double-stranded RNA

“…1A, indicated by arrows). Viral infection of mESCs was confirmed by expression of the LACV gene that encodes the M-segment protein (Gc protein) (29). The infected cells were immunostained monoclonal antibodies against the Gc protein (a gift from Dr. Samantha Soldan, University of Pennsylvania School of Medicine) followed by flow cytometry analysis (29).…”

Orthobunyavirus Entry into Neurons and Other Mammalian Cells Occurs via Clathrin-Mediated Endocytosis and Requires Trafficking into Early Endosomes

“…2A and B) were all significantly decreased by these inhibitors of CME compared to control or DMSO treatments. Furthermore, we modified our FFWI assay for LACV (46,55) to rescue LACV infection in the presence of these inhibitors of CME by inducing virus-to-cell membrane fusion at the plasma membrane (Fig. 4A).…”

“…As previously described (8,55), primary rat neuronal cultures were prepared from embryonic day 17 Sprague Dawley rat pups. Cells were plated on 24-well plates precoated with poly-L-lysine (Sigma), with or without coverslips, at a density of 2 ϫ 10 5 cells per well in neurobasal medium supplemented with B27 (Invitrogen), and they were maintained at 37°C and 5% CO 2 .…”

Systems To Establish Bunyavirus Genome Replication in the Absence of Transcription

“…The expression and reporter constructs for the minigenome and VLP systems of LACV, namely, pTM-LACV_L, pTM-LACV_N, pI.18-LACV_M (encoding the GPs), pHH21-LACV-vMRen, and pTM1-FFLuc, have been described previously (12,19,20). The expression constructs contain the appropriate coding sequences under the control of a T7 promoter and the encephalomyocarditis virus internal ribosome entry site (pTM1 backbone) or under the control of an RNA polymerase II promoter (pI.18 backbone).…”

“…The RNA synthesis activity of the viral polymerase can be measured by using a so-called "minigenome" (or "minireplicon") system, in which L and N expressed from cDNA plasmids encapsidate, replicate, and transcribe a negative-stranded reporter gene containing viral UTRs. If plasmid constructs for the viral glycoproteins (GPs) are cotransfected, the artificial RNPs can be packaged into virus-like particles (VLPs) that are released into the supernatant (9)(10)(11)(12). These VLPs can also infect new cells and express the encoded reporter gene.…”