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1.
Viruses ; 13(2)2021 01 20.
Article in English | MEDLINE | ID: mdl-33498300

ABSTRACT

Alfuy (ALFV) is an attenuated flavivirus related to the Murray Valley encephalitis virus (MVEV). We previously identified markers of attenuation in the envelope (E) protein of the prototype strain (ALFV3929), including the hinge region (E273-277) and lack of glycosylation at E154-156. To further determine the mechanisms of attenuation we assessed ALFV3929 binding to glycosaminoglycans (GAG), a known mechanism of flaviviruses attenuation. Indeed, ALFV3929 exhibited reduced binding to GAG-rich cells in the presence of heparin; however, low-passage ALFV isolates were relatively unaffected. Sequence comparisons between ALFV strains and structural modelling incriminated a positively-charged residue (K327) in ALFV3929 as a GAG-binding motif. Substitution of this residue to the corresponding uncharged residue in MVEV (L), using a previously described chimeric virus containing the prM & E genes of ALFV3929 in the backbone of MVEV (MVEV/ALFV-prME), confirmed a role for K327 in enhanced GAG binding. When the wild type residues at E327, E273-277 and E154-156 of ALFV3929 were replaced with the corresponding residues from virulent MVEV, it revealed each motif contributed to attenuation of ALFV3929, with the E327/E273-277 combination most dominant. These data demonstrate that attenuation of ALFV3929 is multifactorial and provide new insights for the rational design of attenuated flavivirus vaccines.


Subject(s)
Encephalitis Virus, Murray Valley/pathogenicity , Encephalitis Viruses, Japanese/pathogenicity , Encephalitis, Arbovirus/virology , Flavivirus Infections/virology , Viral Envelope Proteins/chemistry , Viral Envelope Proteins/metabolism , Amino Acid Motifs , Animals , Brain/pathology , Brain/virology , Cell Line , Encephalitis Virus, Murray Valley/chemistry , Encephalitis Virus, Murray Valley/metabolism , Encephalitis Viruses, Japanese/chemistry , Encephalitis Viruses, Japanese/growth & development , Encephalitis Viruses, Japanese/metabolism , Encephalitis, Arbovirus/pathology , Flavivirus Infections/pathology , Glycosaminoglycans/metabolism , Glycosylation , Heparin/pharmacology , Mice , Mutation , Protein Domains , Serial Passage , Viral Envelope Proteins/genetics , Viral Plaque Assay , Virulence
2.
mBio ; 11(3)2020 05 19.
Article in English | MEDLINE | ID: mdl-32430465

ABSTRACT

Quantum dots (QDs) possess optical properties of superbright fluorescence, excellent photostability, narrow emission spectra, and optional colors. Labeled with QDs, single molecules/viruses can be rapidly and continuously imaged for a long time, providing more detailed information than when labeled with other fluorophores. While they are widely used to label proteins in single-molecule-tracking studies, QDs have rarely been used to study virus infection, mainly due to a lack of accepted labeling strategies. Here, we report a general method to mildly and readily label enveloped viruses with QDs. Lipid-biotin conjugates were used to recognize and mark viral lipid membranes, and streptavidin-QD conjugates were used to light them up. Such a method allowed enveloped viruses to be labeled in 2 h with specificity and efficiency up to 99% and 98%, respectively. The intact morphology and the native infectivity of viruses were preserved. With the aid of this QD labeling method, we lit wild-type and mutant Japanese encephalitis viruses up, tracked their infection in living Vero cells, and found that H144A and Q258A substitutions in the envelope protein did not affect the virus intracellular trafficking. The lipid-specific QD labeling method described in this study provides a handy and practical tool to readily "see" the viruses and follow their infection, facilitating the widespread use of single-virus tracking and the uncovering of complex infection mechanisms.IMPORTANCE Virus infection in host cells is a complex process comprising a large number of dynamic molecular events. Single-virus tracking is a versatile technique to study these events. To perform this technique, viruses must be fluorescently labeled to be visible to fluorescence microscopes. The quantum dot is a kind of fluorescent tag that has many unique optical properties. It has been widely used to label proteins in single-molecule-tracking studies but rarely used to study virus infection, mainly due to the lack of an accepted labeling method. In this study, we developed a lipid-specific method to readily, mildly, specifically, and efficiently label enveloped viruses with quantum dots by recognizing viral envelope lipids with lipid-biotin conjugates and recognizing these lipid-biotin conjugates with streptavidin-quantum dot conjugates. It is not only applicable to normal viruses, but also competent to label the key protein-mutated viruses and the inactivated highly virulent viruses, providing a powerful tool for single-virus tracking.


Subject(s)
Encephalitis Viruses, Japanese/chemistry , Membrane Lipids/chemistry , Quantum Dots , Single-Cell Analysis/methods , Staining and Labeling/methods , Animals , Biotin/metabolism , Chlorocebus aethiops , Cricetinae , Dogs , Madin Darby Canine Kidney Cells , Microscopy, Fluorescence/methods , Streptavidin/metabolism , Vero Cells
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