Labeling of influenza viruses with synthetic fluorescent and biotin-labeled lipids.

Direct labeling of virus particles is a powerful tool for the visualization of virus-cell interaction events. However, this technique involves the chemical modification of viral proteins that affects viral biological properties. Here we describe an alternative approach of influenza virus labeling that utilizes Function-Spacer-Lipid (FSL) constructs that can be gently inserted into the virus membrane. We assessed whether labeling with fluorescent (fluo-Ad-DOPE) or biotin-labeled (biot-CMG2-DOPE) probes has any deleterious effect on influenza virus hemagglutinin (HA) receptor specificity, neuraminidase (NA) activity, or replicative ability in vitro. Our data clearly show that neither construct significantly affected influenza virus infectivity or viral affinity to sialyl receptors. Neither construct influenced the NA activities of the influenza viruses tested, except the A/Puerto Rico/8/34 (H1N1) strain. Our data indicate that lipid labeling provides a powerful tool to analyze influenza virus infection in vitro.

Fluorescein and radiolabeled Function-Spacer-Lipid constructs allow for simple in vitro and in vivo bioimaging of enveloped virions.

Tools that can aid in vitro and in vivo imaging and also noninvasively determine half-life and biodistribution are required to advance clinical developments. A Function-Spacer-Lipid construct (FSL) incorporating fluorescein (FSL-FLRO4) was used to label vesicular stomatitis virus (VSV), measles virus MV-NIS (MV) and influenza virus (H1N1). The ability of FSL constructs to label these virions was established directly by FACScan of FSL-FLRO4 labeled VSV and MV, and indirectly following labeled H1N1 and MV binding to a cells. FSL-FLRO4 labeling of H1N1 was shown to maintain higher infectivity of the virus when compared with direct fluorescein virus labeling. A novel tyrosine (125)I radioiodinated FSL construct was synthesized (FSL-(125)I) from FSL-tyrosine. This was used to label VSV (VSV-FSL-(125)I), which was infused into the peritoneal cavity of laboratory mice. Bioscanning showed VSV-FSL-(125)I to localize in the liver, spleen and bloodstream in contrast to the free labels FSL-(125)I or (125)I, which localized predominantly in the liver and thyroid respectively. This is a proof-of-principle novel and rapid method for modifying virions and demonstrates the potential of FSL constructs to improve in vivo imaging of virions and noninvasively observe in vivo biodistribution.