Cleavage of Hemagglutinin-Bearing Lentiviral Pseudotypes and Their Use in the Study of Influenza Virus Persistence

Abstract

Influenza A viruses (IAVs) are a major cause of infectious respiratory human diseases and their transmission is dependent upon the environment. However, the role of environmental factors on IAV survival outside the host still raises many questions. In this study, we used lentiviral pseudotypes to study the influence of the hemagglutinin protein in IAV survival. High-titered and cleaved influenza-based lentiviral pseudoparticles, through the use of a combination of two proteases (HAT and TMPRSS2) were produced. Pseudoparticles bearing hemagglutinin proteins derived from different H1N1, H3N2 and H5N1 IAV strains were subjected to various environmental parameters over time and tested for viability through single-cycle infectivity assays. We showed that pseudotypes with different HAs have different persistence profiles in water as previously shown with IAVs. Our results also showed that pseudotypes derived from H1N1 pandemic virus survived longer than those derived from seasonal H1N1 virus from 1999, at high temperature and salinity, as previously shown with their viral counterparts. Similarly, increasing temperature and salinity had a negative effect on the survival of the H3N2 and H5N1 pseudotypes. These results showed that pseudotypes with the same lentiviral core, but which differ in their surface glycoproteins, survived differently outside the host, suggesting a role for the HA in virus stability.

Influenza Virus Segment Composition Influences Viral Stability in the Environment

“…We also introduced non-synonymous mutations in the HA nucleotide sequence to study the impact of a single HA amino acid change on virus persistence. For this purpose, we analyzed the HA amino acid sequences of the HA-NA/Nc99 and HA-NA/Par09 viruses and, based on a preliminary mutation screening performed using a lentiviral pseudo-particles system ( Sawoo et al, 2014 ), we selected five residues in the HA protein that might influence virus particle persistence outside the host ( Figure 2B ). Ten reassortant viruses bearing HA with amino acid substitutions or insertions/deletions were generated after site-directed mutagenesis of the pPol-HA plasmid.…”

Challenges on the development of a pseudotyping assay for Zika glycoproteins

“…The fivefold dilutions of the GFP(+) ZIKV plasmid (2, 0.4, 0.08, 0.016 and 0.0032 µg) with a fixed amount of pNL4.3 backbone () showed no difference between the negative control and the pseudotypes produced with the fivefold dilution. Previous reports have shown that supplementation with viral proteases enhances pseudotype production [35–37]. A tri-plasmid system composed of a fixed concentration of GFP(+) ZIKV and pNL4.3 and increasing amounts of ZIKV NS2B-NS3 containing plasmid was tested ().…”

Survival of Viruses in Water

“…Virus inactivation also seems to be correlated to dissolved oxygen, and increased capsid oxidation has been proposed as an explanation [8]. Studies on influenza virus H5N1 indicated that acidic pH affects hemagglutinin (HA surface protein), which is involved in fusion of the viral membrane with the host membrane [20,41]. In this case, different subtypes carrying different HA would then have different stabilities in relation to the pH.…”

“…In this case, different subtypes carrying different HA would then have different stabilities in relation to the pH. Such differences in HA stability could be explained by their tertiary structures [41]. Surface charges of viruses vary according to the pH: most viruses have a negative charge above a certain threshold pH value (5 for enteric viruses), and a positive one below [2].…”