Lentiviruses inefficiently incorporate human parainfluenza type 3 envelope proteins

Abstract

We have previously shown that the envelope glycoproteins of human parainfluenza type 3 (HPIV3), F and HN, are able to pseudotype lentiviruses, but the titers of these viruses are too low for use in clinical gene transfer. In this study we investigated the cause of these low titers. We compared the mRNA and protein expression levels of HN and F in transfected cells and in cells infected with wild-type HPIV3. Transfected cells contained similar levels of HN and F cytosolic mRNA, but fewer cell-surface HN and F proteins (3.8- and 1.3-fold less, respectively), than cells infected with wild-type HPIV3. To increase expression of HN in transfected cells, we codon-optimized HN and used it to transfect lentivirus producer cells. Cell surface expression of HN, as well as the amount of HN incorporated into virus particles, increased two- to threefold. Virus titers increased 1.2- to 6.4-fold, and the transduction efficiency of polarized MDCK cells via their apical surfaces increased 1.4-fold. Interestingly, even though codon optimization improved the expression levels of HN and virus titers, we found that HPIV3 pseudotyped viruses contained about 14-fold fewer envelope proteins than lentiviruses pseudotyped with the amphotropic envelope protein. Taken together, our findings suggest that titers are low, not because virus producer cells express levels of HPIV3 envelope proteins that are too low, but because too few of these proteins are incorporated by the lentiviruses for them to be able to efficiently transduce cells.

The Hemagglutinin of Bat-Associated Influenza Viruses Is Activated by TMPRSS2 for pH-Dependent Entry into Bat but Not Human Cells

“…We used a VSV-based vector system to study cellular entry of HAL and NAL-bearing particles. VSV pseudotypes allow convenient analysis of entry driven by diverse glycoproteins [28,31,61], although one should keep in mind that due to differences in particle shape and efficiency of glycoprotein incorporation pseudotypes might not adequately mirror all aspects of cellular entry of authentic viruses [62,63]. We found that cell lines frequently used for FLUAV propagation were not susceptible to transduction by HAL and NAL bearing particles, which is in agreement with the finding that replacement of batFLUAV-HAL and -NAL by their FLUAV counterparts is required for spread of batFLUAV in the cell lines studied so far [18,19].…”

Efficient Pseudotyping of Different Retroviral Vectors Using a Novel, Codon-Optimized Gene for Chimeric GALV Envelope

“…Codon optimization was previously used to optimize production of pseudotyped LVVs and γ-RVVs, e.g., with Spike protein from SARS-CoV-1 [ 32 ], parainfluenza type 3 envelope proteins [ 33 ], and a truncated HIV-Env [ 34 ]. In striking contrast, Zucchelli et al observed impaired functionality of codon-optimized RD114-TR, another envelope protein derived from a γ-retrovirus that is commonly used for pseudotyping [ 20 ].…”