TMEM106B is a receptor mediating ACE2-independent SARS-CoV-2 cell entry.

SARS-CoV-2 is associated with broad tissue tropism, a characteristic often determined by the availability of entry receptors on host cells. Here, we show that TMEM106B, a lysosomal transmembrane protein, can serve as an alternative receptor for SARS-CoV-2 entry into angiotensin-converting enzyme 2 (ACE2)-negative cells. Spike substitution E484D increased TMEM106B binding, thereby enhancing TMEM106B-mediated entry. TMEM106B-specific monoclonal antibodies blocked SARS-CoV-2 infection, demonstrating a role of TMEM106B in viral entry. Using X-ray crystallography, cryogenic electron microscopy (cryo-EM), and hydrogen-deuterium exchange mass spectrometry (HDX-MS), we show that the luminal domain (LD) of TMEM106B engages the receptor-binding motif of SARS-CoV-2 spike. Finally, we show that TMEM106B promotes spike-mediated syncytium formation, suggesting a role of TMEM106B in viral fusion. Together, our findings identify an ACE2-independent SARS-CoV-2 infection mechanism that involves cooperative interactions with the receptors heparan sulfate and TMEM106B.

An improved in vitro and in vivo Sindbis virus expression system through host and virus engineering.

The Sindbis viral expression system enables the rapid production of high levels of recombinant protein in mammalian cells; however, this expression is typically limited to transient production due to the cytotoxicity of the virus. Limiting the lethality inherent in the Sindbis virus vector in order to enable long term, sustained expression of recombinant proteins may be possible. In this study, modifications to virus and host have been combined in order to reduce the cytopathic effects. Non-cytopathic replication competent viruses of two Sindbis viral strains, TE and 633, were developed using a non-structural protein (nsP) P726S point mutation in order to obtain persistent heterologous gene expression in infected Baby Hamster Kidney (BHK) cells and Chinese Hamster Ovary (CHO) cells. Cells infected with the P726S variant viruses were able to recover after infection, while cells infected with normal virus died within 3 days. The P726S mutation did not reduce the susceptibility of 5- and 14-day-old mice to 633 and TE viruses in vivo. In addition, animal survival with the P726S variant viruses was increased and GFP expression was sustained for at least 14 days while the 633 and TE infection resulted in short-term GFP expression or an earlier mortality. Modifications to the host BHK and CHO cells themselves were subsequently undertaken by including the anti-apoptotic gene Bcl-2 and a deletion mutant of Bcl-2 (Bcl-2Delta) as another method for limiting the cytopathic effects of the Sindbis virus. The inclusion of anti-apoptotic genes permitted higher production of heterologous GFP protein following Sindbis virus infection, and the combination of the TE-P726S virus and the CHO-Bcl-2Delta cell line showed the greatest improvement in cell survival. Sindbis virus infection also induced ER stress in mammalian cells as detected by increased PERK phosphorylation and ATF4 translation. Overexpression of Parkin, an E3 ubiquitin ligase that can protect cells against agents that induce ER stress, suppressed Sindbis virus-induced cell death in both BHK cells and in vivo studies in mice. Such findings show that viral and host modifications can improve cell survival and production of heterologous proteins, change viral behavior in vitro and in vivo, and assist in the development of new expression or gene delivery vehicles.

Anti-apoptotic genes Aven and E1B-19K enhance performance of BHK cells engineered to express recombinant factor VIII in batch and low perfusion cell culture.

The engineering of production cell lines to express anti-apoptotic genes has been pursued in recent years due to potential process benefits, including enhanced cell survival, increased protein expression, and improved product quality. In this study, a baby hamster kidney cell line secreting recombinant factor VIII (BHK-FVIII) was engineered to express the anti-apoptotic genes Aven and E1B-19K. In high cell density shake flask culture evaluation, 11 clonal cell lines expressing either E1B-19K or a combination of Aven and E1B-19K showed improved survival compared to both parental and blank vector cell line controls. These cell lines exhibited lower caspase-3 activation and reduced Annexin-V binding compared to the controls. Parental and blank vector cell lines were less than 50% viable after 48 h of exposure to thapsigargin while cell lines expressing E1B-19K with or without Aven maintained viabilities approaching 90%. Subsequently, the best Aven-E1B-19K candidate cell line was compared to the parental cell line in 12-L perfusion bioreactor studies. Choosing the appropriate perfusion rates in bioreactors is a bioprocess optimization issue, so the bioreactors were operated at sequentially lower specific perfusion rates, while maintaining a cell density of 2 x 10(7) viable cells/mL. The viability of the parental cell line declined from nearly 100% at a perfusion rate of 0.5 nL/cell/day to below 80% viability, with caspase-3 activity exceeding 15%, at its lower perfusion limit of 0.15 nL/cell/day. In contrast, the Aven-E1B-19K cell line maintained an average viability of 94% and a maximum caspase-3 activity of 2.5% even when subjected to a lower perfusion minimum of 0.1 nL/cell/day. Factor VIII productivity, specific growth rate, and cell size decreased for both cell lines at lower perfusion rates, but the drop in all cases was larger for the parental cell line. Specific consumption of glucose and glutamine and production of lactate were consistently lower for the Aven-E1B-19K culture. Furthermore, the yield of ammonia from glutamine increased for the Aven-E1B-19K cell line relative to the parent to suggest altered metabolic pathways following anti-apoptosis engineering. These results demonstrate that expression of anti-apoptotic genes Aven and E1B-19K can increase the stability and robustness of an industrially relevant BHK-FVIII mammalian cell line over a wide range of perfusion rates.