Biology subjects

Henke, W.

Publications and source records attributed to Henke, W..

3 recordsLinked to original sources

bioRxiv2023

The Role of the Tyrosine-Based Sorting Signals of the ORF3a Protein of SARS-CoV-2 on Intracellular Trafficking, Autophagy, and Apoptosis

The open reading frame 3a (ORF3a) is an accessory transmembrane protein that is important to the pathogenicity of SARS-CoV-2. The cytoplasmic domain of ORF3a has three canonical tyrosine-based sorting signals (Yxx{Phi}; where x is any amino acid and {Phi} is a hydrophobic amino acid with a bulky -R group). They have been implicated in the trafficking of membrane proteins to the cell plasma membrane and to intracellular organelles. Previous studies have indicated that mutation of the 160YSNV163 motif abrogated plasma membrane expression and inhibited ORF3a-induced apoptosis. However, two additional canonical tyrosine-based sorting motifs (211YYQL213, 233YNKI236) exist in the cytoplasmic domain of ORF3a that have not been assessed. We removed all three potential tyrosine-based motifs and systematically restored them to assess the importance of each motif or combination of motifs that restored efficient trafficking to the cell surface and lysosomes. Our results indicate that the Yxx{Phi} motif at position 160 was insufficient for the trafficking of ORF3a to the cell surface. Our studies also showed that ORF3a proteins with an intact Yxx{Phi} at position 211 or at 160 and 211 were most important. We found that ORF3a cell surface expression correlated with the co-localization of ORF3a with LAMP-1 near the cell surface. These results suggest that Yxx{Phi} motifs within the cytoplasmic domain may act cooperatively in ORF3a transport to the plasma membrane and endocytosis to lysosomes. Further, our results indicate that certain tyrosine mutants failed to activate caspase 3 and did not correlate with autophagy functions associated with this protein. IMPORTANCEOpen reading frame 3a (ORF3a) encodes for the largest of the SARS-CoV-2 accessory proteins. While deletion of the ORF3a gene from SARS-CoV-2 results in a virus that replicates slightly less efficiently in cell culture, deletion also results in a virus that is less pathogenic in mouse models of SARS-CoV-2 infections. The ORF3a has been reported to be a viroporin, induces apoptosis and incomplete autophagy in cells. Thus, determining the domains involved in these functions will further our understanding of how this protein influences virus assembly and pathogenesis. Here, we investigated the role of the three potential tyrosine-based sorting signals in the cytoplasmic domain of the ORF3a on intracellular protein trafficking, apoptosis, and in the initiation of autophagy. Our results indicate that more than one Yxx{Phi} motif is required for efficient transport of ORF3a, ORF3a expression resulted in minimal apoptosis, and cell surface expression was not required for autophagy.

cell biology↗

bioRxiv2022

The Envelope Protein of SARS-CoV-2 Inhibits Viral Protein Synthesis and Infectivity of Human Immunodeficiency Virus type 1 (HIV-1)

The human coronavirus SARS-CoV-2 encodes for a small 75 amino acid transmembrane protein known as the envelope (E) protein. The E protein forms an ion channel, like the viroporins from human immunodeficiency virus type 1 (HIV-1) (Vpu) and influenza A virus (M2). Here, we analyzed HIV-1 virus infectivity in the presence of four different {beta}-coronavirus E proteins. We observed that the SARS-CoV-2 and SARS-CoV E proteins reduced HIV-1 yields by approximately 100-fold while MERS-CoV or HCoV-OC43 E proteins restricted HIV-1 infectivity to a lesser extent. This was also reflected in the levels of HIV-1 protein synthesis in cells. Mechanistically, we show that that the E protein neither affected reverse transcription nor genome integration. However, SARS-CoV-2 E protein activated the ER-stress pathway associated with the phosphorylation of eIF-2, which is known to attenuate protein synthesis in cells. Finally, we show that these four E proteins and the SARS-CoV-2 N protein did not significantly down-regulate bone marrow stromal cell antigen 2 (BST-2) while the spike (S) proteins of SARS-CoV and SARS-CoV-2, and HIV-1 Vpu efficiently down-regulated cell surface BST-2 expression. The results of this study show for the first time that viroporins from a heterologous virus can suppress HIV-1 infection. IMPORTANCEThe E protein of coronaviruses is a viroporin that is required for efficient release of infectious virus and for viral pathogenicity. We determined if the E protein from four {beta}-coronaviruses could restrict virus particle infectivity of HIV-1 infection. Our results indicate that the E proteins from SARS-CoV-2 and SARS-CoV potently restricted HIV-1 while those from MERS-CoV and HCoV-OC43 were less restrictive. Substitution of the highly conserved proline in the cytoplasmic domain of SARS-CoV-2 E abrogated the restriction on HIV-1 infection. Mechanistically, the SARS-CoV-2 E protein did not interfere with viral integration or RNA synthesis but rather reduced viral protein synthesis. We show that the E protein-initiated ER stress causing phosphorylation of eIF-2, which is known to attenuate protein synthesis. Companion studies suggest that the E protein also triggers autophagy. These results show for the first time that a viroporin from a coronavirus can restrict infection of another virus.

microbiology↗

bioRxiv2022

SARS-CoV-2 harnesses host translational shutoff and autophagy to optimize virus yields: The role of the envelope (E) protein

The SARS-CoV-2 virion is composed of four structural proteins: spike (S), nucleocapsid (N), membrane (M), and envelope (E). E spans the membrane a single time and is the smallest, yet most enigmatic of the structural proteins. E is conserved among coronaviruses and has an essential role in virus-mediated pathogenesis. We found that ectopic expression of E had deleterious effects on the host cell as it activated stress responses, leading to phosphorylation of the translation initiation factor eIF2 and LC3 lipidation that resulted in host translational shutoff. During infection E is highly expressed although only a small fraction is incorporated into virions, suggesting that E activity is regulated and harnessed by the virus to its benefit. In support of this, we found that the {gamma}1 34.5 protein of herpes simplex virus 1 (HSV-1) prevented deleterious effects of E on the host cell and allowed for E protein accumulation. This observation prompted us to investigate whether other SARS-CoV-2 structural proteins regulate E. We found that the N and M proteins enabled E protein accumulation, whereas S prevented E accumulation. While {gamma}1 34.5 protein prevented deleterious effects of E on the host cells, it had a negative effect on SARS-CoV-2 replication. This negative effect of {gamma}1 34.5 was most likely associated with failure of SARS-CoV-2 to divert the translational machinery and with deregulation of autophagy pathways. Overall, our data suggest that SARS-CoV-2 causes stress responses and subjugates these pathways, including host protein synthesis (phosphorylated eIF2) and autophagy, to support optimal virus production. ImportanceIn 2020, a new {beta}-coronavirus, SARS-CoV-2, entered the human population that has caused a pandemic resulting in 6 million deaths worldwide. Although closely related to SARS-CoV, the mechanisms of SARS-CoV-2 pathogenesis are not fully understood. We found that ectopic expression of the SARS-CoV-2 E protein had detrimental effects on the host cell, causing metabolic alterations including shutoff of protein synthesis and mobilization of cellular resources through autophagy activation. Co-expression of E with viral proteins known to subvert host antiviral responses such as autophagy and translational inhibition, either from SARS-CoV-2 or from heterologous viruses increased cell survival and E protein accumulation. However, such factors were found to negatively impact SARS-CoV-2 infection, as autophagy contributes to formation of viral membrane factories, and translational control offers an advantage for viral gene expression. Overall, SARS-CoV-2 has evolved mechanisms to harness host functions that are essential for virus replication.

microbiology↗