UL148 is a viral endoplasmic reticulum (ER)-resident glycoprotein that contributes to human cytomegalovirus (HCMV) cell tropism. The influence of UL148 on tropism correlates with its potential to promote the expression of glycoprotein O (gO), a viral envelope glycoprotein that participates in a heterotrimeric complex with glycoproteins H and L that is required for infectivity. In an effort to gain insight into the mechanism, we used mass spectrometry to identify proteins that coimmunoprecipitate from infected cells with UL148. This approach led us to identify an interaction between UL148 and SEL1L, a factor that plays key roles in ER-associated degradation (ERAD). In pulse-chase experiments, gO was less stable in cells infected with -null mutant HCMV than during wild-type infection, suggesting a potential functional relevance for the interaction with SEL1L. To investigate whether UL148 regulates gO abundance by influencing ERAD, small interfering RNA (siRNA) silencing of either SEL1L or its partner, Hrd1, was carried out in the context of infection. Knockdown of these ERAD factors strongly enhanced levels of gO but not other viral glycoproteins, and the effect was amplified in the presence of UL148. Furthermore, pharmacological inhibition of ERAD showed similar results. Silencing of SEL1L during infection also stabilized an interaction of gO with the ER lectin OS-9, which likewise suggests that gO is an ERADsubstrate. Taken together, our results identify an intriguing interaction of UL148 with the ERAD machinery and demonstrate that gO behaves as a constitutive ERAD substrate during infection. These findings have implications for understanding the regulation of HCMV cell tropism. Viral glycoproteins in large part determine the cell types that an enveloped virus can infect and hence play crucial roles in transmission and pathogenesis. The glycoprotein H/L heterodimer (gH/gL) is part of the conserved membrane fusion machinery that all herpesviruses use to enter cells. In human cytomegalovirus (HCMV), gH/gL participates in alternative complexes in virions, one of which is a trimer of gH/gL with glycoprotein O (gO). Here, we show that gO is constitutively degraded during infection by the endoplasmic reticulum-associated degradation (ERAD) pathway and that UL148, a viral factor that regulates HCMV cell tropism, interacts with the ERAD machinery and slows gO decay. Since gO is required for cell-free virus to enter new host cells but dispensable for cell-associated spread that resists antibody neutralization, our findings imply that the posttranslational instability of a viral glycoprotein provides a basis for viral mechanisms to modulate tropism and spread.

Eukaryotic cells are equipped with three sensors that respond to the accumulation of misfolded proteins within the lumen of the endoplasmic reticulum (ER) by activating the unfolded protein response (UPR), which functions to resolve proteotoxic stresses involving the secretory pathway. Here, we identify UL148, a viral ER-resident glycoprotein from human cytomegalovirus (HCMV), as an inducer of the UPR. Metabolic labeling results indicate that global mRNA translation is decreased when UL148 expression is induced in uninfected cells. Further, we find that ectopic expression of UL148 is sufficient to activate at least two UPR sensors: the inositol-requiring enzyme-1 (IRE1), as indicated by splicing of mRNA, and the protein kinase R (PKR)-like ER kinase (PERK), as indicated by phosphorylation of the α subunit of eukaryotic initiation factor 2 (eIF2α) and accumulation of activating transcription factor 4 (ATF4). During wild-type HCMV infection, increases in splicing, eIF2α phosphorylation, and accumulation of ATF4 accompany UL148 expression. -null infections, however, show reduced levels of these UPR indicators and decreases in XBP1s abundance and in phosphorylation of PERK and IRE1. Small interfering RNA (siRNA) depletion of PERK dampened the extent of eIF2α phosphorylation and ATF4 induction observed during wild-type infection, implicating PERK as opposed to other eIF2α kinases. A virus with disrupted showed significant 2- to 4-fold decreases during infection in the levels of transcripts canonically regulated by PERK/ATF4 and by the ATF6 pathway. Taken together, our results argue that UL148 is sufficient to activate the UPR when expressed ectopically and that UL148 is an important cause of UPR activation in the context of the HCMV-infected cell. The unfolded protein response (UPR) is an ancient cellular response to ER stress that is of broad importance to viruses. Certain consequences of the UPR, including mRNA degradation and translational shutoff, would presumably be disadvantageous to viruses, while other attributes of the UPR, such as ER expansion and upregulation of protein folding chaperones, might enhance viral replication. Although HCMV is estimated to express well over 150 different viral proteins, we show that the HCMV ER-resident glycoprotein UL148 contributes substantially to the UPR during infection and, moreover, is sufficient to activate the UPR in noninfected cells. Experimental activation of the UPR in mammalian cells is difficult to achieve without the use of toxins. Therefore, UL148 may provide a new tool to investigate fundamental aspects of the UPR. Furthermore, our findings may have implications for understanding the mechanisms underlying the effects of UL148 on HCMV cell tropism and evasion of cell-mediated immunity.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has infected at least 180 million people since its identification as the cause of the current COVID-19 pandemic. The rapid pace of vaccine development has resulted in multiple vaccines already in use worldwide. The contemporaneous emergence of SARS-CoV-2 ‘variants of concern’ (VOC) across diverse geographic locales underscores the need to monitor the efficacy of vaccines being administered globally. All WHO designated VOC carry spike (S) polymorphisms thought to enable escape from neutralizing antibodies. Here, we characterize the neutralizing activity of post-Sputnik V vaccination sera against the ensemble of S mutations present in alpha (B.1.1.7) and beta (B.1.351) VOC. Using de novo generated replication-competent vesicular stomatitis virus expressing various SARS-CoV-2-S in place of VSV-G (rcVSV-CoV2-S), coupled with a clonal 293T-ACE2 + TMPRSS2 + cell line optimized for highly efficient S-mediated infection, we determine that only 1 out of 12 post-vaccination serum samples shows effective neutralization (IC90) of rcVSV-CoV2-S: B.1.351 at full serum strength. The same set of sera efficiently neutralize S from B.1.1.7 and exhibit only moderately reduced activity against S carrying the E484K substitution alone. Taken together, our data suggest that control of some emergent SARS-CoV-2 variants may benefit from updated vaccines.

The on-going coronavirus disease 2019 (COVID-19) pandemic has mobilized a global effort to develop vaccines and therapeutics that inhibit viral entry by inducing or transferring antibodies against the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike glycoprotein (CoV2-S). Phase I/II vaccine clinical trials, monoclonal antibodies, and convalescent sera have all shown promise. However, these efforts often require extensive screening with the live virus under onerous high biocontainment conditions (BSL-3). Virus neutralization assays (VNAs) remain the gold standard for evaluating the anti-viral potency of antibodies and entry inhibitors. The proliferation of pseudotyped virus systems that can be used in BSL-2 compatible VNAs is a positive development. Yet, there is marked variability between VNAs and how the findings are presented, making inter-group comparisons difficult. To address these limitations, we developed a standardized VNA using VSVdeltaG based CoV-2-S pseudotyped particles (CoV2pp) that can be robustly produced at scale. We used our CoV2pp to interrogate the role of exogenous and endogenous proteases in CoV-2-S mediated entry and standardized our VNA based on that understanding. Our CoV2pp VNA showed a strong positive correlation with CoV2-S ELISA and live virus neutralizations in a validated set of patient sera. Our system was subsequently validated by three independent groups as an out-of-the-box VNA. More than 120 patient sera were screened, and we report descriptive statistics for absolute (abs) IC50, IC80, and IC90 values from all positive patient sera. Lastly, we used our CoV2pp in a screen to identify ultrapermissive 293T clones that stably express ACE2 or ACE2+TMPRSS2. When used in combination with our CoV2pp, we can now produce CoV2pp sufficient for 150,000 standardized VNA/week.

Entry of SARS-CoV-2 is facilitated by endogenous and exogenous proteases. These proteases proteolytically activate the SARS-CoV-2 spike glycoprotein and are key modulators of virus tropism. We show that SARS-CoV-2 naïve serum exhibits significant inhibition of SARS-CoV-2 entry. We identify alpha-1-antitrypsin (AAT), and to a lesser degree, alpha-2-macroglobulin (A2M) as highly abundant serum protease inhibitors that potently restrict protease-mediated entry of SARS-CoV-2. AAT inhibition of protease-mediated SARS-CoV-2 entry in vitro occurs at concentrations far below what is present in serum and bronchoalveolar tissues, suggesting that AAT effects are physiologically relevant. Moreover, AAT mutations that have been characterized to affect abundance or function are highly prevalent. In addition to the effects that AAT may have on viral entry itself, we argue that the anti-inflammatory and coagulation regulatory activity of AAT have implications for coronavirus disease 2019 (COVID-19) pathogenicity, SARS-CoV-2 tissue restriction, convalescent plasma therapies, and even potentially AAT therapy.