Biology subjects

Siddiquey, M. N. A.

Publications and source records attributed to Siddiquey, M. N. A..

5 recordsLinked to original sources

bioRxiv2024

A noncanonical glycoprotein H complex enhances cytomegalovirus entry

Human cytomegalovirus (HCMV) causes severe birth defects, lifelong health complications, and $4 billion in annual costs in the United States alone. A major challenge in vaccine design is the incomplete understanding of the diverse protein complexes the virus uses to infect cells. In Herpesviridae, the gH/gL glycoprotein heterodimer is expected to be a basal element of virion cell entry machinery. For HCMV, gH/gL forms a "trimer" with gO and a "pentamer" with UL128, UL130, and UL131A, with each complex binding distinct receptors to enter varied cell types. Here, we reveal a third glycoprotein complex, abundant in HCMV virions, which significantly enhances infection of endothelial cells. In this "3-mer" complex, gH, without gL, associates with UL116 and UL141, an immunoevasin previously known to function in an intracellular role. Cryo-EM reveals the virion-surface 3-mer is structurally unique among Herpesviridae gH complexes, with gH-only scaffolding, UL141-mediated dimerization and a heavily glycosylated UL116 cap. Given that antibodies directed at gH and UL141 each can restrict HCMV replication, our work highlights this virion surface complex as a new target for vaccines and antiviral therapies.

microbiology↗

bioRxiv2020

The human cytomegalovirus protein UL116 interacts with the viral ER resident glycoprotein UL148 and promotes the incorporation of gH/gL complexes into virions

Heterodimers of glycoproteins H (gH) and L (gL) comprise a basal element of the viral membrane fusion machinery conserved across herpesviruses. In human cytomegalovirus (HCMV), a glycoprotein encoded by UL116 noncovalently assembles onto gH at a position similar to that occupied by gL, forming a heterodimer that is incorporated into virions. However, physiological roles for UL116 or its complex with gH remain to be identified. Here, we show that UL116 promotes the expression of gH/gL complexes and is required for the efficient production of infectious cell-free virions. UL116-null mutants show a 10-fold defect in production of infectious cell-free virions from infected fibroblasts and epithelial cells. This defect is accompanied by reduced expression of the two disulfide-linked gH/gL complexes that play crucial roles in viral entry: the heterotrimer of gH/gL with glycoprotein O (gO) and the pentameric complex of gH/gL with UL128, UL130, and UL131. Furthermore, gH/UL116 complexes comprise a substantial constituent of virions since an abundant gH species not covalently linked to other glycoproteins, which has long been observed in the literature, is readily detected from wild-type but not UL116-null virions. Interestingly, UL116 co-immunoprecipitates with UL148, a viral ER resident glycoprotein previously shown to attenuate ER-associated degradation (ERAD) of gO, and we observe elevated levels of UL116 in UL148-null virions. Collectively, our findings suggest that UL116 may serve as a chaperone for gH to support the assembly, maturation, and incorporation of gH/gL complexes into virions. IMPORTANCEHCMV is a betaherpesvirus that causes dangerous opportunistic infections in immunocompromised patients, as well as in the immune-naive fetus and preterm infants. The potential of the virus to enter new host cells is governed in large part by two alternative viral glycoprotein H (gH) / glycoprotein L (gL) complexes that play important roles in entry: gH/gL/gO and gH/gL/UL128-131. A recently identified virion gH complex, comprised of gH bound to UL116, adds a new layer of complexity to the mechanisms that contribute to HCMV infectivity. Here, we show that UL116 promotes the expression of gH/gL complexes, and that UL116 interacts with the viral ER-resident glycoprotein UL148, a factor that supports the expression of gH/gL/gO. Overall, our results suggest that UL116 is a chaperone for gH. These findings have important implications for understanding of HCMV cell tropism as well as for the development of vaccines against the virus.

microbiology↗

bioRxiv2019

A cytomegalovirus immunevasin triggers integrated stress response-dependent reorganization of the endoplasmic reticulum.

Human cytomegalovirus (HCMV) encodes an ER-resident glycoprotein, UL148, which activates the unfolded protein response (UPR) but is fully dispensable for viral replication in cultured cells. Hence, its previously ascribed roles in immune evasion and modulation of viral cell tropism are hypothesized to cause ER stress. Here, we show that UL148 is necessary and sufficient to drive the formation of large ER-derived structures that occupy up to 7% of the infected cell cytosol. The structures are found to be sites where UL148 coalesces with cellular proteins involved in ER quality control, such as Hrd1 and EDEM1. Transmission electron microscopy analyses reveal the structures to be comprised of tortuous, densely packed and apparently collapsed ER membranes that connect to distended cisternae. During induced ectopic expression of UL148-GFP fusion protein, punctate signals traffic to accumulate at prominent structures that exhibit poor recovery of fluorescence after photobleaching. Small molecule blockade of the integrated stress response (ISR) prevents the formation of puncta, leading to a uniform reticular fluorescent signal. Accordingly, ISR inhibition during HCMV infection abolishes the coalescence of UL148 and Hrd1 into discrete structures, which argues that UL148 requires the ISR to cause ER reorganization. Given that UL148 stabilizes immature forms of a receptor binding subunit for a viral envelope glycoprotein complex important for HCMV infectivity, our results imply that stress-dependent ER remodeling contributes to viral cell tropism.\n\nIMPORTANCEPerturbations to ER morphology occur during infection with various intracellular pathogens and in certain genetic disorders. We identify that an HCMV gene product, UL148, profoundly reorganizes the ER during infection, and is sufficient to do so when expressed on its own. Our results reveal that UL148-dependent reorganization of the ER is a prominent feature of HCMV infected cells. Moreover, we find that this example of virally induced organelle remodeling requires the integrated stress response (ISR), a stress adaptation pathway that contributes to a number of disease states. Since ER reorganization accompanies the roles of UL148 in HCMV cell tropism and intracellular retention of the immune cell co-stimulatory ligand CD58, our results may have implications for understanding the mechanisms involved. Furthermore, our findings provide a basis to utilize UL148 as a tool to investigate organelle responses to stress and to identify novel drugs targeting the ISR.

microbiology↗

bioRxiv2018

The human cytomegalovirus ER resident glycoprotein UL148 activates the unfolded protein response

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 markedly decreased when UL148 expression is induced in uninfected cells. Further, we find evidence suggesting 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 Xbp1 mRNA, and the PKR-like ER kinase (PERK), as indicated by phosphorylation of eIF2 and accumulation of ATF4 protein. During wild-type HCMV infection, Xbp-1 splicing, eIF2 phosphorylation and ATF4 accumulation neatly accompanied the onset of UL148 expression. However, the appearance of these UPR indicators was either markedly delayed or absent during UL148-null infections. siRNA depletion of PERK dampened the extent of eIF2 phosphorylation and ATF4 induction observed during wild-type infection, implicating PERK as opposed to other eIF2 kinases. A virus disrupted for UL148 showed statistically 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.\n\nIMPORTANCEThe unfolded protein response (UPR) is an ancient cellular response to ER stress of broad importance to viruses. Certain consequences of the UPR, including mRNA degradation and translational shut-off, 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 at least 200 distinct 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 non-infected 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.

microbiology↗

bioRxiv2018

Human cytomegalovirus tropism modulator UL148 interacts with SEL1L, a cellular factor that governs ER-associated degradation of the viral envelope glycoprotein, gO

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 mechanism, we used mass spectrometry to identify proteins that co-immunoprecipitate 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 a UL148-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, 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 ERAD substrate. 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.

microbiology↗