Biology subjects

von Einem, J.

Publications and source records attributed to von Einem, J..

5 recordsLinked to original sources

bioRxiv2026

Phosphorylation-tuned condensation links HCMV tegument assembly to membrane recruitment

Herpesviruses build complex infectious particles around a protein layer, the tegument, that lacks an ordered architecture. How this apparently amorphous material selectively assembles on the capsid and engages enveloping membranes remains unclear. Here we show that the capsid-anchored human cytomegalovirus protein pp150 forms liquid-like condensates when locally concentrated. Its disordered region recruits soluble tegument proteins and membrane-associated partners, providing a mechanism to couple assembly of the tegument layer to recruitment of the enclosing membrane. Phosphorylation tunes this condensation: phosphomimetic mutations prevent recovery of infectious virus, whereas loss of phosphorylation sites causes aberrant capsid-tegument assemblies and impairs viral replication. Together, these findings identify regulated condensation as a mechanism that couples tegument assembly to membrane recruitment and supports the production of infectious particles, highlighting condensate regulation as a potentially novel point of antiviral intervention.

microbiology↗

bioRxiv2025

Initiation of human cytomegalovirus secondary envelopment requires the gM/gN glycoprotein complex and involves palmitoylation.

Glycoprotein M (gM) of human cytomegalovirus (HCMV) forms a protein complex with glycoprotein N (gN), whose precise function in viral morphogenesis is poorly understood. Both proteins are highly conserved across herpesviruses and likely perform similar functions in virion assembly and egress, with the gM/gN complex being essential for HCMV morphogenesis. To elucidate the function of the gM/gN complex in secondary envelopment, we employed a combination of viral mutants, siRNA knockdown experiments, and ultrastructural analyses. A detailed examination of the virion morphogenesis of a mutant virus with a cysteine-to-serine mutation in the cytoplasmic tail of gN (TB-gN-C123S) showed a defect in the initiation of secondary envelopment, as most capsids in TB-gN-C123S-infected cells were either not in contact with cytoplasmic membranes or, when near membranes, lacked signs of budding. The defect in initiation of secondary envelopment was associated with an accumulation of partially tegumented capsids in the peripheral region of the cytoplasmic viral assembly compartment (cVAC), whereas wild-type virus-infected cells predominantly accumulated enveloped capsids in the central area of the cVAC. Additionally, large protein aggregates were observed within and near the cVAC, often associated with non-enveloped capsids. A comparable ultrastructural phenotype, including altered capsid distribution, envelopment defect, and protein aggregation, was observed in wild-type virus-infected cells treated with siRNA against gM. Indirect immunofluorescence analysis confirmed the altered distribution of capsids in the cVAC in wild-type virus-infected cells with reduced or undetectable gM levels, resembling the pattern observed in TB-gN-C123S-infected cells. Further evidence underscoring the role of the gM/gN glycoprotein complex in viral morphogenesis was obtained by investigating gM- and gN-null mutants, which displayed the same altered capsid distribution observed in TB-gN-C123S infections and after siRNA knockdown of gM. Finally, the inhibition of palmitoylation using 2-bromopalmitate (2-BP) in wild-type virus-infected cells resulted in analogous defects, including an accumulation of partially tegumented capsids in the periphery of the cVAC and protein aggregates associated with capsids. In summary, our findings indicate a crucial role for the gM/gN complex in initiating the secondary envelopment of partially tegumented capsids and highlight the involvement of palmitoylation in this process. Defects in the initiation of secondary envelopment are associated with an altered spatial distribution of capsids within the cVAC. AUTHORS SUMMARYHuman cytomegalovirus (HCMV) is a widespread herpesvirus that can cause severe illness in newborns and immunocompromised individuals. Like other herpesviruses, HCMV assembles its infectious particles through a complex process that includes a final step called "secondary envelopment," in which the virus acquires its envelope. In this study, we investigated the role of two viral proteins, glycoprotein M (gM) and glycoprotein N (gN), which form a conserved complex across herpesviruses. Using genetic mutants, RNA interference, and electron microscopy, we found that the gM/gN complex is crucial for initiating secondary envelopment. When the function of gM or gN was disrupted, or when palmitoylation was chemically blocked, the capsids were unable to initiate budding into membranes, resulting in partially tegumented capsids that accumulated in the outer area of the cytoplasmic viral assembly compartment (cVAC). Our findings highlight the important role of the gM/gN complex and palmitoylation in HCMV assembly and suggest that assembly occurs in a spatially organized manner within the cVAC, offering new insights into how herpesviruses produce infectious particles.

microbiology↗

bioRxiv2024

Human cytomegalovirus deploys molecular mimicry to recruit VPS4A to sites of virus assembly

The AAA-type ATPase VPS4 is recruited by proteins of the endosomal sorting complex required for transport III (ESCRT-III) to catalyse membrane constriction and membrane fission. VPS4A accumulates at the cytoplasmic viral assembly complex (cVAC) of cells infected with human cytomegalovirus (HCMV), the site where nascent virus particles obtain their membrane envelope. Here we show that VPS4A is recruited to the cVAC via interaction with pUL71. Sequence analysis, deep-learning structure prediction, molecular dynamics and mutagenic analysis identify a short peptide motif in the C-terminal region of pUL71 that is necessary and sufficient for the interaction with VPS4A. This motif is predicted to bind the same groove of the N-terminal VPS4A Microtubule-Interacting and Trafficking (MIT) domain as the Type 2 MIT-Interacting Motif (MIM2) of cellular ESCRT-III components, and this viral MIM2-like motif (vMIM2) is conserved across {beta}-herpesvirus pUL71 homologues. However, recruitment of VPS4A by pUL71 is dispensable for HCMV morphogenesis or replication and the function of the conserved vMIM2 during infection remains enigmatic. VPS4-recruitment via a vMIM2 represents a previously unknown mechanism of molecular mimicry in viruses, extending previous observations that herpesviruses encode proteins with structural and functional homology to cellular ESCRT-III components.

cell biology↗

bioRxiv2021

Human Cytomegalovirus uses a Host Stress Response to Balance the Elongation of Saturated/Monounsaturated and Polyunsaturated Very Long Chain Fatty Acids

Stress and virus infection are known to regulate lipid metabolism in cells. Human cytomegalovirus infection induces fatty acid (FA) elongation and increases the cellular abundance of lipids with very long-chain FA tails (VLCFAs). While reprogramming of metabolism can be stress-related, the role of stress in HCMV reprogramming of lipid metabolism is poorly understood. In this study, we engineered cells to knockout PKR-like ER kinase (PERK) in the ER stress pathway and measured lipid changes using lipidomics to determine if PERK is needed for lipid changes associated with HCMV infection. We found that in HCMV-infected cells, PERK promotes the increase in the levels of phospholipids with saturated FA (SFA) and monounsaturated FA (MUFA) VLCFAs tails. Consistent with the SFA/MUFA lipidome changes, PERK enhances the protein levels of FA elongase 7 (ELOVL7), which elongates SFA and MUFA VLCFAs. Additionally, we found that increases in the elongation of polyunsaturated fatty acids (PUFAs) associated with HCMV infection was independent of PERK and that lipids with PUFA tails accumulated in HCMV-infected PERK knockout cells. Consistent with the PUFA lipidome changes, the protein levels of ELOVL5, which elongates PUFAs, are increased by HCMV infection through a PERK-independent mechanism. These observations show that PERK differentially regulates ELOVL7 and ELOVL5, creating a balance between the synthesis of lipids with SFA/MUFA tails and PUFA tails. Additionally, we found that PERK was necessary for virus replication and the infectivity of released viral progeny. Overall, our findings indicate that PERK--and more broadly, ER stress--may be necessary for membrane biogenesis needed to generate infectious HCMV virions. IMPORTANCEHCMV is a common herpesvirus that establishes lifelong persistent infections. While infection is asymptomatic in most people, HCMV causes life-threatening illnesses in immunocompromised people, including transplant recipients and cancer patients. Additionally, HCMV infection is a leading cause of congenital disabilities. HCMV replication relies on lipid synthesis. Here, we demonstrated that the ER stress mediator, PERK, controls fatty acid (FA) elongation and cellular abundance of several types of lipids following HCMV infection. Specifically, we found that PERK promotes FA elongase 7 synthesis of lipids with saturated/monounsaturated very long-chain FA tails which are important for building the viral membrane of infectious HCMV virions. Overall, our study shows that PERK is an essential host factor that supports HCMV replication and promotes lipidome changes caused by HCMV infection.

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↗