The BICD2 dynein cargo adaptor binds to the HPV16 L2 capsid protein and promotes HPV infection.

During entry, human papillomavirus (HPV) traffics from the endosome to the trans Golgi network (TGN) and Golgi and then the nucleus to cause infection. Although dynein is thought to play a role in HPV infection, how this host motor recruits the virus to support infection and which entry step(s) requires dynein are unclear. Here we show that the dynein cargo adaptor BICD2 binds to the HPV L2 capsid protein during entry, recruiting HPV to dynein for transport of the virus along the endosome-TGN/Golgi axis to promote infection. In the absence of BICD2 function, HPV accumulates in the endosome and TGN and infection is inhibited. Cell-based and in vitro binding studies identified a short segment near the C-terminus of L2 that can directly interact with BICD2. Our results reveal the molecular basis by which the dynein motor captures HPV to promote infection and identify this virus as a novel cargo of the BICD2 dynein adaptor.

Autophagy of the ER: the secretome finds the lysosome.

Lysosomal degradation of the endoplasmic reticulum (ER) and its components through the autophagy pathway has emerged as a major regulator of ER proteostasis. Commonly referred to as ER-phagy and ER-to-lysosome-associated degradation (ERLAD), how the ER is targeted to the lysosome has been recently clarified by a growing number of studies. Here, we summarize the discoveries of the molecular components required for lysosomal degradation of the ER and their proposed mechanisms of action. Additionally, we discuss how cells employ these machineries to create the different routes of ER-lysosome-associated degradation. Further, we review the role of ER-phagy in viral infection pathways, as well as the implication of ER-phagy in human disease. In sum, we provide a comprehensive overview of the current field of ER-phagy.

The atlastin ER morphogenic proteins promote formation of a membrane penetration site during non-enveloped virus entry.

During entry, non-enveloped viruses penetrate a host membrane to cause infection, although how this is accomplished remains enigmatic. Polyomaviruses (PyVs) are non-enveloped DNA viruses that penetrate the endoplasmic reticulum (ER) membrane to reach the cytosol en route to the nucleus for infection. To penetrate the ER membrane, the prototype PyV simian virus 40 (SV40) induces formation of ER-escape sites, called foci, composed of repeating units of multi-tubular ER junctions where the virus is thought to exit. How SV40 triggers formation of the ER-foci harboring these multi-tubular ER junctions is unclear. Here, we show that the ER morphogenic atlastin 2 (ATL2) and ATL3 membrane proteins play critical roles in SV40 infection. Mechanistically, ATL3 mobilizes to the ER-foci where it deploys its GTPase-dependent membrane fusion activity to promote formation of multi-tubular ER junctions within the ER-foci. ATL3 also engages an SV40-containing membrane penetration complex. By contrast, ATL2 does not reorganize to the ER-foci. Instead, it supports the reticular ER morphology critical for the integrity of the ATL3-dependent membrane complex. Our findings illuminate how two host factors play distinct roles in the formation of an essential membrane penetration site for a non-enveloped virus. IMPORTANCE Membrane penetration by non-enveloped viruses, a critical infection step, remains enigmatic. The non-enveloped PyV simian virus 40 (SV40) penetrates the endoplasmic reticulum (ER) membrane to reach the cytosol en route for infection. During ER-to-cytosol membrane penetration, SV40 triggers formation of ER-associated structures (called ER-foci) that function as the membrane penetration sites. Here, we discover a role of the ATL ER membrane proteins-known to shape the ER morphology-during SV40-induced ER-foci formation. These findings illuminate how a non-enveloped virus hijacks host components to construct a membrane penetration structure.

How host ER membrane chaperones and morphogenic proteins support virus infection.

The multi-functional endoplasmic reticulum (ER) is exploited by viruses to cause infection. Morphologically, this organelle is a highly interconnected membranous network consisting of sheets and tubules whose levels are dynamic, changing in response to cellular conditions. Functionally, the ER is responsible for protein synthesis, folding, secretion and degradation, as well as Ca2+ homeostasis and lipid biosynthesis, with each event catalyzed by defined ER factors. Strikingly, these ER host factors are hijacked by viruses to support different infection steps, including entry, translation, replication, assembly and egress. Although the full repertoire of these ER factors that are hijacked is unknown, recent studies have uncovered several ER membrane machineries that are exploited by viruses - ranging from polyomavirus to flavivirus and coronavirus - to facilitate different steps of their life cycle. These discoveries should provide better understanding of virus infection mechanisms, potentially leading to the development of more effective anti-viral therapies.

Nutrient-dependent regulation of ß-cell proinsulin content.

Insulin is made from proinsulin, but the extent to which fasting/feeding controls the homeostatically regulated proinsulin pool in pancreatic ß-cells remains largely unknown. Here, we first examined ß-cell lines (INS1E and Min6, which proliferate slowly and are routinely fed fresh medium every 2-3 days) and found that the proinsulin pool size responds to each feeding within 1 to 2 h, affected both by the quantity of fresh nutrients and the frequency with which they are provided. We observed no effect of nutrient feeding on the overall rate of proinsulin turnover as quantified from cycloheximide-chase experiments. We show that nutrient feeding is primarily linked to rapid dephosphorylation of translation initiation factor eIF2α, presaging increased proinsulin levels (and thereafter, insulin levels), followed by its rephosphorylation during the ensuing hours that correspond to a fall in proinsulin levels. The decline of proinsulin levels is blunted by the integrated stress response inhibitor, ISRIB, or by inhibition of eIF2α rephosphorylation with a general control nonderepressible 2 (not PERK) kinase inhibitor. In addition, we demonstrate that amino acids contribute importantly to the proinsulin pool; mass spectrometry shows that ß-cells avidly consume extracellular glutamine, serine, and cysteine. Finally, we show that in both rodent and human pancreatic islets, fresh nutrient availability dynamically increases preproinsulin, which can be quantified without pulse-labeling. Thus, the proinsulin available for insulin biosynthesis is rhythmically controlled by fasting/feeding cycles.

Reticulons promote formation of ER-derived double-membrane vesicles that facilitate SARS-CoV-2 replication.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), the etiologic agent for the global COVID-19 pandemic, triggers the formation of endoplasmic reticulum (ER)-derived replication organelles, including double-membrane vesicles (DMVs), in the host cell to support viral replication. Here, we clarify how SARS-CoV-2 hijacks host factors to construct the DMVs. We show that the ER morphogenic proteins reticulon-3 (RTN3) and RTN4 help drive DMV formation, enabling viral replication, which leads to productive infection. Different SARS-CoV-2 variants, including the delta variant, use the RTN-dependent pathway to promote infection. Mechanistically, our results reveal that the membrane-embedded reticulon homology domain (RHD) of the RTNs is sufficient to functionally support viral replication and physically engage NSP3 and NSP4, two viral non-structural membrane proteins known to induce DMV formation. Our findings thus identify the ER morphogenic RTN3 and RTN4 membrane proteins as host factors that help promote the biogenesis of SARS-CoV-2-induced DMVs, which can act as viral replication platforms.

HPV is a cargo for the COPI sorting complex during virus entry.

During entry, human papillomavirus (HPV) traffics from the cell surface to the endosome and then to the trans-Golgi network (TGN) and Golgi apparatus. HPV must transit across the TGN/Golgi and exit these compartments to reach the nucleus to cause infection, although how these steps are accomplished is unclear. Combining cellular fractionation, unbiased proteomics, and gene knockdown strategies, we identified the coat protein complex I (COPI), a highly conserved protein complex that facilitates retrograde trafficking of cellular cargos, as a host factor required for HPV infection. Upon TGN/Golgi arrival, the cytoplasmic segment of HPV L2 binds directly to COPI. COPI depletion causes the accumulation of HPV in the TGN/Golgi, resembling the fate of a COPI binding-defective L2 mutant. We propose that the L2-COPI interaction drives HPV trafficking through the TGN and Golgi stacks during virus entry. This shows that an incoming virus is a cargo of the COPI complex.

Integration of ER protein quality control mechanisms defines ß cell function and ER architecture.

Three principal ER quality-control mechanisms, namely, the unfolded protein response, ER-associated degradation (ERAD), and ER-phagy are each important for the maintenance of ER homeostasis, yet how they are integrated to regulate ER homeostasis and organellar architecture in vivo is largely unclear. Here we report intricate crosstalk among the 3 pathways, centered around the SEL1L-HRD1 protein complex of ERAD, in the regulation of organellar organization in ß cells. SEL1L-HRD1 ERAD deficiency in ß cells triggers activation of autophagy, at least in part, via IRE1α (an endogenous ERAD substrate). In the absence of functional SEL1L-HRD1 ERAD, proinsulin is retained in the ER as high molecular weight conformers, which are subsequently cleared via ER-phagy. A combined loss of both SEL1L and autophagy in ß cells leads to diabetes in mice shortly after weaning, with premature death by approximately 11 weeks of age, associated with marked ER retention of proinsulin and ß cell loss. Using focused ion beam scanning electron microscopy powered by deep-learning automated image segmentation and 3D reconstruction, our data demonstrate a profound organellar restructuring with a massive expansion of ER volume and network in ß cells lacking both SEL1L and autophagy. These data reveal at an unprecedented detail the intimate crosstalk among the 3 ER quality-control mechanisms in the dynamic regulation of organellar architecture and ß cell function.

Non-enveloped virus membrane penetration: New advances leading to new insights.

Host cell membranes pose a particular challenge for non-enveloped viruses. Whereas enveloped viruses enter cells by fusing their lipid envelopes with the cellular membrane, non-enveloped viruses generally must (1) enter cells via endocytosis, then (2) penetrate the cellular endomembrane to reach the cytosol. Only then can the viruses begin to replicate (or transit to the nucleus to replicate). Although membrane penetration of non-enveloped viruses is a crucial entry step, many of the precise molecular details of this process remain unclear. Recent findings have begun to untangle the various mechanisms by which non-enveloped viral proteins disrupt and penetrate cellular endomembranes. Specifically, high-resolution microscopy studies have revealed precise conformational changes in viral proteins that enable penetration, while biochemical studies have identified key host proteins that promote viral penetration and transport. This brief article summarizes new discoveries in the membrane penetration process for three of the most intensely studied families of non-enveloped viruses: reoviruses, papillomaviruses, and polyomaviruses.

Components of the LINC and NPC complexes coordinately target and translocate a virus into the nucleus to promote infection.

Nuclear entry represents the final and decisive infection step for most DNA viruses, although how this is accomplished by some viruses is unclear. Polyomavirus SV40 transports from the cell surface through the endosome, the endoplasmic reticulum, and the cytosol from where it enters the nucleus to cause infection. Here we elucidate the nuclear entry mechanism of SV40. Our results show that cytosol-localized SV40 is targeted to the nuclear envelope by directly engaging Nesprin-2 of the linker of nucleoskeleton and cytoskeleton (LINC) nuclear membrane complex. Additionally, we identify the NUP188 subunit of the nuclear pore complex (NPC) as a new Nesprin-2-interacting partner. This physical proximity positions the NPC to capture SV40 upon release from Nesprin-2, enabling the channel to facilitate nuclear translocation of the virus. Strikingly, SV40 disassembles during nuclear entry, generating a viral genome-VP1-VP3 subcomplex that efficiently crosses the NPC to enter the nucleus. Our results reveal how two major nuclear membrane protein complexes are exploited to promote targeting and translocation of a virus into the nucleus.

Proteasomal degradation of WT proinsulin in pancreatic beta cells.

Preproinsulin entry into the endoplasmic reticulum yields proinsulin, and its subsequent delivery to the distal secretory pathway leads to processing, storage, and secretion of mature insulin. Multiple groups have reported that treatment of pancreatic beta cell lines, rodent pancreatic islets, or human islets with proteasome inhibitors leads to diminished proinsulin and insulin protein levels, diminished glucose-stimulated insulin secretion, and changes in beta-cell gene expression that ultimately lead to beta-cell death. However, these studies have mostly examined treatment times far beyond that needed to achieve acute proteasomal inhibition. Here, we report that although proteasomal inhibition immediately downregulates new proinsulin biosynthesis, it nevertheless acutely increases beta-cell proinsulin levels in pancreatic beta cell lines, rodent pancreatic islets, and human islets, indicating rescue of a pool of recently synthesized WT INS gene product that would otherwise be routed to proteasomal disposal. Our pharmacological evidence suggests that this disposal most likely reflects ongoing endoplasmic reticulum-associated protein degradation. However, we found that within 60 min after proteasomal inhibition, intracellular proinsulin levels begin to fall in conjunction with increased phosphorylation of eukaryotic initiation factor 2 alpha, which can be inhibited by blocking the general control nonderepressible 2 kinase. Together, these data demonstrate that a meaningful subfraction of newly synthesized INS gene product undergoes rapid proteasomal disposal. We propose that free amino acids derived from proteasomal proteolysis may potentially participate in suppressing general control nonderepressible 2 kinase activity to maintain ongoing proinsulin biosynthesis.

A specific EMC subunit supports Dengue virus infection by promoting virus membrane fusion essential for cytosolic genome delivery.

Dengue virus (DENV) represents the most common human arboviral infection, yet its cellular entry mechanism remains unclear. The multi-subunit endoplasmic reticulum membrane complex (EMC) supports DENV infection, in part, by assisting the biosynthesis of viral proteins critical for downstream replication steps. Intriguingly, the EMC has also been shown to act at an earlier step prior to viral protein biogenesis, although this event is not well-defined. Here we demonstrate that the EMC subunit EMC4 promotes fusion of the DENV and endosomal membranes during entry, enabling delivery of the viral genome into the cytosol which is then targeted to the ER for viral protein biosynthesis. We also found that EMC4 mediates ER-to-endosome transfer of phosphatidylserine, a phospholipid whose presence in the endosome facilitates DENV-endosomal membrane fusion. These findings clarify the EMC-dependent DENV early entry step, suggesting a mechanism by which an ER-localized host factor can regulate viral fusion at the endosome.

The ER transmembrane protein PGRMC1 recruits misfolded proteins for reticulophagic clearance.

ER-specific autophagy (reticulophagy) has emerged as a critical degradative route for misfolded secretory proteins. Our previous work showed that RTN3 (reticulon 3) drives reticulophagic clearance of disease-causing mutant prohormones. How RTN3, a protein residing on the cytosolic leaflet of the ER bilayer, recruits these lumenally-localized cargos has remained a mystery. To address this question, we used an unbiased proteomics approach to identify RTN3-interacting partners. We discovered that RTN3 recruits misfolded prohormones for lysosomal degradation through the ER transmembrane protein PGRMC1. RTN3 complexes with PGRMC1, which directly binds to misfolded prohormones via its distal ER lumenal domain. Cargos for the RTN3-PGRMC1 degradative axis include mutant POMC (proopiomelanocortin) and proinsulin, each of which oligomerizes in the ER during misfolding, entrapping their wild-type counterparts, leading to secretion defects. Although reticulophagy is thought to degrade large protein aggregates, PGRMC1 instead selectively recruits and promotes degradation of only small oligomers of the mutant prohormones. Of physiological importance, genetic or pharmacological inactivation of PGRMC1 in pancreatic ß-cells expressing both wild-type and mutant proinsulin impairs mutant proinsulin turnover and promotes trafficking of wild-type proinsulin. These findings pinpoint PGRMC1 as a possible intervention point for diseases caused by ER protein retention.

Lunapark-dependent formation of a virus-induced ER exit site contains multi-tubular ER junctions that promote viral ER-to-cytosol escape.

Viruses rearrange host membranes to support different entry steps. Polyomavirus simian virus 40 (SV40) reorganizes the endoplasmic reticulum (ER) membrane to generate focus structures that enable virus ER-to-cytosol escape, a decisive infection step. The molecular architecture of the ER exit site that might illuminate why it is ideally suited for membrane penetration is unknown. Here 3D focused ion beam scanning electron microscopy (FIB-SEM) reconstruction reveals that the ER focus structure consists of multi-tubular ER junctions where SV40 preferentially localizes, suggesting that tubular branch points are virus ER-to-cytosol penetration sites. Functional analysis demonstrates that lunapark-an ER membrane protein that typically stabilizes three-way ER junctions-relocates to the ER foci, where it supports focus formation, leading to SV40 ER escape and infection. Our results reveal how a virus repurposes the activity of an ER membrane protein to form a virus-induced ER substructure required for membrane escape and suggest that ER tubular junctions are vulnerable sites exploited by viruses for membrane penetration.

PGRMC1 acts as a size-selective cargo receptor to drive ER-phagic clearance of mutant prohormones.

The reticulon-3 (RTN3)-driven targeting complex promotes clearance of misfolded prohormones from the endoplasmic reticulum (ER) for lysosomal destruction by ER-phagy. Because RTN3 resides in the cytosolic leaflet of the ER bilayer, the mechanism of selecting misfolded prohormones as ER-phagy cargo on the luminal side of the ER membrane remains unknown. Here we identify the ER transmembrane protein PGRMC1 as an RTN3-binding partner. Via its luminal domain, PGRMC1 captures misfolded prohormones, targeting them for RTN3-dependent ER-phagy. PGRMC1 selects cargos that are smaller than the large size of other reported ER-phagy substrates. Cargos for PGRMC1 include mutant proinsulins that block secretion of wildtype proinsulin through dominant-negative interactions within the ER, causing insulin-deficiency. Chemical perturbation of PGRMC1 partially restores WT insulin storage by preventing ER-phagic degradation of WT and mutant proinsulin. Thus, PGRMC1 acts as a size-selective cargo receptor during RTN3-dependent ER-phagy, and is a potential therapeutic target for diabetes.

Distinct states of proinsulin misfolding in MIDY.

A precondition for efficient proinsulin export from the endoplasmic reticulum (ER) is that proinsulin meets ER quality control folding requirements, including formation of the Cys(B19)-Cys(A20) "interchain" disulfide bond, facilitating formation of the Cys(B7)-Cys(A7) bridge. The third proinsulin disulfide, Cys(A6)-Cys(A11), is not required for anterograde trafficking, i.e., a "lose-A6/A11" mutant [Cys(A6), Cys(A11) both converted to Ser] is well secreted. Nevertheless, an unpaired Cys(A11) can participate in disulfide mispairings, causing ER retention of proinsulin. Among the many missense mutations causing the syndrome of Mutant INS gene-induced Diabetes of Youth (MIDY), all seem to exhibit perturbed proinsulin disulfide bond formation. Here, we have examined a series of seven MIDY mutants [including G(B8)V, Y(B26)C, L(A16)P, H(B5)D, V(B18)A, R(Cpep + 2)C, E(A4)K], six of which are essentially completely blocked in export from the ER in pancreatic ß-cells. Three of these mutants, however, must disrupt the Cys(A6)-Cys(A11) pairing to expose a critical unpaired cysteine thiol perturbation of proinsulin folding and ER export, because when introduced into the proinsulin lose-A6/A11 background, these mutants exhibit native-like disulfide bonding and improved trafficking. This maneuver also ameliorates dominant-negative blockade of export of co-expressed wild-type proinsulin. A growing molecular understanding of proinsulin misfolding may permit allele-specific pharmacological targeting for some MIDY mutants.

How DNA and RNA Viruses Exploit Host Chaperones to Promote Infection.

To initiate infection, a virus enters a host cell typically via receptor-dependent endocytosis. It then penetrates a subcellular membrane, reaching a destination that supports transcription, translation, and replication of the viral genome. These steps lead to assembly and morphogenesis of the new viral progeny. The mature virus finally exits the host cell to begin the next infection cycle. Strikingly, viruses hijack host molecular chaperones to accomplish these distinct entry steps. Here we highlight how DNA viruses, including polyomavirus and the human papillomavirus, exploit soluble and membrane-associated chaperones to enter a cell, penetrating and escaping an intracellular membrane en route for infection. We also describe the mechanism by which RNA viruses-including flavivirus and coronavirus-co-opt cytosolic and organelle-selective chaperones to promote viral endocytosis, protein biosynthesis, replication, and assembly. These examples underscore the importance of host chaperones during virus infection, potentially revealing novel antiviral strategies to combat virus-induced diseases.

Normal and defective pathways in biogenesis and maintenance of the insulin storage pool.

Both basal and glucose-stimulated insulin release occur primarily by insulin secretory granule exocytosis from pancreatic ß cells, and both are needed to maintain normoglycemia. Loss of insulin-secreting ß cells, accompanied by abnormal glucose tolerance, may involve simple exhaustion of insulin reserves (which, by immunostaining, appears as a loss of ß cell identity), or ß cell dedifferentiation, or ß cell death. While various sensing and signaling defects can result in diminished insulin secretion, somewhat less attention has been paid to diabetes risk caused by insufficiency in the biosynthetic generation and maintenance of the total insulin granule storage pool. This Review offers an overview of insulin biosynthesis, beginning with the preproinsulin mRNA (translation and translocation into the ER), proinsulin folding and export from the ER, and delivery via the Golgi complex to secretory granules for conversion to insulin and ultimate hormone storage. All of these steps are needed for generation and maintenance of the total insulin granule pool, and defects in any of these steps may, weakly or strongly, perturb glycemic control. The foregoing considerations have obvious potential relevance to the pathogenesis of type 2 diabetes and some forms of monogenic diabetes; conceivably, several of these concepts might also have implications for ß cell failure in type 1 diabetes.

ER functions are exploited by viruses to support distinct stages of their life cycle.

The endoplasmic reticulum (ER), with its expansive membranous system and a vast network of chaperones, enzymes, sensors, and ion channels, orchestrates diverse cellular functions, ranging from protein synthesis, folding, secretion, and degradation to lipid biogenesis and calcium homeostasis. Strikingly, some of the functions of the ER are exploited by viruses to promote their life cycles. During entry, viruses must penetrate a host membrane and reach an intracellular destination to express and replicate their genomes. These events lead to the assembly of new viral progenies that exit the host cell, thereby initiating further rounds of infection. In this review, we highlight how three distinct viruses - polyomavirus, flavivirus, and coronavirus - co-opt key functions of the ER to cause infection. We anticipate that illuminating this virus-ER interplay will provide rational therapeutic approaches to combat the virus-induced diseases.