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Herpes simplex viruses (HSVs) are unusual in that unlike most enveloped viruses, they require at least four entry glycoproteins, gB, gD, gH, and gL, for entry into target cells in addition to a cellular receptor for gD. The dissection of the herpes simplex virus 1 (HSV-1) entry mechanism is complicated by the presence of more than a dozen proteins on the viral envelope. To investigate HSV-1 entry requirements in a simplified system, we generated vesicular stomatitis virus (VSV) virions pseudotyped with HSV-1 essential entry glycoproteins gB, gD, gH, and gL but lacking the native VSV fusogen G. These virions, referred to here as VSVΔG-BHLD virions, infected a cell line expressing a gD receptor, demonstrating for the first time that the four essential entry glycoproteins of HSV-1 are not only required but also sufficient for cell entry. To our knowledge, this is the first time the VSV pseudotyping system has been successfully extended beyond two proteins. Entry of pseudotyped virions required a gD receptor and was inhibited by HSV-1 specific anti-gB or anti-gH/gL neutralizing antibodies, which suggests that membrane fusion during the entry of the pseudotyped virions shares common requirements with the membrane fusion involved in HSV-1 entry and HSV-1-mediated syncytium formation. The HSV pseudotyping system established in this study presents a novel tool for systematic exploration of the HSV entry and membrane fusion mechanisms. IMPORTANCE Herpes simplex viruses (HSVs) are human pathogens that can cause cold sores, genital herpes, and blindness. No vaccines or preventatives are available. HSV entry into cells—a prerequisite for a successful infection—is a complex process that involves multiple viral and host proteins and occurs by different routes. Detailed mechanistic knowledge of the HSV entry is important for understanding its pathogenesis and would benefit antiviral and vaccine development, yet the presence of more than a dozen proteins on the viral envelope complicates the dissection of the HSV entry mechanisms. In this study, we generated heterologous virions displaying the four essential entry proteins of HSV-1 and showed that they are capable of cell entry and, like HSV-1, require all four entry glycoproteins along with a gD receptor. This HSV pseudotyping system pioneered in this work opens doors for future systematic exploration of the herpesvirus entry mechanisms.

Herpesvirus entry into cells is mediated by the viral fusogen gB, which is thought to refold from the prefusion to the postfusion form in a series of large conformational changes that energetically couple refolding to membrane fusion. In contrast to most viral fusogens, gB requires a conserved heterodimer, gH/gL, as well as other nonconserved proteins. In a further mechanistic twist, gB-mediated cell-cell fusion appears restricted by its intraviral or cytoplasmic domain (cytodomain) because mutations within it result in a hyperfusogenic phenotype. Here, we characterized a panel of hyperfusogenic HSV-1 gB cytodomain mutants and show that they are fully functional in cell-cell fusion at shorter coincubation times and at lower temperatures than those for wild-type (WT) gB, which suggests that these mutations reduce the kinetic energy barrier to fusion. Despite this, the mutants require both gH/gL and gD. We confirm previous observations that the gH cytotail is an essential component of the cell-cell fusion mechanism and show that the N-terminal portion of the gH cytotail is critical for this process. Moreover, the fusion levels achieved by all gB constructs, WT and mutant, were proportionate to the length of the gH cytotail. Putting these results together, we propose that the gH cytotail, in addition to the gH/gL ectodomain, plays an essential role in gB activation, potentially acting as a “wedge” to release the gB cytodomain “clamp” and enable gB activation. IMPORTANCE Herpesviruses infect their hosts for life and cause a substantial disease burden. Herpes simplex viruses cause oral and genital sores as well as rare yet severe encephalitis and a panoply of ocular ailments. Infection initiates when the viral envelope fuses with the host cell membrane in a process orchestrated by the viral fusogen gB, assisted by the viral glycoproteins gH, gL, and gD and a cellular gD receptor. This process is more complicated than that of most other viruses and is subject to multiple regulatory inputs. Antiviral and vaccine development would benefit from a detailed mechanistic knowledge of this process and how it is regulated.

All enveloped viruses must catalyze the fusion of their surrounding membrane with that of their target cell. In herpesviruses, fusion is powered by a large structural rearrangement of the trimeric fusogen gB, with help from viral glycoproteins gD and gH/gL. Although the structure of the postfusion gB ectodomain has been solved, the architecture of the membrane-interacting portion of gB - its membrane proximal region (MPR), transmembrane domain (TMD), and cytoplasmic domain (CTD) - is unknown. These domains both facilitate and regulate fusion, but the lack of structural information on them has hindered reconstruction of the gB refolding process. To fill this gap, we have crystallized full-length, postfusion gB in a detergent-amphipol environment. The resulting structure is the first to encompass the membrane-spanning portion of a viral fusogen in its postfusion conformation. In each protomer of the gB trimer, the MPR is a lipid embedded helix that forms a platform beneath the ectodomain. The TMD helices traverse the membrane at an angle, creating a flexible conduit that could transmit the status of the regulatory CTD below. In the CTD, the three protomers form a shallow basket bounded by a long helix that angles upward toward the membrane. While the remainder of the CTD is unstructured in these crystals, electron spin resonance (ESR) spectroscopy data indicate that many of these distal residues form an amphipathic helix embedded in the outer bilayer. Protein/protein and protein/membrane contacts may enable the CTD to restrain fusion by acting as a clamp that braces against the bilayer. These results thus present a possible mechanism for regulation of gB mediated fusion and a strategy for capturing another critical gB form - the elusive prefusion conformation.

Pharmacological inhibitors of cysteine proteases have provided useful insights into the regulation of calpain activity in erythrocytes. However, the precise biological function of calpain activity in erythrocytes remains poorly understood. Erythrocytes express calpain-1, an isoform regulated by calpastatin, the endogenous inhibitor of calpains. In the present study, we investigated the function of calpain-1 in mature erythrocytes using our calpain-1-null [KO (knockout)] mouse model. The calpain-1 gene deletion results in improved erythrocyte deformability without any measurable effect on erythrocyte lifespan in vivo. The calcium-induced sphero-echinocyte shape transition is compromised in the KO erythrocytes. Erythrocyte membrane proteins ankyrin, band 3, protein 4.1R, adducin and dematin are degraded in the calcium-loaded normal erythrocytes but not in the KO erythrocytes. In contrast, the integrity of spectrin and its state of phosphorylation are not affected in the calcium-loaded erythrocytes of either genotype. To assess the functional consequences of attenuated cytoskeletal remodelling in the KO erythrocytes, the activity of major membrane transporters was measured. The activity of the K+–Cl− co-transporter and the Gardos channel was significantly reduced in the KO erythrocytes. Similarly, the basal activity of the calcium pump was reduced in the absence of calmodulin in the KO erythrocyte membrane. Interestingly, the calmodulin-stimulated calcium pump activity was significantly elevated in the KO erythrocytes, implying a wider range of pump regulation by calcium and calmodulin. Taken together, and with the atomic force microscopy of the skeletal network, the results of the present study provide the first evidence for the physiological function of calpain-1 in erythrocytes with therapeutic implications for calcium imbalance pathologies such as sickle cell disease.

The double-stranded DNA polyomavirus Merkel cell polyomavirus (MCV) causes Merkel cell carcinoma, an aggressive but rare human skin cancer that most often affects the immunosuppressed and elderly. As in other polyomaviruses, the large T-antigen of MCV recognizes the viral origin of replication by binding repeating G(A/G)GGC pentamers. The spacing, number, orientation, and necessity of repeats for viral replication differ, however, from other family members such as SV40 and murine polyomavirus. We report here the 2.9 Å crystal structure of the MCV large T-antigen origin binding domain in complex with a DNA fragment from the MCV origin of replication. Consistent with replication data showing that three of the G(A/G)GGC-like binding sites near the center of the origin are required for replication, the crystal structure contains three copies of the origin-binding domain. This stoichiometry was verified using isothermal titration calorimetry. The affinity for G(A/G)GGC-containing dsDNA was found to be ~740 nM, approximately 8-fold weaker than the equivalent domain in SV40 for the analogous region of the SV40 origin. The difference in affinity is partially attributable to DNA-binding residue Lys 331 (Arg 154 in SV40). In contrast to SV40, a small protein-protein interface is observed between MCV origin-binding domains when bound to the central region of the origin. This protein-protein interface is reminiscent of that seen in bovine papilloma virus E1 protein. Mutational analysis indicates, however, that this interface contributes little to the DNA binding energy.