Characterization of Vesicular Stomatitis Virus Pseudotypes Bearing Essential Entry Glycoproteins gB, gD, gH, and gL of Herpes Simplex Virus 1.

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.

Calpain-1 knockout reveals broad effects on erythrocyte deformability and physiology.

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.

Asymmetric assembly of Merkel cell polyomavirus large T-antigen origin binding domains at the viral origin.

The double-stranded DNA polyomavirus Merkel cell polyomavirus (MCV) causes Merkel cell carcinoma, an aggressive but rare human skin cancer that most often affects immunosuppressed and elderly persons. 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 (OBD) 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 OBD. This stoichiometry was verified using isothermal titration calorimetry. The affinity for G(A/G)GGC-containing double-stranded DNA 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 Lys331 (Arg154 in SV40). In contrast to SV40, a small protein-protein interface is observed between MCV OBDs 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 DNA binding energy.