Morphogenesis of Hepatitis E Virus.

Hepatitis E virus, a leading cause of acute hepatitis worldwide, has been recognized as non-enveloped virus since its discovery in the 1980s. However, the recent identification of lipid membrane-associated form termed as "quasi-enveloped" HEV has changed this long-held notion. Both naked HEV and quasi-enveloped HEV play important roles in the pathogenesis of hepatitis E. However, the biogenesis and the mechanisms underlying the composition, biogenesis regulation, and functions of the novel quasi-enveloped virions remain enigmatic. In this chapter, we highlight the most recent discoveries on the dual life cycle of these two different types of virions, and further discuss the implication of the quasi-envelopment in our understanding of the molecular biology of HEV.

Two pan-SARS-CoV-2 nanobodies and their multivalent derivatives effectively prevent Omicron infections in mice.

With the widespread vaccinations against coronavirus disease 2019 (COVID-19), we are witnessing gradually waning neutralizing antibodies and increasing cases of breakthrough infections, necessitating the development of drugs aside from vaccines, particularly ones that can be administered outside of hospitals. Here, we present two cross-reactive nanobodies (R14 and S43) and their multivalent derivatives, including decameric ones (fused to the immunoglobulin M [IgM] Fc) that maintain potent neutralizing activity against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) after aerosolization and display not only pan-SARS-CoV-2 but also varied pan-sarbecovirus activities. Through respiratory administration to mice, monovalent and decameric R14 significantly reduce the lung viral RNAs at low dose and display potent pre- and post-exposure protection. Furthermore, structural studies reveal the neutralizing mechanisms of R14 and S43 and the multiple inhibition effects that the multivalent derivatives exert. Our work demonstrates promising convenient drug candidates via respiratory administration against SARS-CoV-2 infection, which can contribute to containing the COVID-19 pandemic.

Host Cell Receptors Implicated in the Cellular Tropism of BVDV.

Bovine viral diarrhea virus (BVDV) is one of the most hazardous viruses, which causes huge economic losses in the cattle industry around the world. In recent years, there has been a continuous increase in the diversity of pestivirus worldwide. As a member of the genus Pestivirus in the Flaviviridae family, BVDV has a wide range of host animals including cattle, goat, sheep, pig, camel and other cloven-hoofed animals, and it has multi-tissue tropism as well. The recognition of their permissive cells by viruses via interaction with the cellular receptors is a prerequisite for successful infection. So far, little is known about the cellular receptors essential for BVDV entry and their detailed functions during BVDV infection. Thus, discovery of the cellular receptors involved in the entry of BVDV and other pestiviruses is significant for development of the novel intervention. The viral envelope glycoprotein Erns and E2 are crucial determinants of the cellular tropism of BVDV. The cellular proteins bound with Erns and E2 potentially participate in BVDV entry, and their abundance might determine the cellular tropism of BVDV. Here, we summarize current knowledge regarding the cellular molecules have been described for BVDV entry, such as, complement regulatory protein 46 (CD46), heparan sulfate (HS), the low-density lipoprotein (LDL) receptor, and a disintegrin and metalloproteinase 17 (ADAM17). Furthermore, we focus on their implications of the recently identified cellular receptors for pestiviruses in BVDV life cycle. This knowledge provides a theoretical basis for BVDV prevention and treatment by targeting the cellular receptors essential for BVDV infection.

Generation and Characterization of a Nanobody Against SARS-CoV.

The sudden emergence of severe acute respiratory syndrome coronavirus (SARS-CoV) has caused global panic in 2003, and the risk of SARS-CoV outbreak still exists. However, no specific antiviral drug or vaccine is available; thus, the development of therapeutic antibodies against SARS-CoV is needed. In this study, a nanobody phage-displayed library was constructed from peripheral blood mononuclear cells of alpacas immunized with the recombinant receptor-binding domain (RBD) of SARS-CoV. Four positive clones were selected after four rounds of bio-panning and subjected to recombinant expression in E. coli. Further biological identification demonstrated that one of the nanobodies, S14, showed high affinity to SARS-CoV RBD and potent neutralization activity at the picomole level against SARS-CoV pseudovirus. A competitive inhibition assay showed that S14 blocked the binding of SARS-CoV RBD to either soluble or cell-expressed angiotensin-converting enzyme 2 (ACE2). In summary, we developed a novel nanobody targeting SARS-CoV RBD, which might be useful for the development of therapeutics against SARS.

MicroRNA-379 mediates pigmentation, migration and proliferation of melanocytes by targeting the insulin-like growth factor 1 receptor.

Melanogenesis, migration and proliferation of melanocytes are important factors that determine the hair colours of mammals. MicroRNAs (miRNAs) have been shown to be closely related to these processes. In melanocytes of alpacas, insulin-like growth factor 1 (IGF1) has been shown to improve melanogenesis through the cyclic AMP (cAMP) pathway. miR-379 was predicted to target insulin-like growth factor (IGF) receptor 1 (IGF1R), which binds to IGF1. Therefore, we hypothesized that miR-379 could mediate melanogenesis, migration and proliferation of melanocytes. Here, we report that miR-379 was highly expressed in alpaca melanocytes. Subsequent overexpression of miR-379 in alpaca melanocytes led to the generation of the phenotype of melanogenesis, proliferation and migration. In addition, the expression of genes related to these phenotypes in melanocytes was detected. Our results showed that miR-379 targets IGF1R in melanocytes. The overexpression of miR-379 stimulated dendrite extension or elongation and limited the perinuclear distribution of melanin, but inhibited melanogenesis via cAMP response element (CRE)-binding protein (CREB)/microphthalmia-associated transcription factor (MITF) pathway. miR-379 attenuated melanocyte migration by downregulating the focal adhesion kinase (FAK) and enhanced melanocyte proliferation by upregulating protein kinase B (AKT). These observations suggest the involvement of miR-379 in the physiological regulation of melanocytes, mediated by targeting IGF1R on insulin receptor (IR) compensation and subsequent crosstalk.

A high affinity nanobody against endothelin receptor type B: a new approach to the treatment of melanoma.

The aim of the study was to produce a single-domain antibody (nanobody) specific for endothelin receptor type B (EDNRB) which has high expression in melanoma. Cultured human melanoma cells were used as antigens to immunize alpacas. After antibody generation was verified in alpaca serum, total RNA was extracted from alpaca lymphocytes and the target VHH fragment was amplified by two-step PCR, cloned in the pCANTAB5E phagemid vector, and used to transform Escherichia coli TG1 cells to obtain a phage-display nanobody library, which was enriched by panning. The results indicated successful construction of a phage-display anti-human melanoma A375 nanobodies library with a size of 1.2 × 108/ml and insertion rate of 80%. After screening, eight positive clones of anti-EDNRB nanobodies were used to infect E. coli HB2151 for production of soluble nanobodies, which were identified by ELISA. Finally, we obtained a high-affinity anti-EDNRB nanobody, which consisted of 119 amino acids (molecular weight: 12.97 kDa) with 22 amino acids in CDR3 and had good affinity in vitro. The results suggest that the nanobody may be potentially used for the treatment of human melanoma.

Synaptotagmin-4 promotes dendrite extension and melanogenesis in alpaca melanocytes by regulating Ca2+ influx via TRPM1 channels.

Synaptotagmin-4 (SYT4) is a membrane protein that regulates membrane traffic in neurons in a calcium-dependent or calcium-independent manner. In melanocytes, the intracellular free calcium ion (Ca2+ ) may be important for dendrite growth and melanogenesis. Mammalian melanocytes originating from neural crest cells produce melanins. Therefore, we predicted that SYT4 might play a role in melanogenesis and the dendrite morphology of melanocytes. To investigate whether SYT4 is involved in melanocyte physiology, SYT4 was overexpressed in alpaca melanocytes and B16-F10 cells. The results showed that SYT4 overexpression resulted in a phenotype consistent with melanogenesis and dendrite extension. At the molecular level, SYT4 interacted with extracellular regulated MAP kinase (ERK) to decrease p-ERK activity, which negatively regulated CREB expression. Furthermore, cyclic AMP-responsive element-binding protein (CREB) was upregulated and caused the downregulation of the expression of melanogenic regulatory proteins, including microphthalmia-associated transcription factor (MITF), tyrosinase (TYR), tyrosinase-related protein-1 (TYRP1), dopachrome tautomerase (DCT), and transient receptor potential melastatin 1 (TRPM1). Intracellular free Ca2+ promoted the upregulation of calcium/calmodulin dependent protein kinase IV (CAMK4) expression, which phosphorylated CREB (p-CREB). In turn, p-CREB participated in the transcription of MITF. These results demonstrated that SYT4 promoted melanogenesis through dendrite extension and tyrosinase activity, during which the regulation of Ca2+ influx via the TRPM1 channel was a key factor. SIGNIFICANCE OF THE STUDY: Intracellular Ca2+ is important for the function and survival of melanocytes and melanoma cells. SYT4 stimulated melanogenesis through calcium. These results provide evidence that SYT4 regulates Ca2+ influx through TRPM1 to cause melanogenesis and axonal elongation in alpaca melanocytes and further suggesting that the growth and metastasis of melanoma is controlled by the inhibited expression of SYT4 in melanoma cells.

Knockdown of microRNA­143­5p by STTM technology affects eumelanin and pheomelanin production in melanocytes.

MicroRNAs (miRNAs) serve various roles in the regulation of melanogenesis in mammalian melanocytes that contribute to the development of hair color. The manipulation of the melanocyte action is a new target for genetic improvement. Short tandem target mimic (STTM) is a potent approach for silencing miRNAs in plants and animals. To investigate the function of miR­143­5p in melanogenesis, STTM was used to block the expression of miR­143­5p (STTM­miR­143­5p). The molecular analysis and luciferase reporter assay identified myosin Va gene (MYO5A) as one of the miR­143­5p targets. STTM­miR­143­5p overexpression resulted in an increased expression of downstream melanogenesis genes including microphthalmia­associated transcription factor (MITF), tyrosinase family members [tyrosinase (TYR) and tyrosinase­related protein 1 (TYRP1)], melanophilin (MLPH), and Rab27a, thereby contributing to melanocyte pigmentation by promoting total alkali­soluble melanogenesis (ASM) and eumelanin (EM) contents; conversely, STTM­miR­143­5p overexpression resulted in decreased expression of the tyrosinase­related protein 2 (TYRP2)/dopachrome tautomerase (DCT), which is responsible for decreased pheomelanin (PM) content in mouse melanocytes. The results indicated that melanin production in melanocytes could be increased by manipulating miR­143­5p expression using STTM which resulted in ASM and EM production.

Cyclin-dependent kinase 5 regulates proliferation, migration, tyrosinase activity, and melanin production in B16-F10 melanoma cells via the essential regulator p-CREB.

Melanoma is an aggressive cancer with increasing incidence and a growing lifetime risk that arises from normal melanocytes or their precursors. A thorough understanding of the molecular mechanism of melanomagenesis and melanoma biology is essential for the diagnosis, prognostication, and therapy of melanoma. Cyclin-dependent protein kinase 5 (Cdk5) is one of the proteins highly expressed in B16-F10 melanoma cells that controls melanoma cell motility, invasiveness, and metastatic spread and might be a promising novel therapeutic target. The effect of Cdk5 on proliferation and migration, which are important for carcinogenesis, has not been reported. In the current study, we found that siRNA-mediated knockdown of Cdk5 in B16-F10 melanoma cells inhibited melanoma cell proliferation through downregulation of the CaMK4-p-CREB pathway, inhibited migration through downregulation of p-CREB, integrin beta 1, and integrin beta 5, and also inhibited tyrosinase activity and melanin production through p-CREB-MITF regulation. The results indicate that Cdk5 controls melanoma development, with an essential regulatory role for p-CREB.