Utilization of Recombinant Baculovirus Expression System to Produce the RBD Domain of SARS-CoV-2 Spike Protein

Continuous outbreaks of viral diseases in humans facilitates a need for the rapid development of viral test kits and vaccines. These require expression systems to produce a pure and high yield of target viral proteins. We utilized a baculovirus–silkworm expression system to produce the receptor binding domain (RBD) of the SARS-CoV-2 spike protein. First, we had to develop a strategy for constructing a recombinant baculovirus for RBD expression. For this, the coding region of the Bombyx mori cypovirus (BmCPV) polyhedron was assembled with the Bombyx mori nuclear polyhedrosis virus (BmNPV) promoter. We demonstrated that the recombinant baculovirus has the ability to form polyhedrons within host silkworm cells. In addition, the encapsulated BVs are able to infect silkworms by ingestion and induce foreign protein expression. In this way, we utilized this novel system to obtain a high yield of the target foreign protein, the RBD of the SARS-CoV-2 S protein. However, the viral infection rate of our recombinant BV needs to be improved. Our study shed light on developing a highly efficient expression system for the production of antigens and subsequent immunoassays and vaccines

A new cell culture model to genetically dissect the complete human papillomavirus life cycle

<div><p>Herein, we describe a novel infection model that achieves highly efficient infection of primary keratinocytes with human papillomavirus type 16 (HPV16). This cell culture model does not depend on immortalization and is amenable to extensive genetic analyses. In monolayer cell culture, the early but not late promoter was active and yielded a spliced viral transcript pattern similar to HPV16-immortalized keratinocytes. However, relative levels of the E8^E2 transcript increased over time post infection suggesting the expression of this viral repressor is regulated independently of other early proteins and that it may be important for the shift from the establishment to the maintenance phase of the viral life cycle. Both the early and the late promoter were strongly activated when infected cells were subjected to differentiation by growth in methylcellulose. When grown as organotypic raft cultures, HPV16-infected cells expressed late E1^E4 and L1 proteins and replication foci were detected, suggesting that they supported the completion of the viral life cycle. As a proof of principle that the infection system may be used for genetic dissection of viral factors, we analyzed E1, E6 and E7 translation termination linker mutant virus for establishment of infection and genome maintenance. E1 but not E6 and E7 was essential to establish infection. Furthermore, E6 but not E7 was required for episomal genome maintenance. Primary keratinocytes infected with wild type HPV16 immortalized, whereas keratinocytes infected with E6 and E7 knockout virus began to senesce 25 to 35 days post infection. The novel infection model provides a powerful genetic tool to study the role of viral proteins throughout the viral life cycle but especially for immediate early events and enables us to compare low- and high-risk HPV types in the context of infection.</p></div

Efficient infection of primary keratinocytes.

<p><b>(A)</b> HFK cells were infected with EdU-labeled HPV16 pseudovirus using ECM-to-cell transfer. At 40 hpi, cells were fixed and processed for the detection of EdU-labeled DNA (red), PML (green), lamin A/C (blue). A representative image of HFK cells infected with EdU-labeled pseudovirus is shown. <b>(B)</b> Quantification of HFK cells from two different donors containing nuclear EdU-labeled pseudogenome at 40hpi. EdU-labeled viral pseudogenome number was counted manually in z-stacks spanning the whole nucleus for each cell as expressed as percent of analyzed cells. 72 & 60 and 64 & 77 cells were analyzed for HFK1 and HFK2, respectively. <b>(C)</b> HFKs from three different donors were infected with HPV16 via ECM-to-cell transfer or by direct binding to cells. At 72 hpi, RNA was isolated and E1^E4 transcript was measured by RT-qPCR. The data are presented as fold changes relative to cells infected by direct virion binding. <b>(D and E)</b> E7 and E1^E4 transcript levels increase with prolonged exposure to virus-loaded ECM in primary HFK (D) and HTE (E). <b>(F)</b> RT-qPCR analysis of viral transcripts at 6 dpi of HFK with wt and E1-TTL mutant HPV16 virus. Unless otherwise noted, all results are based on three biological replicates. HFK were maintained and infected in the presence of 10 μM Y-27632 for experiments shown in panels A, B, D, and F.</p

E6 but not E7 is required for episomal genome maintenance in monolayer cells.

<p><b>(A)</b> E6 and E1^E4 transcript levels at 6 dpi of HFK with wt, E6-, and E7-TTL mutant virus. <b>(B)</b> E6 protein detection using the ArborVita Onco<i>E6</i>^<i>™</i> Cervical Test. The test is described in detail in Material and Methods. Note the absence of the E6-specific band in samples derived from control and E6-TTL mutant virus infected HFK. <b>(C)</b> E7 protein detection by western blot. Note the absence of E7 protein in samples derived from HFK infected with E7-TTL mutant virus. Samples from two different HPV16-immortalized HFK lines were analyzed. Ctrl: extracts from mock-infected HFK. <b>(D)</b> E6 transcript levels at 27 to 33 dpi with the indicated virus. The samples were taken at the last passage before control, E6-, and E7-TTL virus infected HFK underwent senescence. We also obtained samples from HFK infected with wt HPV16, which were the only cells to become immortalized, one passage later. *: indicates that these cells had senesced. <b>(E)</b> Quantification of viral genome by qPCR isolated from HFK at 27–33 dpi with respective virus and expressed as percent of ß-actin DNA levels. <b>(F)</b> Viral genome remains episomal in HFK infected with wt and E7-TTL but not with E6-TTL virus. DNA isolated from HFK infected with respective virus was subjected to exonuclease 5 treatment. Resistant DNA was quantified by qPCR. Primers for amplification of HPV16, mitochondrial (mt) DNA and 18S DNA were used.</p

Genome amplification and capsid protein expression in HPV16-infected HFK subjected to organotypic raft culture.

<p><b>(A)</b> Transcription profile of viral transcripts from rafts derived from HPV16-immortalized and -infected HFK. The data shown are fold changes normalized to E6 transcript levels. Error bars represent SEM of three independent experiments. <b>(B)</b> HPV16 genome is enriched in organotypic rafts over pEGFPN1 plasmid. <b>(C)</b> Thin cuts of organotypic rafts generated from HPV16-immortalized and -infected HFK cells subjected to HPV16-specific FISH. <b>(D and E)</b> E1^E4 (D) and L1 (E) protein are expressed in rafts grown from HPV16-infected HFK. Nuclei were stained with Dapi.</p

Differentiation activates both viral promoters and results in genome amplification.

<p>HFKs maintained and infected in the presence of 10 μM Y-27632 for 5 days with HPV16 were subjected for 24–48 h to differentiation by plating in MC. <b>(A)</b> RT-qPCR analysis of loricrin transcript level in cells grown in monolayer and MC. The data shown are fold changes relative to uninfected HFK cells grown in monolayer. Error bars represent SEM of three independent experiments. <b>(B)</b> Western blot for keratin 10 in cells grown in monolayer and MC. <b>(C)</b> Expression of E7 and E1^E4 in HPV16 infected HFKs. The data shown are fold changes relative to cells grown in monolayer. Error bars represent SEM of three independent experiments. <b>(D)</b> Relative expression levels of individual ORFs in differentiated HPV16 infected cells. The data shown are fold changes normalized to E6 transcript levels in infected cells grown in monolayer. Error bars represent SEM of four biological replicates. <b>(E)</b> Read depths maps of viral transcripts isolated from HPV16-infected HFK grown in monolayer and MC. <b>(F)</b> Expression of E6/E7 and E1^E4 in HPV16 immortalized HFKs. The data shown are fold changes relative to cells grown in monolayer. Error bars represent SEM of six independent experiments. <b>(G)</b> Southern blot for viral genome isolated from HPV16-infected HFK grown in monolayer and MC.</p

Analysis of viral transcripts by next generation RNA sequencing.

<p><b>(A)</b> Relative expression levels of individual viral ORFs in HPV16-infected HFK at 2 and 7 dpi. Cells grown in the presence of 10 μM Y-27632 were infected with HPV16 quasivirions via ECM-to-cell transfer and transcripts were quantified by RT-qPCR. The data shown are fold changes normalized to E6 transcript levels at 2 dpi of HFK. <b>(B)</b> RT-qPCR analysis of viral transcripts isolated from HPV16-immortalized HFKs and normalized to transcript levels in HPV16-infected HFKs at 10 dpi. <b>(C)</b> Schematic representation of the HPV16 genome and its ORFs. <b>(D)</b> Read depth maps of viral transcripts isolated from HPV16-infected HFK at 2, 4 and 7 dpi and from HPV-immortalized HFK of representative samples. <b>(E)</b> Detailed analysis of splice junctions of viral transcripts isolated from HFK at 7dpi. Each curved line represents a splice junction derived from individual reads that connects splice donor and acceptor sites.</p

Immunofluorescent detection of cellular markers in organotypic rafts grown from HPV16-infected HFK.

<p>Thin cuts of organotypic rafts were stained for MCM7 <b>(A)</b>, PCNA <b>(B)</b>, and p53 <b>(C)</b>. The lower panels show a merge with the Dapi stain to highlight nuclei.</p