Search

It is being assumed that human papillomavirus (HPV) utilizes three different modes of replication during its life cycle: initial amplification during the establishment phase, genome maintenance, and differentiation-induced amplification. However, HPV16 initial amplification was never formally proven due to a lack of an infection model.

HPV genome maintenance is currently thought to be achieved by regulating the expression and activity of the viral replication factors E1 and E2. In addition, the E8^E2 repressor has been shown to be important for restricting genome copy numbers by competing with E1 and E2 for binding to the viral origin of replication and by recruiting repressor complexes.

The current model for human papillomavirus (HPV) replication is comprised of three modes of replication. Following infectious delivery, the viral genome is amplified during the establishment phase to reach up to some hundred copies per cell. HPV genome copy number remains constant during the maintenance stage. Differentiation of infected cells induces HPV genome amplification. Using highly sensitive in situ hybridization (DNAscope) and freshly HPV16-infected as well as established HPV16-positive cell lines, we observed that viral genome is amplified in each S phase of undifferentiated keratinocytes cultured as monolayers. Nuclear viral genome copy number is reset to pre-S phase levels during mitosis. The majority of viral genome fails to tether to host chromosomes and is lost to the cytosol. Cytosolic viral genome gradually decreases during cell cycle progression. Loss of cytosolic genome is blocked in presence of NH4Cl or other drugs interfering with lysosomal acidification, suggesting the involvement of autophagy in viral genome degradation. These observations were also made with HPV31 cell lines obtained from patient samples. Cytosolic viral genome was not detected in UMSCC47 cells carrying integrated HPV16 DNA. Analyses of organotypic raft cultures derived from keratinocytes harboring episomal HPV16 revealed the presence of cytosolic viral genome as well. We conclude that HPV maintains viral genome copy number by balancing viral genome amplification during S phase with loss of viral genome that is lost to the cytosol during mitosis. It seems plausible that restrictions to viral genome tethering to mitotic chromosomes resets genome copy number in each cell cycle.IMPORTANCEHPV genome maintenance is currently being thought to be achieved by regulating expression and activity of viral replication factors E1 and E2. In addition, the E8^E2 repressor has been shown to be important for restricting genome copy number by competing with E1 and E2 for binding to the viral origin of replication and by recruitment of repressor complexes. Herein, we demonstrate that HPV viral genome is amplified in each S phase. Nuclear genome copy number is reset during mitosis by a failure of the majority of genomes to tether to mitotic chromosomes. Rather, they accumulate in the cytoplasm of freshly divided cells. Cytosolic viral DNA is quickly degraded in G1 in a lysosome dependent manner contributing to the genome copy reset. Our data imply that the mode of replication during establishment and maintenance is the same and further suggest that restrictions to genome tethering significantly contributes to viral genome maintenance.

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.