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Protein ubiquitination is a highly conserved post-translational modification affecting various biological processes including viral propagation. Ubiquitination has multiple effects on viral propagation, including viral genome uncoating, viral replication, and immune evasion. Ubiquitination of viral proteins is triggered by the ubiquitin-proteasome system (UPS). This involves the covalent attachment of the highly conserved 76 amino acid residue ubiquitin protein to target proteins by the consecutive actions of E1, E2 and E3 enzymes, and the 26S proteasome that together form a multiprotein complex that degrades target proteins. The UPS is the primary cytosolic proteolytic machinery for the selective degradation of various forms of proteins including viral proteins, thereby limiting viral growth in host cells. To combat this host anti-viral machinery, viruses have evolved the ability to employ or subvert the UPS to inactivate or degrade cellular proteins to favour viral propagation. This review highlights our current knowledge on the different roles of the UPS during viral propagation., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Bombyx mori nucleopolyhedrovirus (BmNPV) is a major viral agent that causes deadly grasserie disease in silkworms, while BmNPV DNA polymerase (BmNPV-pol), encoded by ORF53 gene, plays a central role in viral DNA replication. Efficacy studies of BmNPV-POL are limited because of poor heterologous protein expression in E. coli. Here, we redesigned the BmNPV-pol to preferentially match codon frequencies of E. coli without altering the amino acid sequence. Following de novo synthesis, codon-optimized BmNPV-pol (co-BmNPV-pol) gene was cloned into pET32a and pGEX-4T-2 vector. The expression of co-BmNPV-POL in E. coli was significantly increased when BmNPV-POL was fused with GST protein rather than a His-tag. The co-BmNPV-POL fusion proteins were isolated using GST affinity chromatography and Mono Q iron exchange chromatography. Protein purity and identity were confirmed by western blot and MALDI-TOF analyses. The biological activity of purified proteins was measured on a poly(dA)/oligo(dT) primer/template. The specific polymerasing activity of the recombinant BmNPV-POL was 6,329 units/mg at optimal conditions. Thus, a large amount of purified protein as a soluble form with high activity would provide many benefits for the functional research and application of BmNPV-POL.

Orf68 (ac68) of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is identified to be an early gene, but its transcription start site remains unknown. The coding sequence of ac68 overlaps 280-bp leading sequence and 159-bp coding sequence of lef3 (ac67). In this study, the transcription start site of ac68 was determined by 5' RACE analysis to be 18 nucleotides upstream from the start codon. In order to investigate the effect of ac68 deletion on virus propagation, we generated a bacmid with an ac68 knockout by deleting 360-bp inside the ac68 gene, which also deleted 220-bp leading sequence of lef3. Production of infectious budded virus and formation of nucleocapsids and occlusion bodies exhibited wild-type patterns of virus propagation in Sf-9 cells infected with the mutant bacmid. The result demonstrated that ac68 was not an essential gene for viral propagation which was confirmed by further deletion of ac68, and disruption of the lef3 leading sequence did not affect viral propagation. Ac68 was the second auxiliary gene discovered besides Ac133 (alk-exo) among the 30 core genes of AcMNPV.

Orf94 (Bm94) of Bombyx mori nucleopolyhedrovirus (BmNPV) potentially encodes 424-amino acids with a predicted molecular weight of 49.4 kDa, but its function remains unknown. Blast search results revealed that Bm94 homologues exist in 10 completely sequenced Lepidopteron NPVs with identities ranging from 95 to 37%. Results of our recent study showed that Bm94 was transcribed from 12 to 72 h and the corresponding protein was detected from 24 to 72 h post-infection. Furthermore, Western blot analysis revealed that Bm94 was present in occlusion-derived virus (ODV) and in total protein from BmNPV-infected BmN cells, but not in budded virus. Immunofluorescence analysis revealed that the protein located primarily in the cytoplasm and was also present in the nucleus in the later infection. In conclusion, these results together indicated that Bm94 was a late gene, which distributed both in the cytoplasm and in the nucleus, and was identified to be a component of BmNPV ODV.

Orf101 ( Bm101) of Bombyx mori nucleopolyhedrovirus (BmNPV) is a highly conserved gene in lepidopteran nucleopolyhedroviruses, but its function remains unknown. In this study, Bm101 was characterized. Transcripts of Bm101 were detected from 24 through 96 h post infection (h p.i.) by RT-PCR. The corresponding protein was also detected from 24 to 96 h p.i. in BmNPV-infected BmN cells by Western blot analysis using a polyclonal antibody against Bm101. Western blot assay of occlusion-derived virus and budded virus (BV) preparations revealed that Bm101 encodes a 28-kDa structural protein that is associated with BV and is located in the envelope fraction of budded virions. In addition, confocal analysis showed that the protein was localized in the cytosol and cytoplasmic membrane in virus-infected cells. In conclusion, the available data suggest that Bm101 is a functional ORF of BmNPV and encodes a protein expressed in the late stage of the infection cycle that is associated with the BV envelope. [ABSTRACT FROM AUTHOR]

Nucleolar proteins facilitate the replication of certain human and animal viruses through interaction with viral proteins. In this study, an interaction between nervous necrosis virus capsid protein and nucleolar phosphoprotein B23 was identified using in vitro experimental approaches. The capsid protein binds to B23 early during the viral infection and accumulates in the nucleus, particularly in the nucleolus. However, over the course of the infection B23 is redistributed from the nucleoli to the nucleoplasm. siRNA-mediated knockdown of B23 reduced viral replication and cytopathic effect. Thus, B23 targets capsid protein to the nucleus and facilitates NNV replication. The results provide the first demonstration that nucleolar protein B23 has a direct role in the nodavirus replication process. [ABSTRACT FROM AUTHOR]