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The Pivotal Roles of US3 Protein in Cell-to-Cell Spread and Virion Nuclear Egress of Duck Plague Virus.

ABSTRACT: The duck plague virus (DPV) US3 protein, a homolog of the herpes simplex virus-1 (HSV-1) US3 protein that is reported to be critical for viral replication, has been minimally studied. Therefore, to investigate the function of the DPV US3 protein, we used scarless Red recombination technology based on an infectious bacterial artificial chromosome (BAC) containing the DPV Chinese virulent strain (CHv) genome and successfully constructed and rescued a US3-deleted mutant and the corresponding revertant virus (BAC-CHv-?US3 and BAC-CHv-?US3R, respectively). For viral growth characteristics, compared to the parental and revertant viruses, the US3-deleted mutant showed an approximately 100-fold reduction in viral titers but no significant reduction in genome copies, indicating that the US3-deleted mutant exhibited decreased viral replication but not decreased viral DNA generation. In addition, the US3-deleted mutant formed viral plaques that were 33% smaller on average than those formed by the parental and revertant viruses, demonstrating that US3 protein affected the viral cell-to-cell spread of DPV. Finally, the results of electron microscopy showed that the deletion of US3 resulted in a large number of virions accumulating in the nucleus and perinuclear space, thus blocking virion nuclear egress. In this study, we found that the DPV US3 protein played pivotal roles in viral replication by promoting viral cell-to-cell spread and virion nuclear egress, which may provide some references for research on the function of the DPV US3 protein.

SUBMITTER: Deng L

PROVIDER: S-EPMC7189242 | biostudies-literature | 2020 Apr

REPOSITORIES: biostudies-literature

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The Pivotal Roles of US3 Protein in Cell-to-Cell Spread and Virion Nuclear Egress of Duck Plague Virus.

Deng Liyao L Wang Mingshu M Cheng Anchun A Yang Qiao Q Wu Ying Y Jia Renyong R Chen Shun S Zhu Dekang D Liu Mafeng M Zhao Xinxin X Zhang Shaqiu S Huang Juan J Ou Xumin X Mao Sai S Zhang Ling L Liu Yunya Y Yu Yanling Y Tian Bin B Pan Leichang L Rehman Mujeeb Ur MU Chen Xiaoyue X

Scientific reports 20200428 1

The duck plague virus (DPV) US3 protein, a homolog of the herpes simplex virus-1 (HSV-1) US3 protein that is reported to be critical for viral replication, has been minimally studied. Therefore, to investigate the function of the DPV US3 protein, we used scarless Red recombination technology based on an infectious bacterial artificial chromosome (BAC) containing the DPV Chinese virulent strain (CHv) genome and successfully constructed and rescued a US3-deleted mutant and the corresponding revert ...[more]

PMID: 32346128

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Project description:Viral proteins pUL16 and pUL21 are required for efficient nuclear egress of herpes simplex virus 2 capsids. To better understand the role of these proteins in nuclear egress, we established whether nuclear egress complex (NEC) distribution and/or function was altered in the absence of either pUL16 or pUL21. NEC distribution in cells infected with pUL16-deficient viruses was indistinguishable from that observed in cells infected with wild-type viruses. In contrast, NEC distribution was aberrant in cells infected with pUL21-deficient virus and, instead, showed some similarity to the aberrant NEC distribution pattern observed in cells infected with pUs3-deficient virus. These results indicated that pUL16 plays a role in nuclear egress that is distinct from that of pUL21 and pUs3. Higher-resolution examination of nuclear envelope ultrastructure in cells infected with pUL21-deficient viruses by transmission electron microscopy showed different types of nuclear envelope perturbations, including some that were not observed in cells infected with pUs3 deficient virus. The formation of the nuclear envelope perturbations observed in pUL21-deficient virus infections was dependent on a functional NEC, revealing a novel role for pUL21 in regulating NEC activity. The results of comparisons of nuclear envelope ultrastructure in cells infected with viruses lacking pUs3, pUL16, or both pUs3 and pUL16 were consistent with a role for pUL16 in advance of primary capsid envelopment and shed new light on how pUs3 functions in nuclear egress.IMPORTANCE The membrane deformation activity of the herpesvirus nuclear egress complex (NEC) allows capsids to transit through both nuclear membranes into the cytoplasm. NEC activity must be precisely controlled during viral infection, and yet our knowledge of how NEC activity is controlled is incomplete. To determine how pUL16 and pUL21, two viral proteins required for nuclear egress of herpes simplex virus 2, function in nuclear egress, we examined how the lack of each protein impacted NEC distribution. These analyses revealed a function of pUL16 in nuclear egress distinct from that of pUL21, uncovered a novel role for pUL21 in regulating NEC activity, and shed new light on how a viral kinase, pUs3, regulates nuclear egress. Nuclear egress of capsids is required for all herpesviruses. A complete understanding of all aspects of nuclear egress, including how viral NEC activity is controlled, may yield strategies to disrupt this process and aid the development of herpes-specific antiviral therapies.

Project description:Duck plague virus (DPV) is a representative pathogen transmitted among aquatic animals that causes gross lesions and immune inhibition in geese and ducks. The mechanism of organ tropism and innate immune evasion of DPV has not been completely deciphered due to a lack of cell models to study the innate immune manipulation and pathogenicity of aquatic viruses. In the present study, we isolated five types of duck primary cells [duck embryo fibroblasts (DEFs), neurons, astrocytes, peripheral blood mononuclear cells (PBMCs), and monocytes/macrophages] to identify appropriate cell models for DPV, using tropism infection and innate immunologic assays. Cells responded differently to stimulation with DNA viruses or RNA virus analogs. DPV infection exhibited broad tropism, as the recombinant virulent strain (CHv-GFP) infected DEFs, neurons, astrocytes, and monocytes/macrophages, but not the PBMCs, as the expression of EGFP was negligible. The basal levels of innate immunity molecules were highest in monocytes/macrophages and lower in DEFs and astrocytes. Conversely, the titer and genomic copy number of the attenuated virus strain was higher in DEFs and astrocytes than in neurons and monocytes/macrophages. The titer and genomic copy number of the attenuated virus strain were higher compared with the virulent strain in DEFs, neurons, and astrocytes. The innate immune response was not significantly induced by either DPV strain in DEFs, neurons, or astrocytes. The virulent strain persistently infected monocytes/macrophages, but the attenuated strain did so abortively, and this was accompanied by the phenomenon of innate immune inhibition and activation by the virulent and attenuated strains, respectively. Blockage of IFNAR signaling promoted replication of the attenuated strain. Pre-activation of IFNAR signaling inhibited infection by the virulent strain. The selection assay results indicated that induction of innate immunity plays an essential role in controlling DPV infection, and monocytes/macrophages are an important cell model for further investigations. Our study provided practical methods for isolating and culturing duck primary cells, and our results will facilitate further investigations of organ tropism, innate immune responses, latent infection, and the effectiveness of antiviral drugs for treating DPV and potentially other aerial bird pathogens.

Dataset Information

The Pivotal Roles of US3 Protein in Cell-to-Cell Spread and Virion Nuclear Egress of Duck Plague Virus.

Publications

The Pivotal Roles of US3 Protein in Cell-to-Cell Spread and Virion Nuclear Egress of Duck Plague Virus.

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