Interaction between Two Iridovirus Core Proteins and Their Effects on Ranavirus (RGV) Replication in Cells from Different Species

Zeng, Xiao-Tao; Zhang, Qi-Ya

doi:10.3390/v11050416

Cited by 12 publications

(17 citation statements)

References 36 publications

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“…Co-IP assays were performed as previously described with minor modifications [ 20 ]. Briefly, HEK293T cells seeded in 10 cm dishes were cotransfected with 7.5 μg of pcDNA3.1- S - HA and 7.5 μg of pcDNA3.1- ZDHHC5 - 3Flag or other indicated plasmids.…”

Section: Methodsmentioning

confidence: 99%

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The interactions of ZDHHC5/GOLGA7 with SARS-CoV-2 spike (S) protein and their effects on S protein’s subcellular localization, palmitoylation and pseudovirus entry

Zeng

Cheng

2021

Virol J

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Background Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) protein determines virus entry and the palmitoylation of S protein affects virus infection. An acyltransferase complex ZDHHC5/GOGAL7 that interacts with S protein was detected by affinity purification mass spectrometry (AP-MS). However, the palmitoylated cysteine residues of S protein, the effects of ZDHHC5 or GOLGA7 knockout on S protein’s subcellular localization, palmitoylation, pseudovirus entry and the enzyme for depalmitoylation of S protein are not clear. Methods The palmitoylated cysteine residues of S protein were identified by acyl-biotin exchange (ABE) assays. The interactions between S protein and host proteins were analyzed by co-immunoprecipitation (co-IP) assays. Subcellular localizations of S protein and host proteins were analyzed by fluorescence microscopy. ZDHHC5 or GOGAL7 gene was edited by CRISPR-Cas9. The entry efficiencies of SARS-CoV-2 pseudovirus into A549 and Hela cells were analyzed by measuring the activity of Renilla luciferase. Results In this investigation, all ten cysteine residues in the endodomain of S protein were palmitoylated. The interaction of S protein with ZDHHC5 or GOLGA7 was confirmed. The interaction and colocalization of S protein with ZDHHC5 or GOLGA7 were independent of the ten cysteine residues in the endodomain of S protein. The interaction between S protein and ZDHHC5 was independent of the enzymatic activity and the PDZ-binding domain of ZDHHC5. Three cell lines HEK293T, A549 and Hela lacking ZDHHC5 or GOLGA7 were constructed. Furthermore, S proteins still interacted with one host protein in HEK293T cells lacking the other. ZDHHC5 or GOLGA7 knockout had no significant effect on S protein’s subcellular localization or palmitoylation, but significantly decreased the entry efficiencies of SARS-CoV-2 pseudovirus into A549 and Hela cells, while varying degrees of entry efficiencies may be linked to the cell types. Additionally, the S protein interacted with the depalmitoylase APT2. Conclusions ZDHHC5 and GOLGA7 played important roles in SARS-CoV-2 pseudovirus entry, but the reason why the two host proteins affected pseudovirus entry remains to be further explored. This study extends the knowledge about the interactions between SARS-CoV-2 S protein and host proteins and probably provides a reference for the corresponding antiviral methods.

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Section: Methodsmentioning

confidence: 99%

“…Western blot analysis was performed as described previously [ 20 ]. Protein samples were resolved by SDS-PAGE, followed by electroblotting to polyvinylidene difluoride (PVDF) membranes.…”

Section: Methodsmentioning

confidence: 99%

The interactions of ZDHHC5/GOLGA7 with SARS-CoV-2 spike (S) protein and their effects on S protein’s subcellular localization, palmitoylation and pseudovirus entry

Zeng

Cheng

2021

Virol J

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“…Subcellular localization can provide clues for protein biological function [ 51 ], and different subcellular localizations suggest different functions. As previously reported, viral proteins such as ICP0 encoded by HSV-1 and core protein 91R encoded by Rana grylio virus (RGV) were localized at the nucleus, which has a nuclear localization signal (NLS), and play an important role in promoting the successful onset of lytic infection and productive reactivation of viral genomes from latency or virus genome replication [ 32 , 52 , 53 , 54 ]. Here, 52L of CaHV were observed localized at the nucleus and predicted containing an NLS, and probably have similar functions.…”

Section: Discussionmentioning

confidence: 99%

“…Epithelioma papulosum cyprinid (EPC) cells were maintained in Medium 199 supplemented with 10% fetal bovine serum (FBS) at 25 °C [ 31 ], and used for transfection and fluorescence observation. Human embryonic kidney (HEK293T) cells were cultured under an atmosphere of 5% CO 2 (37 °C) in fresh Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% FBS [ 32 ], and used for transfection and ubiquitination detection. Ubiquitin, E1, and E2 enzymes were purchased from Boston Biochem.…”

Section: Methodsmentioning

confidence: 99%

Structural and Functional Diversity among Five RING Finger Proteins from Carassius Auratus Herpesvirus (CaHV)

Wang

Fei

Zhang

et al. 2021

Viruses

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Carassius auratus herpesvirus (CaHV) has been identified as a high-virulence pathogenic virus that infects aquatic animals, but the key factor for virus–host interaction is still unclear. Five Really interesting new genes (RING) finger proteins (39L, 52L, 131R, 136L, and 143R) of CaHV were screened to determine structural diversity. RING finger proteins were also predicted in other known fish herpesviruses, with an arrangement and number similar to CaHV. We performed multifaceted analyses of the proteins, including protein sizes, skeleton structures, subcellular localizations, and ubiquitination activities, to determine their precise roles in virus–host interactions. The five proteins were overexpressed and detected different levels of ubiquitination activities, and 143R showed the highest activity. Then, the prokaryotic expressed and purified full-length proteins (131R and 136L), RING domain isolates (131R12–43 and 136L45–87), and RING domain-deleted mutants (131RΔ12–43 and 136LΔ45–87) were prepared to detect their activities through ubiquitination assays. The results indicate that both full-length proteins and their isolates have activities that catalyze ubiquitination, and the full-length proteins possess higher activity than the isolates, but RING domain-deleted mutants lose their activities. Furthermore, the activities of the five proteins were verified as E3 ubiquitin ligase activity, showing that the RING domains determine the ubiquitination activity. These proteins present different subcellular localization. RING domain-deleted mutants showed similar subcellular localization with their full-length proteins, and all the isolates diffused in whole cells. The current results indicate that the sequence outside the RING domain determines subcellular localization and the level of ubiquitination activity, suggesting that the RING finger proteins of fish herpesviruses might have diverse functions in virus–host interaction.

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“…Ranavirus genome replication has been reported to occur in two-stages involving the nucleus and cytoplasm [ 10 ]. Although several viral proteins involved in ranavirus DNA replication have been predicted or investigated [ 11 , 12 , 13 ], the mechanism of ranavirus DNA replication still needs to be explored. Andrias davidianus ranavirus (ADRV) was isolated from diseased Chinese giant salamanders, which have a genome size of 106.7 kbp with 101 predicted open reading frames (ORFs) [ 6 ].…”

Section: Introductionmentioning

confidence: 99%

Andrias davidianus Ranavirus (ADRV) Genome Replicate Efficiently by Engaging Cellular Mismatch Repair Protein MSH2

Fei

Wang

et al. 2022

Viruses

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As nucleocytoplasmic large DNA viruses, replication of ranaviruses (genus Ranavirus, family Iridoviridae) involves a series of viral and host proteins. We have described that the replication and transcription machinery of Andrias davidianus ranavirus (ADRV) which was isolated from the Chinese giant salamander contained host factors. Here, a new host factor, the MutS homolog 2 (MSH2), was proved as an important protein that participated in ADRV infection. Expression of MSH2 was stable during ADRV infection in cultured cells and it localized at the cytoplasmic viral factories and colocalized with virus nascent DNA, indicating its possible role in virus genome replication. Investigation of the viral proteins that interacted with MSH2 by co-immunoprecipitation showed that A. davidianus MSH2 can interact with ADRV-35L (possible components associated with virus transcription), ADRV-47L (virus DNA polymerase), and ADRV-98R. Further knockdown MSH2 expression by RNAi significantly reduced the late gene expression of ADRV. Additionally, MSH2 knockout by CRISPR/Cas9 significantly reduced viral titers, genome replication, and late gene transcription of ADRV. Thus, the current study proved that ADRV can engage cellular MSH2 for its efficient genome replication and late gene transcription, which provided new information for understanding the roles of host factors in ranavirus replication and transcription.

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Interaction between Two Iridovirus Core Proteins and Their Effects on Ranavirus (RGV) Replication in Cells from Different Species

Cited by 12 publications

References 36 publications

The interactions of ZDHHC5/GOLGA7 with SARS-CoV-2 spike (S) protein and their effects on S protein’s subcellular localization, palmitoylation and pseudovirus entry

The interactions of ZDHHC5/GOLGA7 with SARS-CoV-2 spike (S) protein and their effects on S protein’s subcellular localization, palmitoylation and pseudovirus entry

Structural and Functional Diversity among Five RING Finger Proteins from Carassius Auratus Herpesvirus (CaHV)

Andrias davidianus Ranavirus (ADRV) Genome Replicate Efficiently by Engaging Cellular Mismatch Repair Protein MSH2

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