Complementation of the human adenovirus type 5 VA RNAI defect by the Vaccinia virus E3L protein and serotype-specific VA RNAIs

Inturi, Raviteja; Kamel, Wael; Akusjärvi, Göran; Punga, Tanel

doi:10.1016/j.virol.2015.07.002

Cited by 5 publications

(15 citation statements)

References 64 publications

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“…Infected 911 cells were fixed with fixing solution (95% methanol in PBS) at 42 hpi, followed by incubation with mouse anti-adenovirus monoclonal antibody (EMD Millipore, MAB8052, 1:50). The cells were washed three times with 1 ml of PBS, followed by incubation with fluorescent dye-conjugated secondary antibodies as described earlier (68). After the final washing steps, the expression of the adenovirus capsid proteins was analyzed using a fluorescence microscope.…”

Section: Discussionmentioning

confidence: 99%

“…The primers ZNF622_Fw (5=-TGATTCTGCCTTCTGGTGCC-3=) and ZNF622_Rev (5=-CACGGCCTTCCGATTTTTGG-3=) were used. The data analysis and the HPRT1 oligonucleotides have been described previously (46,68).…”

Section: Methodsmentioning

confidence: 99%

“…Indirect immunofluorescence assay. Indirect immunofluorescence assay was carried out as described previously (68). Briefly, stable U2OS cell lines [KOϩZNF622(wt)-HA, KOϩZNF622(1-359), and KOϩZNF622(109-477)] were infected with HAdV-pVII-Flag (1 FFU/cell) or mock infected (infection medium without virus) for 48 h. Cells were fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) for 15 min and permeabilized with 0.1% Triton X-100 in PBS-T (PBS plus 0.01% Tween 20) for 15 min at room temperature.…”

Section: Methodsmentioning

confidence: 99%

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Cellular Zinc Finger Protein 622 Hinders Human Adenovirus Lytic Growth and Limits Binding of the Viral pVII Protein to Virus DNA

Mun

Punga

2019

J Virol

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Human adenovirus (HAdV) encodes a multifunctional DNA-binding protein pVII, which is involved in virus DNA packaging and extracellular immune signaling regulation. Although the pVII is an essential viral protein, its exact role in the virus life cycle and interplay with cellular proteins have remained to a large extent unclear. We have recently identified the cellular zinc finger protein 622 (ZNF622) as a potential pVII-interacting protein. In this study, we describe the functional consequences of the ZNF622-pVII interplay and the role of ZNF622 in the HAdV life cycle. ZNF622 protein expression increased, and it accumulated similarly to the pVII protein in the nuclei of virus-infected cells. The lack of the ZNF622 protein specifically increased pVII binding to viral DNA in the infected cells and elevated the pVII protein levels in the purified virions. In addition, ZNF622 knockout cells showed an increased cell lysis and enhanced accumulation of the infectious virus particles. Protein interaction studies revealed that ZNF622 forms a trimeric complex with the pVII protein and the cellular histone chaperon protein nucleophosmin 1 (NPM1). The integrity of this complex is important since ZNF622 mutations and NPM1 deficiency changed pVII ability to bind viral DNA. Collectively, our results implicate that ZNF622 may act as a cellular antiviral protein hindering lytic HAdV growth and limiting pVII protein binding to viral DNA.IMPORTANCEHuman adenoviruses (HAdVs) are common human pathogens causing a wide range of acute infections. To counteract viral pathogenicity, cells encode a variety of antiviral proteins and noncoding RNAs to block virus growth. In this study, we show that the cellular zinc finger protein 622 (ZNF622) interacts with an essential HAdV protein known as pVII. This mutual interaction limits pVII binding to viral DNA. Further, ZNF622 has a role in HAdV life cycle since the lack of ZNF622 correlates with increased lysis of the infected cells and accumulation of the infectious virions. Together, our study reveals a novel cellular antiviral protein ZNF622, which may impede lytic HAdV growth.

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Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Cellular Zinc Finger Protein 622 Hinders Human Adenovirus Lytic Growth and Limits Binding of the Viral pVII Protein to Virus DNA

Mun

Punga

2019

J Virol

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“…The HAdV-pVII-Flag virus was amplified in 911 cells and purified by CsCl gradient centrifugation. Virus infections and titrations were done as described previously (65). The multiplicity of infection (MOI) (fluorescence-forming units [FFU]/per cell) is indicated in the respective figure legends.…”

Section: Methodsmentioning

confidence: 99%

“…RNA extraction and qRT-PCR. Total RNA extraction, cDNA synthesis with random primers, and reverse transcription-quantitative PCRs (qRT-PCRs) were performed as previously described (65). The following MKRN1 primers were used: tp575 (5=-GCAGCAAGGGATGACTTTGT-3) and tp576 (5=-TGTATTTA TGGAGACCGCTGC-3).…”

Section: Methodsmentioning

confidence: 99%

Human Adenovirus Infection Causes Cellular E3 Ubiquitin Ligase MKRN1 Degradation Involving the Viral Core Protein pVII

Inturi

Mun

Singethan

et al. 2018

J Virol

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Human adenoviruses (HAdVs) are common human pathogens encoding a highly abundant histone-like core protein, VII, which is involved in nuclear delivery and protection of viral DNA as well as in sequestering immune danger signals in infected cells. The molecular details of how protein VII acts as a multifunctional protein have remained to a large extent enigmatic. Here we report the identification of several cellular proteins interacting with the precursor pVII protein. We show that the cellular E3 ubiquitin ligase MKRN1 is a novel precursor pVII-interacting protein in HAdV-C5-infected cells. Surprisingly, the endogenous MKRN1 protein underwent proteasomal degradation during the late phase of HAdV-C5 infection in various human cell lines. MKRN1 protein degradation occurred independently of the HAdV E1B55K and E4orf6 proteins. We provide experimental evidence that the precursor pVII protein binding enhances MKRN1 self-ubiquitination, whereas the processed mature VII protein is deficient in this function. Based on these data, we propose that the pVII protein binding promotes MKRN1 self-ubiquitination, followed by proteasomal degradation of the MKRN1 protein, in HAdV-C5-infected cells. In addition, we show that measles virus and vesicular stomatitis virus infections reduce the MKRN1 protein accumulation in the recipient cells. Taken together, our results expand the functional repertoire of the HAdV-C5 precursor pVII protein in lytic virus infection and highlight MKRN1 as a potential common target during different virus infections. Human adenoviruses (HAdVs) are common pathogens causing a wide range of diseases. To achieve pathogenicity, HAdVs have to counteract a variety of host cell antiviral defense systems, which would otherwise hamper virus replication. In this study, we show that the HAdV-C5 histone-like core protein pVII binds to and promotes self-ubiquitination of a cellular E3 ubiquitin ligase named MKRN1. This mutual interaction between the pVII and MKRN1 proteins may prime MKRN1 for proteasomal degradation, because the MKRN1 protein is efficiently degraded during the late phase of HAdV-C5 infection. Since MKRN1 protein accumulation is also reduced in measles virus- and vesicular stomatitis virus-infected cells, our results signify the general strategy of viruses to target MKRN1.

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Efficacy of recombinant adenovirus expressing a fusion gene from GM-CSF and Epstein-Barr virus LMP2A in a mouse tumor model

Xue

Yang

et al. 2017

Human Vaccines & Immunotherapeutics

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In this study, purified GM-CSF and LMP2A mRNAs were amplified by PCR. Then, the GM-CSF and LMP2A sequences were connected by the polypeptide linker (GlySer) using gene splicing by overlap extension. The constructed fusion gene GC2A was inserted into the adenovirus vector. Then the recombinant vector was introduced into HEK 293T cells by calcium phosphate transfection to package the adenovirus. The levels of antibodies against the GM-CSF and LMP2Afusion proteins were measured by ELISA, and the CTL activity of the mouse splenic lymphocytes was determined by lactate dehydrogenase (LDH) release assay. Immunotherapy of mouse tumor (EBV-positive epithelial tumor cell line (GT39)) tissues was performed, and their morphologies were assessed. Finally, the data of each group were analyzed using SPSS 11.5 statistical software. The recombinant adenovirus could replicate in HEK 293Tcells and induce humoral and cellular immune responses in the mice. The maximum dose resulted in an antibody titer of 18500 (184.5 ± 8.7 pg/ml). At an effector: target ratio of 40:1, maximum specific lysis was observed which was approximately three times that detected in the control immunized mice. The tumor inhibition rate was approximately 76% compared with the control groups, indicating the presence of significant differences among the groups. Tumor-infiltrating lymphocytes were detected by hematoxylin-eosin (HE) staining. The recombinant adenovirus induced humoral and cellular immune responses and inhibited tumor growth in mice. It provided a theoretical basis and candidate vaccine for further preclinical trials.

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Complementation of the human adenovirus type 5 VA RNAI defect by the Vaccinia virus E3L protein and serotype-specific VA RNAIs

Cited by 5 publications

References 64 publications

Cellular Zinc Finger Protein 622 Hinders Human Adenovirus Lytic Growth and Limits Binding of the Viral pVII Protein to Virus DNA

Cellular Zinc Finger Protein 622 Hinders Human Adenovirus Lytic Growth and Limits Binding of the Viral pVII Protein to Virus DNA

Human Adenovirus Infection Causes Cellular E3 Ubiquitin Ligase MKRN1 Degradation Involving the Viral Core Protein pVII

Efficacy of recombinant adenovirus expressing a fusion gene from GM-CSF and Epstein-Barr virus LMP2A in a mouse tumor model

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