Triad of human cellular proteins, IRF2, FAM111A, and RFC3, restrict replication of orthopoxvirus SPI-1 host-range mutants

Panda, Debasis; Fernández, Daniel; Lal, Madhu; Buehler, Eugen; Moss, Bernard

doi:10.1073/pnas.1700678114

Cited by 29 publications

(54 citation statements)

References 54 publications

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“…Supporting this idea, an unbiased FAM111A interactome analysis revealed the Replication Factor C (RFC) complex, the main cellular PCNA loader 8 , but not PCNA itself as a major replication fork-associated cellular FAM111A binding partner ( Fig. 1h), in agreement with genetic data indicating a link between FAM111A and RFC subunits in restricting viral replication 9 . Co-immunoprecipitation experiments suggested that FAM111A associates with the RFC complex through interaction with the RFC1 subunit independently of the PIP box, involving a region of the extended regulatory N-terminal portion of human RFC1 that encompasses its BRCT domain 8 (Fig.…”

supporting

confidence: 75%

“…Although both RFC1 and RPB1 emerge as attractive candidate FAM111 substrates based on our findings, we have so far been unable to conclusively establish such relationships. While the biological functions of FAM111 proteases are not well understood, our finding that FAM111 proteolytic activity is dispensable for normal cell growth but powerfully suppresses DNA replication, transcription and cell viability once elevated is well aligned with the putative role of FAM111A as a host restriction factor suppressing Simian Virus 40 (SV40) and poxvirus gene expression and DNA replication 5,6,9 . When properly controlled, these features could provide an important contribution towards a general line of defense against invading viruses and their propagation.…”

mentioning

confidence: 66%

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Gain-of-function mutations amplify cytotoxic FAM111 protease activity in human genetic disorders

Hoffmann

Pentakota

Mund

et al. 2020

Preprint

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Dominant missense mutations in the human serine protease FAM111A underlie perinatally lethal gracile bone dysplasia and Kenny-Caffey syndrome 1-3 , yet how FAM111A mutations lead to disease is not known. We show that FAM111A proteolytic activity suppresses DNA replication and transcription by displacing key effectors of these processes from chromatin, triggering rapid programmed cell death by Caspase-dependent apoptosis to potently undermine cell viability. Patient-associated point mutations in FAM111A exacerbate these phenotypes by hyperactivating its intrinsic protease activity. Moreover, FAM111A forms a complex with the uncharacterized homologous serine protease FAM111B, point mutations in which cause a hereditary fibrosing poikiloderma syndrome 4 , and we demonstrate that disease-associated FAM111B mutants display amplified proteolytic activity and phenocopy the cellular impact of deregulated FAM111A catalytic activity. Thus, patient-associated FAM111A and FAM111B mutations may drive multisystem disorders via a common gain-offunction mechanism that relieves inhibitory constraints on their protease activities to powerfully undermine cellular fitness. Main textDominant missense mutations in human FAM111A underlie Kenny-Caffey syndrome and gracile bone dysplasia, which present with a broad spectrum of severe growth abnormalities with variable expressivity, including thin and brittle bones, dwarfism, facial dysmorphism and splenic hypoplasia 1-3 . FAM111A is a prospective host restriction factor that has been linked to DNA replication 5-7 but whose precise cellular function remains poorly understood. In addition to a PCNA-binding PIP box, FAM111A contains a functionally uncharacterized C-terminal serine protease domain, which

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supporting

confidence: 75%

mentioning

confidence: 66%

Gain-of-function mutations amplify cytotoxic FAM111 protease activity in human genetic disorders

Hoffmann

Pentakota

Mund

et al. 2020

Preprint

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“…However, the VACV proteins A20 and D4 form a complex with the VACV DNA polymerase to provide a similar function (18). Nevertheless, a possible role for PCNA in replication of VACV was suggested indirectly by a report that two human PCNA-associated proteins are inhibitors of a host range mutant (104). To ascertain whether to pursue this lead in the future, we investigated the effect of the specific PCNA inhibitor T2AA (105).…”

Section: Discussionmentioning

confidence: 99%

Identification of Vaccinia Virus Replisome and Transcriptome Proteins by Isolation of Proteins on Nascent DNA Coupled with Mass Spectrometry

Senkevich

Katsafanas

Weisberg

et al. 2017

J Virol

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Poxviruses replicate within the cytoplasm and encode proteins for DNA and mRNA synthesis. To investigate poxvirus replication and transcription from a new perspective, we incorporated 5-ethynyl-2=-deoxyuridine (EdU) into nascent DNA in cells infected with vaccinia virus (VACV). The EdU-labeled DNA was conjugated to fluor-or biotin-azide and visualized by confocal, superresolution, and transmission electron microscopy. Nuclear labeling decreased dramatically after infection, accompanied by intense labeling of cytoplasmic foci. The nascent DNA colocalized with the VACV single-stranded DNA binding protein I3 in multiple puncta throughout the interior of factories, which were surrounded by endoplasmic reticulum. Complexes containing EdU-biotin-labeled DNA cross-linked to proteins were captured on streptavidin beads. After elution and proteolysis, the peptides were analyzed by mass spectrometry to identify proteins associated with nascent DNA. The known viral replication proteins, a telomere binding protein, and a protein kinase were associated with nascent DNA, as were the DNA-dependent RNA polymerase and intermediate-and late-stage transcription initiation and elongation factors, plus the capping and methylating enzymes. These results suggested that the replicating pool of DNA is transcribed and that few if any additional viral proteins directly engaged in replication and transcription remain to be discovered. Among the host proteins identified by mass spectrometry, topoisomerases II␣ and II␤ and PCNA were noteworthy. The association of the topoisomerases with nascent DNA was dependent on expression of the viral DNA ligase, in accord with previous proteomic studies. Further investigations are needed to determine possible roles for PCNA and other host proteins detected. IMPORTANCE Poxviruses, unlike many well-characterized animal DNA viruses, replicate entirely within the cytoplasm of animal cells, raising questions regarding the relative roles of viral and host proteins. We adapted newly developed procedures for click chemistry and iPOND (Isolation of proteins on nascent DNA) to investigate vaccinia virus (VACV), the prototype poxvirus. Nuclear DNA synthesis ceased almost immediately following VACV infection, followed swiftly by the synthesis of viral DNA within discrete cytoplasmic foci. All viral proteins known from genetic and proteomic studies to be required for poxvirus DNA replication were identified in the complexes containing nascent DNA. The additional detection of the viral DNAdependent RNA polymerase and intermediate and late transcription factors provided evidence for a temporal coupling of replication and transcription. Further studies are needed to assess the potential roles of host proteins, including topoisomerases II␣ and II␤ and PCNA, which were found associated with nascent DNA.

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“…RNAi screening was conducted using the Ambion Silencer® Select Human Druggable Genome siRNA Library Version 4 as described previously [63] and the HTRF assay for NS1 antigen was performed as described above. The HTRF signal was for each unique non-overlapping siRNA against the target genes was normalized to a negative control targeting siRNA.…”

Section: Sirna Screeningmentioning

confidence: 99%

An Integrated Systems Biology Approach Identifies the Proteasome as a Critical Host Machinery for ZIKV and DENV Replication

Song¹,

Lee

Pan³

et al. 2020

Preprint

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The Zika (ZIKV) and dengue (DENV) flaviviruses exhibit similar replicative processes but distinct clinical outcomes. A systematic understanding of virus-host protein-protein interaction networks can reveal cellular pathways critical to viral replication and disease pathogenesis. Here we employed three independent systems biology approaches toward this goal. First, protein array analysis of direct interactions between individual ZIKV/DENV viral proteins and 20,240 human proteins revealed multiple conserved cellular pathways and protein complexes, including proteasome complexes. Second, an RNAi screen of 10,415 druggable genes to identify host proteins required for ZIKV infection uncovered proteasome proteins. Third, a high-throughput screening of 6,016 bioactive compounds for ZIKV inhibitors yielded 134 effective compounds, including six proteasome inhibitors that suppress both ZIKV and DENV replication. Integrative analyses of these orthogonal datasets pinpoints proteasome as critical host machinery for ZIKV/DENV replication. Our study provides multi-omics datasets for further studies of flavivirus-host interactions, disease pathogenesis, and new drug targets.

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Triad of human cellular proteins, IRF2, FAM111A, and RFC3, restrict replication of orthopoxvirus SPI-1 host-range mutants

Cited by 29 publications

References 54 publications

Gain-of-function mutations amplify cytotoxic FAM111 protease activity in human genetic disorders

Gain-of-function mutations amplify cytotoxic FAM111 protease activity in human genetic disorders

Identification of Vaccinia Virus Replisome and Transcriptome Proteins by Isolation of Proteins on Nascent DNA Coupled with Mass Spectrometry

An Integrated Systems Biology Approach Identifies the Proteasome as a Critical Host Machinery for ZIKV and DENV Replication

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