Human cytomegalovirus (HCMV) is an important pathogen with multiple immune evasion strategies, including virally facilitated degradation of host antiviral restriction factors. Here, we describe a multiplexed approach to discover proteins with innate immune function on the basis of active degradation by the proteasome or lysosome during early-phase HCMV infection. Using three orthogonal proteomic/transcriptomic screens to quantify protein degradation, with high confidence we identified 35 proteins enriched in antiviral restriction factors. A final screen employed a comprehensive panel of viral mutants to predict viral genes that target >250 human proteins. This approach revealed that helicase-like transcription factor (HLTF), a DNA helicase important in DNA repair, potently inhibits early viral gene expression but is rapidly degraded during infection. The functionally unknown HCMV protein UL145 facilitates HLTF degradation by recruiting the Cullin4 E3 ligase complex. Our approach and data will enable further identifications of innate pathways targeted by HCMV and other viruses.

• Klíčová slova
• host-pathogen interaction, immune evasion, innate immunity, lysosome, proteasome, protein degradation, pulsed SILAC, quantitative proteomics, restriction factor, tandem mass tag,
• MeSH
• cytomegalovirové infekce genetika   imunologie   virologie   MeSH
• Cytomegalovirus genetika   imunologie   fyziologie   MeSH
• DNA vazebné proteiny chemie   genetika   imunologie   MeSH
• imunitní únik MeSH
• lidé MeSH
• proteiny chemie   genetika   imunologie   MeSH
• proteomika MeSH
• stabilita proteinů MeSH
• transkripční faktory chemie   genetika   imunologie   MeSH
• virové proteiny chemie   genetika   imunologie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• DNA vazebné proteiny MeSH
• HLTF protein, human MeSH Prohlížeč
• proteiny MeSH
• transkripční faktory MeSH
• virové proteiny MeSH

Successful and accurate completion of the replication of damage-containing DNA requires mainly recombination and RAD18-dependent DNA damage tolerance pathways. RAD18 governs at least two distinct mechanisms: translesion synthesis (TLS) and template switching (TS)-dependent pathways. Whereas TS is mainly error-free, TLS can work in an error-prone manner and, as such, the regulation of these pathways requires tight control to prevent DNA errors and potentially oncogenic transformation and tumorigenesis. In humans, the PCNA-associated recombination inhibitor (PARI) protein has recently been shown to inhibit homologous recombination (HR) events. Here, we describe a biochemical mechanism in which PARI functions as an HR regulator after replication fork stalling and during double-strand break repair. In our reconstituted biochemical system, we show that PARI inhibits DNA repair synthesis during recombination events in a PCNA interaction-dependent way but independently of its UvrD-like helicase domain. In accordance, we demonstrate that PARI inhibits HR in vivo, and its knockdown suppresses the UV sensitivity of RAD18-depleted cells. Our data reveal a novel human regulatory mechanism that limits the extent of HR and represents a new potential target for anticancer therapy.

• MeSH
• aminokyselinové motivy MeSH
• DNA vazebné proteiny chemie   metabolismus   fyziologie   MeSH
• DNA-polymerasa III antagonisté a inhibitory   MeSH
• DNA biosyntéza   MeSH
• HEK293 buňky MeSH
• lidé MeSH
• rekombinační oprava DNA * MeSH
• ubikvitinligasy fyziologie   MeSH
• ultrafialové záření MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• DNA vazebné proteiny MeSH
• DNA-polymerasa III MeSH
• DNA MeSH
• PARPBP protein, human MeSH Prohlížeč
• RAD18 protein, human MeSH Prohlížeč
• ubikvitinligasy MeSH

Stalling of replication forks at unrepaired DNA lesions can result in discontinuities opposite the damage in the newly synthesized DNA strand. Translesion synthesis or facilitating the copy from the newly synthesized strand of the sister duplex by template switching can overcome such discontinuities. During template switch, a new primer-template junction has to be formed and two mechanisms, including replication fork reversal and D-loop formation have been suggested. Genetic evidence indicates a major role for yeast Rad5 in template switch and that both Rad5 and its human orthologue, Helicase-like transcription factor (HLTF), a potential tumour suppressor can facilitate replication fork reversal. This study demonstrates the ability of HLTF and Rad5 to form a D-loop without requiring ATP binding and/or hydrolysis. We also show that this strand-pairing activity is independent of RAD51 in vitro and is not mechanistically related to that of another member of the SWI/SNF family, RAD54. In addition, the 3'-end of the invading strand in the D-loop can serve as a primer and is extended by DNA polymerase. Our data indicate that HLTF is involved in a RAD51-independent D-loop branch of template switch pathway that can promote repair of gaps formed during replication of damaged DNA.

• MeSH
• adenosintrifosfatasy metabolismus   MeSH
• DNA vazebné proteiny MeSH
• DNA-helikasy metabolismus   MeSH
• DNA chemie   metabolismus   MeSH
• forkhead transkripční faktory metabolismus   MeSH
• genetické matrice MeSH
• jaderné proteiny metabolismus   MeSH
• lidé MeSH
• poškození DNA * MeSH
• rekombinasa Rad51 metabolismus   MeSH
• replikace DNA * MeSH
• replikační protein A metabolismus   MeSH
• Saccharomyces cerevisiae - proteiny metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• adenosintrifosfatasy MeSH
• DNA vazebné proteiny MeSH
• DNA-helikasy MeSH
• DNA MeSH
• forkhead transkripční faktory MeSH
• FOXN2 protein, human MeSH Prohlížeč
• jaderné proteiny MeSH
• RAD5 protein, S cerevisiae MeSH Prohlížeč
• RAD54L protein, human MeSH Prohlížeč
• rekombinasa Rad51 MeSH
• replikační protein A MeSH
• Saccharomyces cerevisiae - proteiny MeSH