Sirtuins Are Evolutionarily Conserved Viral Restriction Factors

ABSTRACT The seven human sirtuins are a family of ubiquitously expressed and evolutionarily conserved NAD+-dependent deacylases/mono-ADP ribosyltransferases that regulate numerous cellular and organismal functions, including metabolism, cell cycle, and longevity. Here, we report the discovery that all seven sirtuins have broad-range antiviral properties. We demonstrate that small interfering RNA (siRNA)-mediated knockdown of individual sirtuins and drug-mediated inhibition of sirtuin enzymatic activity increase the production of virus progeny in infected human cells. This impact on virus growth is observed for both DNA and RNA viruses. Importantly, sirtuin-activating drugs inhibit the replication of diverse viruses, as we demonstrate for human cytomegalovirus, a slowly replicating DNA virus, and influenza A (H1N1) virus, an RNA virus that multiplies rapidly. Furthermore, sirtuin defense functions are evolutionarily conserved, since CobB, the sirtuin homologue in Escherichia coli, protects against bacteriophages. Altogether, our findings establish sirtuins as broad-spectrum and evolutionarily conserved components of the immune defense system, providing a framework for elucidating a new set of host cell defense mechanisms and developing sirtuin modulators with antiviral activity. IMPORTANCE We live in a sea of viruses, some of which are human pathogens. These pathogenic viruses exhibit numerous differences: DNA or RNA genomes, enveloped or naked virions, nuclear or cytoplasmic replication, diverse disease symptoms, etc. Most antiviral drugs target specific viral proteins. Consequently, they often work for only one virus, and their efficacy can be compromised by the rapid evolution of resistant variants. There is a need for the identification of host proteins with broad-spectrum antiviral functions, which provide effective targets for therapeutic treatments that limit the evolution of viral resistance. Here, we report that sirtuins present such an opportunity for the development of broad-spectrum antiviral treatments, since our findings highlight these enzymes as ancient defense factors that protect against a variety of viral pathogens. We live in a sea of viruses, some of which are human pathogens. These pathogenic viruses exhibit numerous differences: DNA or RNA genomes, enveloped or naked virions, nuclear or cytoplasmic replication, diverse disease symptoms, etc. Most antiviral drugs target specific viral proteins. Consequently, they often work for only one virus, and their efficacy can be compromised by the rapid evolution of resistant variants. There is a need for the identification of host proteins with broad-spectrum antiviral functions, which provide effective targets for therapeutic treatments that limit the evolution of viral resistance. Here, we report that sirtuins present such an opportunity for the development of broad-spectrum antiviral treatments, since our findings highlight these enzymes as ancient defense factors that protect against a variety of viral pathogens.

[1]  Yi Zhang,et al.  The First Identification of Lysine Malonylation Substrates and Its Regulatory Enzyme* , 2011, Molecular & Cellular Proteomics.

[2]  J. Baur,et al.  Are sirtuins viable targets for improving healthspan and lifespan? , 2012, Nature Reviews Drug Discovery.

[3]  B. Wanner,et al.  One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[4]  W. Munyon,et al.  Temperature-sensitive mutants of herpes simplex virus type 1 defective in lysis but not in transformation , 1975, Journal of virology.

[5]  Sarah L. Grady,et al.  Divergent Effects of Human Cytomegalovirus and Herpes Simplex Virus-1 on Cellular Metabolism , 2011, PLoS pathogens.

[6]  David Sinclair,et al.  Sirtuins in mammals: insights into their biological function. , 2007, The Biochemical journal.

[7]  R. Mostoslavsky,et al.  Sirtuins, metabolism, and DNA repair. , 2014, Current opinion in genetics & development.

[8]  S. S. Koh,et al.  Hepatitis B virus X (HBX) protein upregulates β-catenin in a human hepatic cell line by sequestering SIRT1 deacetylase. , 2012, Oncology reports.

[9]  J. Drake,et al.  The Bacteriophage T4 Rapid-Lysis Genes and Their Mutational Proclivities , 2011, Journal of bacteriology.

[10]  H. Mori,et al.  Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection , 2006, Molecular systems biology.

[11]  G. Kochs,et al.  Interferon, Mx, and viral countermeasures , 2007, Cytokine & Growth Factor Reviews.

[12]  J. Hoffmann,et al.  The Drosophila systemic immune response: sensing and signalling during bacterial and fungal infections , 2007, Nature Reviews Immunology.

[13]  Xiaoling Xu,et al.  Hepatic-specific disruption of SIRT6 in mice results in fatty liver formation due to enhanced glycolysis and triglyceride synthesis. , 2010, Cell metabolism.

[14]  Thomas Shenk,et al.  Dynamics of the Cellular Metabolome during Human Cytomegalovirus Infection , 2006, PLoS pathogens.

[15]  René Thomas,et al.  Genetics and physiology of defective lysogeny in K12 (λ): Studies of early mutants☆ , 1966 .

[16]  Qing Xu,et al.  Hepatocyte-specific deletion of SIRT1 alters fatty acid metabolism and results in hepatic steatosis and inflammation. , 2009, Cell metabolism.

[17]  M. Levrero,et al.  Nuclear HBx binds the HBV minichromosome and modifies the epigenetic regulation of cccDNA function , 2009, Proceedings of the National Academy of Sciences.

[18]  A. Levine,et al.  Monoclonal antibodies which recognize native and denatured forms of the adenovirus DNA-binding protein. , 1983, Virology.

[19]  T. Shenk,et al.  UL26-Deficient Human Cytomegalovirus Produces Virions with Hypophosphorylated pp28 Tegument Protein That Is Unstable within Newly Infected Cells , 2006, Journal of Virology.

[20]  Ileana M Cristea,et al.  Sirtuin 7 Plays a Role in Ribosome Biogenesis and Protein Synthesis* , 2013, Molecular & Cellular Proteomics.

[21]  S. Showalter,et al.  Monoclonal antibodies to herpes simplex virus type 1 proteins, including the immediate-early protein ICP 4 , 1981, Infection and immunity.

[22]  Alexander Varvak,et al.  Multiple regulatory layers of SREBP1/2 by SIRT6. , 2013, Cell reports.

[23]  J. Denu,et al.  Regulation of Glycolytic Enzyme Phosphoglycerate Mutase-1 by Sirt1 Protein-mediated Deacetylation♦ , 2011, The Journal of Biological Chemistry.

[24]  P. Distefano,et al.  Inhibition of SIRT1 Catalytic Activity Increases p53 Acetylation but Does Not Alter Cell Survival following DNA Damage , 2006, Molecular and Cellular Biology.

[25]  I. Cristea,et al.  Increased expression of LDL receptor-related protein 1 during human cytomegalovirus infection reduces virion cholesterol and infectivity. , 2012, Cell host & microbe.

[26]  R. Mostoslavsky,et al.  Sirt6 Regulates Tnf-Alpha Secretion Through Hydrolysis of Long-Chain Fatty Acyl Lysine , 2013 .

[27]  R. Roberts,et al.  The evolution of the type I interferons. , 1998, Journal of interferon & cytokine research : the official journal of the International Society for Interferon and Cytokine Research.

[28]  David Y. Huang,et al.  3,4',5-Trihydroxy-trans-stilbene (resveratrol) inhibits human cytomegalovirus replication and virus-induced cellular signaling. , 2004, Antiviral research.

[29]  W. Britt Manifestations of human cytomegalovirus infection: proposed mechanisms of acute and chronic disease. , 2008, Current topics in microbiology and immunology.

[30]  Shou-Jiang Gao,et al.  Activation of Kaposi's Sarcoma-Associated Herpesvirus (KSHV) by Inhibitors of Class III Histone Deacetylases: Identification of Sirtuin 1 as a Regulator of the KSHV Life Cycle , 2014, Journal of Virology.

[31]  A. Poulsen,et al.  SIRT1 Modulating Compounds from High-Throughput Screening as Anti-Inflammatory and Insulin-Sensitizing Agents , 2006, Journal of biomolecular screening.

[32]  D. Sinclair,et al.  Mammalian sirtuins: biological insights and disease relevance. , 2010, Annual review of pathology.

[33]  W. Greene,et al.  Human immunodeficiency virus type 1 Tat protein inhibits the SIRT1 deacetylase and induces T cell hyperactivation. , 2008, Cell host & microbe.

[34]  L. Marraffini,et al.  CRISPR interference: RNA-directed adaptive immunity in bacteria and archaea , 2010, Nature Reviews Genetics.

[35]  R. Roberts,et al.  The Evolution of the Type I Interferons1 , 1998 .

[36]  Qibin Zhang,et al.  Temporal Proteome and Lipidome Profiles Reveal Hepatitis C Virus-Associated Reprogramming of Hepatocellular Metabolism and Bioenergetics , 2010, PLoS pathogens.

[37]  A. Seluanov,et al.  SIRT6 Promotes DNA Repair Under Stress by Activating PARP1 , 2011, Science.

[38]  Izumi Horikawa,et al.  Evolutionarily conserved and nonconserved cellular localizations and functions of human SIRT proteins. , 2005, Molecular biology of the cell.

[39]  Orian S. Shirihai,et al.  The Histone Deacetylase Sirt6 Regulates Glucose Homeostasis via Hif1α , 2010, Cell.

[40]  D. Sharp,et al.  Isolation and Characterization of Influenza A Virus (PR8 Strain) , 1943, The Journal of Immunology.

[41]  Frank Fischer,et al.  An acetylome peptide microarray reveals specificities and deacetylation substrates for all human sirtuin isoforms , 2013, Nature Communications.

[42]  Robert V Farese,et al.  SIRT 3 regulates mitochondrial fatty-acid oxidation by reversible enzyme deacetylation , 2010 .

[43]  T. Shenk,et al.  Human cytomegalovirus IE1 and IE2 proteins block apoptosis. , 1995, Journal of virology.

[44]  L. Nencioni,et al.  Inhibition of influenza A virus replication by resveratrol. , 2005, The Journal of infectious diseases.

[45]  Johan Auwerx,et al.  Sirt5 Is a NAD-Dependent Protein Lysine Demalonylase and Desuccinylase , 2011, Science.

[46]  Yi Wang,et al.  Sirt6 regulates TNFα secretion via hydrolysis of long chain fatty acyl lysine , 2013, Nature.

[47]  Angelika Pedal,et al.  SIRT1 Regulates HIV Transcription via Tat Deacetylation , 2005, PLoS biology.

[48]  M. Casadaban,et al.  Transposition and fusion of the lac genes to selected promoters in Escherichia coli using bacteriophage lambda and Mu. , 1976, Journal of molecular biology.

[49]  Juan Chen,et al.  Sirtuin 1 Regulates Hepatitis B Virus Transcription and Replication by Targeting Transcription Factor AP-1 , 2013, Journal of Virology.

[50]  N. Jones,et al.  Isolation of deletion and substitution mutants of adenovirus type 5 , 1978, Cell.

[51]  F. Jacob,et al.  Genetics and physiology of defective lysogeny in K12 (lambda): studies of early mutants. , 1966, Virology.

[52]  Xiao-Jiang Feng,et al.  Systems-level metabolic flux profiling identifies fatty acid synthesis as a target for antiviral therapy , 2008, Nature Biotechnology.

[53]  I. Cristea,et al.  A Targeted Spatial-Temporal Proteomics Approach Implicates Multiple Cellular Trafficking Pathways in Human Cytomegalovirus Virion Maturation* , 2009, Molecular & Cellular Proteomics.

[54]  B. Cullen,et al.  Is RNA interference a physiologically relevant innate antiviral immune response in mammals? , 2013, Cell host & microbe.

[55]  I. Wang,et al.  Lysis Timing and Bacteriophage Fitness , 2006, Genetics.

[56]  R. Marmorstein,et al.  Structure and substrate binding properties of cobB, a Sir2 homolog protein deacetylase from Escherichia coli. , 2004, Journal of molecular biology.

[57]  C. Hagemeier,et al.  Inhibition of human cytomegalovirus replication by small interfering RNAs. , 2004, The Journal of general virology.

[58]  F. Alt,et al.  SIRT4 Inhibits Glutamate Dehydrogenase and Opposes the Effects of Calorie Restriction in Pancreatic β Cells , 2006, Cell.

[59]  P. Puigserver,et al.  Conserved role of SIRT1 orthologs in fasting-dependent inhibition of the lipid/cholesterol regulator SREBP. , 2010, Genes & development.

[60]  H. Mori,et al.  Complete set of ORF clones of Escherichia coli ASKA library (a complete set of E. coli K-12 ORF archive): unique resources for biological research. , 2006, DNA research : an international journal for rapid publication of reports on genes and genomes.

[61]  D. Herranz,et al.  SIRT1 stabilizes PML promoting its sumoylation , 2011, Cell Death and Differentiation.

[62]  J. Rabinowitz,et al.  Saturated Very Long Chain Fatty Acids Are Required for the Production of Infectious Human Cytomegalovirus Progeny , 2013, PLoS pathogens.

[63]  L. Guarente,et al.  SIRT1 deacetylates and positively regulates the nuclear receptor LXR. , 2007, Molecular cell.

[64]  Oksana Gavrilova,et al.  SIRT6 Deficiency Results in Severe Hypoglycemia by Enhancing Both Basal and Insulin-stimulated Glucose Uptake in Mice* , 2010, The Journal of Biological Chemistry.

[65]  Eric Verdin,et al.  Mammalian Sir2 Homolog SIRT3 Regulates Global Mitochondrial Lysine Acetylation , 2007, Molecular and Cellular Biology.