Intricate Roles of Mammalian Sirtuins in Defense against Viral Pathogens
For a number of years, sirtuin enzymes have been appreciated as effective “sensors” of the cellular environment to rapidly transmit information to diverse cellular pathways. Much effort was placed into exploring their roles in human cancers and aging. However, a growing body of literature brings these enzymes to the spotlight in the field of virology. Here, we discuss sirtuin functions in the context of viral infection, which provide regulatory points for therapeutic intervention against pathogens.
多年来，去乙酰化酶一直被认为是细胞环境的有效“传感器” ，可以将信息快速传递到不同的细胞途径。人们投入了大量精力来探索它们在人类癌症和衰老中的作用。然而，越来越多的文献将这些酶带到了病毒学领域的聚光灯下。在这里，我们讨论 sirtuin 在病毒感染的背景下的功能，这为针对病原体的治疗干预提供了调节点。
The discovery of host proteins that provide defense against viral pathogens is a research area of significant interest, being relevant both for understanding basic mechanisms of host defense and for developing alternative antiviral therapeutics. Given that most currently available antiviral drugs target virally encoded proteins, they tend to be specific for certain types of viral infections and to have their efficacy compromised by drug-resistant viral strains. Recent studies have pointed to mammalian sirtuins (SIRTs) as important defense factors against viruses. As the Escherichia coli sirtuin homolog also functions in defense against bacteriophages (1), studying sirtuin function holds promise to have a broad impact in the field of infectious disease.
发现能够抵御病毒病原体的宿主蛋白是一个重要的研究领域，对于理解宿主防御的基本机制和开发替代性抗病毒疗法都是相关的。鉴于目前大多数可用的抗病毒药物针对病毒编码的蛋白质，它们往往对某些类型的病毒感染具有特异性，并且其疗效受到耐药病毒株的影响。最近的研究指出哺乳动物去乙酰化酶(SIRTs)是抵抗病毒的重要防御因子。由于大肠桿菌 sirtuin 同源序列也具有防御噬菌体的功能，研究 sirtuin 功能有望在传染病领域产生广泛的影响。
The seven mammalian SIRTs (SIRT1 to SIRT7) are ubiquitously expressed in most cells and tissues and are evolutionarily conserved. Sirtuins are NAD+-dependent enzymes, primarily known as lysine deacetylases, and derive their name from the founder of the family, the Saccharomyces cerevisiae transcriptional regulator Sir2 protein. Although their functions remain to be fully characterized, a growing body of literature indicates that SIRTs impact a wide range of cellular pathways. This breadth of function is partly derived from their diverse intracellular localizations, with SIRT1, SIRT6, and SIRT7 being nuclear; SIRT2 being cytoplasmic; and SIRT3, SIRT4, and SIRT5 being predominantly mitochondrial (Fig. 1). Equally important is the still-evolving understanding that, in addition to their deacetylase activity, these proteins can have alternative enzymatic activities, including ADP ribosylation (SIRT1, SIRT4, and SIRT6), desuccinylation and demalonylation (SIRT5), delipoylation (SIRT4), and demyristoylation and depalmitoylation (SIRT6) (reviewed in reference 2). Given these diverse activities and localizations, sirtuins are core regulators of transcription and metabolism. Therefore, sirtuins have the ability to control numerous cellular pathways required throughout the viral life cycle. Here, we highlight recent research on characterizing sirtuin functions during viral infection and place these findings into the broader context of developing improved antiviral therapeutics.
7种哺乳动物 SIRTs (SIRT1 to SIRT7)在大多数细胞和组织中普遍表达，在进化上是保守的。Sirtuins 是 NAD + 依赖的酶，主要被称为赖氨酸脱乙酰酶，它们的名字来自于家族的创始人，酿酒酵母转录调节因子 Sir2蛋白。虽然它们的功能仍然有待完整的描述，但越来越多的文献表明 sirt 影响广泛的细胞通路。这种功能的广度部分来源于它们不同的细胞内定位，其中 SIRT1，SIRT6和 SIRT7是细胞核，SIRT2是细胞质，而 SIRT3，SIRT4和 SIRT5主要是线粒体(图1)。同样重要的是仍在进化的理解，除了它们的去乙酰化酶活性，这些蛋白质可以有替代的酶活性，包括 ADP 核糖基化(SIRT1，SIRT4，和 SIRT6) ，去 uccinylation 和 demalonylation (SIRT5) ，delipylation (SIRT4) ，和 demyristoylation 和 demitioylation (SIRT6)。鉴于这些不同的活动和定位，去乙酰化酶是转录和代谢的核心调节因子。因此，去乙酰化酶能够控制病毒生命周期中所需的许多细胞通路。在这里，我们强调最近的研究表征 sirtuin 功能在病毒感染和地方这些发现进入更广泛的背景下发展改进抗病毒治疗。
FIG 1 图1
Enzymatic activity and subcellular localization of mammalian sirtuins. SIRT1 (deacetylase), SIRT6 (demyristoylase and depalmitoylase), and SIRT7 (deacetylase) are nuclear; SIRT2 (deacetylase and demyristoylase) is predominantly cytoplasmic; SIRT3 (deacetylase), SIRT4 (ADP-ribosyltransferase and lipoamidase), and SIRT5 (desuccinylase and demalonylase) are mitochondrial. In the nucleus, the role of SIRT1 is depicted during HIV and HBV infections. On the right, virus-induced changes to metabolic pathways are shown for several viruses, along with mitochondrial sirtuin substrates that have critical roles in the dysregulated pathways. Ac-CoA, acetyl coenzyme A; PDH, pyruvate dehydrogenase; MDH, malate dehydrogenase; GDH, glutamate dehydrogenase; Lipo, lipoyl; Ac, acetyl; Su, succinyl; AR, ADP-ribose.
哺乳动物去乙酰化酶的酶活性和亚细胞定位。SIRT1(deacetylase)、 SIRT6(demyristoylase and depalmitolase)和 SIRT7(deacetylase)为核，SIRT2(deacetylase and demyristoylase)为胞质，SIRT3(deacetylase)、 SIRT4(adp- 核糖基转移酶和脂肪酰胺酶)和 SIRT5(desuccinamide 和 demyricolase)为线粒体。在细胞核中，SIRT1在 HIV 和 HBV 感染期间的作用被描述。右图显示，病毒诱导的几种病毒代谢途径的变化，以及线粒体去乙酰化酶底物在失调途径中的关键作用。乙酰辅酶A; PDH，丙酮酸脱氢酶; MDH，苹果酸脱氢酶; GDH，谷氨酸脱氢酶; Lipo，脂酰; Ac，乙酰; Su，琥珀酰; AR，adp 核糖。
ROLES OF SIRTUINS IN GENE EXPRESSION DURING VIRAL INFECTION
Sirtuins are known to regulate gene expression primarily by controlling the modification status of histones and transcription factors. In the nucleus, the SIRT1-mediated removal of acetyl groups from lysine residues on histones (H3K9ac, H3K14ac, or H4K16ac) modulates heterochromatin formation, thereby impacting gene expression (reviewed in reference 2). SIRT6 deacetylates H3K9ac, targeting NF-κB-dependent gene expression, while H3K18ac deacetylation by SIRT7 promotes oncogenic transformation. Although primarily cytoplasmic, SIRT2 was shown to regulate DNA compaction through deacetylation of H4K16ac during the G2/M transition. Sirtuins also target important transcription factors, such as p53, NF-κB, and FOXO1, directly affecting the expression of their target genes. Through these actions, sirtuins may be able to impact the outcome of viral infection by modulating both host and viral gene expression. In turn, viruses are known to manipulate host epigenetic and transcription machineries by hijacking SIRT-regulated pathways. This has been well illustrated during infection with human immunodeficiency virus (HIV). SIRT1 was shown to deacetylate the HIV protein Tat, enabling HIV transactivation (3). However, Tat can also inhibit SIRT1, activating NF-κB-responsive gene transcription and promoting CD4+ T-cell hyperactivation that is favorable for viral replication (4, 5). Another elegant mechanism aimed at modulating gene expression is seen with influenza A virus, which has evolved to encode NS1, a protein mimic of histone H3 tail that can suppress transcription of antiviral genes (6). As NS1 can be acetylated and methylated at the lysine site equivalent to the mammalian histone H3K4 (6), it remains to be investigated if these modifications are modulated by sirtuins. Moreover, sirtuin inhibition was shown to increase influenza A virus titers, while activation reduced titers (1).
已知 Sirtuins 主要通过控制组蛋白和转录因子的修饰状态来调节基因表达。在细胞核中，sirt1介导的去除组蛋白上赖氨酸残基(H3K9ac、 H3K14ac 或 H4K16ac)上的乙酰基可以调节异染色质的形成，从而影响基因的表达(参考文献2)。SIRT6去乙酰化 H3K9ac，靶向 nf-κb 依赖基因的表达，而 H3K18ac 去乙酰化通过 SIRT7促进肿瘤转化。虽然 SIRT2主要是细胞质，但是在 g 2/m 转换期间，它通过脱乙酰化 H4K16ac 来调节 DNA 的紧密化。Sirtuins 还靶向重要的转录因子，如 p53、 NF-κB 和 FOXO1，直接影响其靶基因的表达。通过这些作用，去乙酰化酶可能通过调节宿主和病毒基因的表达而影响病毒感染的结果。反过来，我们知道病毒通过劫持 sirt 调节的通路来操纵宿主的表观遗传和转录机制。这在感染人类免疫缺陷病毒病毒(HIV)的过程中得到了很好的说明。SIRT1被证明能够使 HIV 蛋白 Tat 脱乙酰化，从而使 HIV 转录激活(3)。然而，Tat 也可以抑制 SIRT1，激活 nf-κb 反应基因转录，促进 CD4 + t 细胞过度激活，这有利于病毒复制(4,5)。另一个旨在调节基因表达的优雅机制是 a 型流感病毒，该病毒进化到编码 NS1，一种类似组蛋白 H3尾部的蛋白，可以抑制抗病毒基因的转录(6)。由于 NS1可以在赖氨酸位点乙酰化和甲基化，相当于哺乳动物组蛋白 H3K4(6) ，如果这些修饰被去乙酰化酶调控，它仍然有待研究。此外，去乙酰化酶抑制剂可以提高 a 型流感病毒的滴度，而激活可以降低滴度(1)。
Through their modulation of histone modifications on viral nucleosomes, sirtuins can also be critical regulatory hubs during infections with DNA viruses. Chromatin assembly around viral DNA has been documented for several nuclear-replicating viruses, including hepatitis B virus (HBV), human cytomegalovirus (HCMV), varicella-zoster virus (VZV), human papillomavirus (HPV), and herpes simplex virus 1 (HSV-1). As a result, transcription from the viral genome can be regulated by histone modifications. For example, recruitment of SIRT1 onto HBV covalently closed circular DNA (cccDNA) was observed late in infection, coinciding with a decline in viral replication (7).
通过调节病毒核小体上的组蛋白修饰，去乙酰化酶也可以成为 DNA 病毒感染过程中的重要调控枢纽。已有文献报道几种核复制病毒的染色质组装，包括乙型肝炎病毒病毒(HBV)、人巨细胞病毒(HCMV)、水痘-带状疱疹病毒(VZV)、人类乳突病毒病毒(HPV)和单纯疱疹病毒病毒1型(HSV-1)。因此，病毒基因组的转录可以被组蛋白修饰所调控。例如，在感染后期观察到 SIRT1在 HBV 共价闭合环状 DNA (cccDNA)上的补充，与病毒复制下降相吻合(7)。
Histone modifications on viral nucleosomes are also finely tuned in a temporal manner during HCMV infection. HCMV nucleosomal structures were shown to have elevated transcriptionally active marks late in infection compared to prominent repressive patterns early in infection. Additionally, H4K16 acetylation, associated with cell cycle progression and transcriptional repression, and a known substrate of SIRT1 and SIRT2, decreased during HCMV infection (8). Reduction in the levels of these SIRTs was reported to trigger increased HCMV yields (1). Because sirtuins can deacetylate histones on both viral and host chromatin, their effect on viral replication needs further exploration from the intertwined perspectives of the virus and host. As an example of viral genome regulation, reactivation of Kaposi’s sarcoma-associated herpesvirus (KSHV) was observed upon treatment with sirtuin inhibitors; this increased active (H3K4me3) and decreased repressive (H3K27me3) histone marks at the viral transcription activator promoter (9).
病毒核小体上的组蛋白修饰在 HCMV 感染过程中也以时间的方式进行微调。HCMV 核小体结构在感染后期表现为转录活性增高，而在感染早期表现为明显的抑制模式。此外，H4K16乙酰化与细胞周期进程和转录阻遏，以及一个已知的底物 SIRT1和 SIRT2，减少 HCMV 感染(8)。减少这些水平的 sirt 被报道触发 HCMV 产量增加(1)。由于去乙酰化去乙酰化组蛋白可以同时作用于病毒和宿主染色质，其对病毒复制的影响需要从病毒和宿主的角度进一步探讨。作为病毒基因组调控的一个例子，卡波济肉瘤相关疱疹病毒(KSHV)在用去乙酰化酶抑制剂治疗时被观察到重新激活，在病毒转录激活剂启动子(9)处出现活跃(H3K4me3)和抑制(H3K27me3)组蛋白标记。
Along with chromatin regulation, DNA viruses can modulate the activity of various host transcription factors through manipulation of sirtuin levels and their interactions. For example, HSV-1 inhibits apoptosis to promote neuronal cell survival by modulating SIRT1 interaction and colocalization with p53 (10). Since multiple deacetylases can target important transcription factors, it remains to be determined whether sirtuins’ roles are redundant or dependent on specific conditions, such as cell type. From the host perspective, sirtuins may represent a first line of defense against viral infection. SIRT1 was demonstrated to promote sumoylation and stabilization of PML in vesicular stomatitis virus (VSV)-infected cells, which explains the increase in VSV titers upon SIRT1 knockout (11).
除了染色质调控外，DNA 病毒还可以通过调控去乙酰化酶水平及其相互作用来调控宿主转录因子的活性。例如，HSV-1通过调节 SIRT1的相互作用和与 p53(10)的共定位来抑制凋亡以促进神经细胞的存活。由于多个去乙酰化酶可以靶向重要的转录因子，因此去乙酰化酶的作用是多余的还是依赖于特定的条件，如细胞类型，还有待确定。从宿主的角度来看，去乙酰化酶可能是抵抗病毒感染的第一道防线。SIRT1被证明能促进感染水疱性口膜炎病毒的细胞中 PML 的 sumo 化和稳定化，这解释了 SIRT1基因敲除后 VSV 滴度的增加。
The diversity and abundance of sirtuin substrates, which include both histone and nonhistone proteins, introduce complexity to the interpretation of their functions during infection. This illustrates the need for further insight into the spatial and temporal sirtuin-mediated events during infection, helping to define mechanisms through which hosts and viruses battle for control over sirtuin functions to either inhibit or promote viral replication.
去乙酰化酶底物的多样性和丰富性，包括组蛋白和非组蛋白，为解释它们在感染过程中的功能带来了复杂性。这说明需要进一步深入了解感染过程中 sirtuin 介导的空间和时间事件，帮助确定宿主和病毒通过何种机制争夺对 sirtuin 功能的控制，以抑制或促进病毒复制。
SIRTUIN REGULATION OF HOST CELL METABOLISM DURING VIRAL INFECTION
Among the human sirtuins, the three mitochondrial SIRTs (SIRT3, SIRT4, and SIRT5) act as sensors of their environment to regulate essential metabolic pathways, many of which are known to be altered during viral infection. SIRT3 is a major mitochondrial deacetylase with numerous substrates, having the ability to impact a wide range of critical metabolic processes, such as fatty acid oxidation and the tricarboxylic acid (TCA) cycle (12). SIRT4 was identified as an ADP-ribosyltransferase (13) and more recently as a lipoamidase that inhibits the pyruvate dehydrogenase complex (14). In addition to acting as a weak deacetylase, SIRT5 was characterized as a desuccinylase and demalonylase, regulating many metabolic pathways, including the urea cycle via its substrate CPS1 (15, 16). Although the investigations of mitochondrial sirtuin functions during viral infection have only recently emerged and are still limited, the interface between SIRT functions, dynamic changes in metabolism, and viral infection holds significant promise to understanding both the biology and pathogenicity of infections. Indeed, small interfering RNA (siRNA)-mediated knockdown of each mitochondrial sirtuin was reported to lead to increased viral titers for several DNA viruses (1). The observed variation in the magnitude of the viral titers is likely indicative of the different metabolic needs of the tested viruses and the specific sirtuin regulation of those pathways.
One of the metabolic signatures of viral infection is the increase in glucose uptake and glycolytic flux. Using mass spectrometry, an elegant example was shown during infection with HCMV, where the efflux of additional carbon is shuttled through the TCA cycle to increase lipid biosynthesis, required for the envelopment of infectious virions (17, 18). Influenza virus infection is also known to increase glycolytic flux at 12 h postinfection, the point of virus release and onset of virus-mediated apoptosis, which is necessary for viral replication (19). Highlighting the global importance of this metabolic pathway during infection, dengue virus infection was also shown to increase glycolytic flux and nucleotide synthesis, both critical for viral replication (20). Sirtuins are optimally positioned to regulate enzymes of the glycolytic and TCA cycle pathways, including the pyruvate dehydrogenase complex under the control of SIRT3, SIRT4, and SIRT5 (12, 14, 16). Malate dehydrogenase in the TCA cycle is a substrate of both SIRT3 and SIRT5 for deacetylation and desuccinylation, respectively, pointing to possible sirtuin cross talk and fine-tuned regulation of metabolic processes (12, 15). Another metabolic reprogramming signature of infection is the anapleurotic influx into the TCA cycle, requiring glutamate dehydrogenase, a substrate of both SIRT4 and SIRT5 (13, 15, 17, 18). This need for glutamine as a carbon source for the TCA cycle was reported for both HCMV and vaccinia virus infections (17, 18, 20).
Given these findings regarding sirtuin functions and virus-induced metabolic changes, better understanding of viral metabolic signatures promises to aid the development of improved antiviral therapies. These therapies will likely need to be tailored to each virus. For example, even closely related viruses, such as the herpesviruses HCMV and HSV-1, trigger different metabolic changes. This knowledge was essential for the understanding that nucleotide analogs, but not fatty acid synthesis inhibitors, are effective against HSV-1 but not HCMV and vice versa (18). Even more nuanced, this metabolic reprogramming can vary by cell type. In a metabolomics study of HIV-1 infection in CD4+ T cells and model macrophages, increased glucose uptake and glycolytic flux were observed only in CD4+ T cells, while macrophages had striking reductions in both uptake and flux (21). These studies demonstrate that within one virus family, or even one type of viral infection, divergent metabolic responses can arise, illustrating the need for tailored antiviral therapeutics. Sirtuins are promising targets for this because of their specific connection to numerous metabolic processes impacted during infection.
鉴于这些关于 sirtuin 功能和病毒引起的代谢变化的发现，更好地了解病毒代谢特征有助于改进抗病毒疗法的开发。这些疗法可能需要针对每种病毒量身定做。例如，即使是与之密切相关的病毒，如人类疱疹病毒(HCMV)和 HSV-1，也会引发不同的代谢变化。这些知识对于了解核苷酸类似物，而不是脂肪酸合成抑制剂，对 HSV-1有效，但对 HCMV 无效，反之亦然。更微妙的是，这种新陈代谢的重编程可能因细胞类型而异。在 CD4 + t 细胞和模型巨噬细胞感染 HIV-1的代谢组学研究中，只有 CD4 + t 细胞的葡萄糖摄取和糖酵解通量增加，而巨噬细胞的摄取和通量都显著减少(21)。这些研究表明，在一个病毒家族中，甚至在一种病毒感染中，都可能产生不同的代谢反应，这说明需要量身定制的抗病毒治疗。去乙酰化酶是很有希望的目标，因为它们与感染期间影响的许多代谢过程有特定的联系。
In summary, sirtuins offer a remarkably rich diversity of regulatory points. Their NAD+-dependent activities allow them to transmit information about changes in the environment to major cellular pathways for rapid and effective responses. With the discovery that all sirtuins can impact the replication of DNA and RNA viruses, we expect to learn more about their universal roles in the immune response, the stress response, and other types of defense responses utilized by an organism to prevent disease caused by pathogens. Therefore, being able to target sirtuins provides a valuable antiviral therapeutic strategy. Maybe the simplest way to control this family of enzymes is through regulation of NAD+ levels. It is possible that certain viruses have already acquired this capacity; for example, HSV-1 infection leads to reduced NAD+ levels, likely inhibiting sirtuins (18). However, simply regulating NAD+ levels is probably not preferred. From a host defense and clinical perspective, the simultaneous regulation of all sirtuins could lead to global stress responses and cytotoxicity. Additionally, from a virus perspective, the transcriptional functions of certain sirtuins appear necessary for the regulation of viral gene expression. So, the fine-tuning of sirtuin regulation at the level of one enzyme or one given enzyme function may be the more promising way forward to take advantage of sirtuin defense properties. However, to achieve this level of precision in sirtuin regulation, it is critical to first understand the basic mechanisms for their housekeeping and antiviral functions. The constantly evolving knowledge regarding the sirtuin catalytic activities indicates that there is still a way to go before fully understanding their repertoire of functions. The integration of molecular and cellular biology with modern proteomic and metabolomic approaches can provide mechanistic insights into sirtuin functions. There is great value in future studies aimed at defining sirtuin substrates in the context of infection and the unique epigenetic and metabolic reprogramming by different viruses. To bridge these findings with clinical applications, such studies would benefit from considering sirtuin cross talk and roles in other human diseases, including cancers. Furthermore, as sirtuins may impact the susceptibility of a host to viral or bacterial infections, it will be informative to further investigate their roles during superinfection or infection with opportunistic pathogens.
总之，去乙酰化酶提供了非常丰富的调节点。它们依赖 NAD + 的活动使它们能够将环境变化的信息传递给主要的细胞通路，以便迅速有效地作出反应。随着发现所有去乙酰化酶都能影响 DNA 和 RNA 病毒的复制，我们希望更多地了解它们在免疫反应、应激反应以及其他类型的防御反应中的普遍作用，这些反应是有机体用来防止病原体引起的疾病的。因此，靶向去乙酰化酶提供了一种有价值的抗病毒治疗策略。也许控制这一系列酶最简单的方法就是调节 NAD + 的水平。有可能某些病毒已经获得了这种能力; 例如，HSV-1感染导致 NAD + 水平降低，可能抑制了去乙酰化酶(18)。然而，简单地调节 NAD + 水平可能并不是首选。从宿主防御和临床的角度来看，所有去乙酰化酶同时调节可能导致整体应激反应和细胞毒性。此外，从病毒的角度来看，某些去乙酰化酶的转录功能对于调节病毒基因的表达是必要的。因此，在一种酶或一种给定的酶功能水平上微调去乙酰化酶的调节可能是更有希望的利用去乙酰化酶防御特性的方法。然而，要达到这种水平的 sirtuin 调节的精确度，首先必须了解其内务和抗病毒功能的基本机制。关于去乙酰化酶催化活动的不断发展的知识表明，要充分了解它们的功能还有很长的路要走。分子和细胞生物学与现代蛋白质组学和代谢组学方法的结合可以为去乙酰化酶的功能提供机制性的见解。在定义去乙酰化酶基因在感染和不同病毒独特的表观遗传和代谢重编程方面的研究具有很大的价值。为了将这些发现与临床应用联系起来，考虑 sirtuin 相声和其他人类疾病，包括癌症中的作用，这些研究将受益匪浅。此外，由于去乙酰化酶可能影响宿主对病毒或细菌感染的敏感性，进一步研究它们在重复感染或机会致病菌感染中的作用将是有益的。