Herpes Simplex Viruses: Mechanisms of DNA Replication

Herpes simplex virus (HSV) encodes seven proteins necessary for viral DNA synthesis—UL9 (origin-binding protein), ICP8 (single-strand DNA [ssDNA]-binding protein), UL30/UL42 (polymerase), and UL5/UL8/UL52 (helicase/primase). It is our intention to provide an up-to-date analysis of our understanding of the structures of these replication proteins and how they function during HSV replication. The potential roles of host repair and recombination proteins will also be discussed.The Herpesviridae are a large family of double-stranded DNA viruses responsible for many human and veterinary diseases. Although members of this family differ in tissue tropism and many aspects of their interactions with their hosts, the mechanisms by which they replicate their DNA during productive (“lytic”) infection are largely conserved. The molecular mechanisms involved in herpesvirus DNA replication and its regulation are of interest as they provide important models for the study of eukaryotic DNA replication. Many of the replication proteins encoded by herpesviruses represent functional analogs of the eukaryotic DNA replication machinery, with informative similarities and differences. In addition, viral enzymes involved in DNA replication have provided a rich store of useful targets for antiviral therapy. This work will focus primarily on DNA replication of herpes simplex virus 1 and 2 (HSV-1 and HSV-2), but will refer, on occasion, to findings from other herpesviruses. Because this work is intended to update the work on HSV DNA replication written for the previous edition of DNA Replication and Human Disease (Weller and Coen 2006), it draws primarily from work published during the last 6 years. Topics covered in detail in the previous work by the authors (Weller and Coen 2006) and reviewed elsewhere will be summarized only briefly (see Coen 2009; Weller 2010; Livingston and Kyratsous 2011; Ward and Weller 2011; Weitzman and Weller 2011).     

Cidofovir Inhibits Genome Encapsidation and Affects Morphogenesis during the Replication of Vaccinia Virus

Cidofovir (CDV) is one of the most effective antiorthopoxvirus drugs, and it is widely accepted that viral DNA replication is the main target of its activity. In the present study, we report a detailed analysis of CDV effects on the replicative cycles of distinct vaccinia virus (VACV) strains: Cantagalo virus, VACV-IOC, and VACV-WR. We show that despite the approximately 90% inhibition of production of virus progeny, virus DNA accumulation was reduced only 30%, and late gene expression and genome resolution were unaltered. The level of proteolytic cleavage of the major core proteins was diminished in CDV-treated cells. Electron microscopic analysis of virus-infected cells in the presence of CDV revealed reductions as great as 3.5-fold in the number of mature forms of virus particles, along with a 3.2-fold increase in the number of spherical immature particles. A detailed analysis of purified virions recovered from CDV-treated cells demonstrated the accumulation of unprocessed p4a and p4b and nearly 67% inhibition of DNA encapsidation. However, these effects of CDV on virus morphogenesis resulted from a primary effect on virus DNA synthesis, which led to later defects in genome encapsidation and virus assembly. Analysis of virus DNA by atomic force microscopy revealed that viral cytoplasmic DNA synthesized in the presence of CDV had an altered structure, forming aggregates with increased strand overlapping not observed in the absence of the drug. These aberrant DNA aggregations were not encapsidated into virus particles.Vaccinia virus (VACV) is the prototypical member of the Poxviridae, a family of large DNA-containing viruses. During infection, a cascade of temporally regulated viral gene expression occurs exclusively in the cell cytoplasm, where viral DNA replication takes place. DNA replication is essential for the onset of the intermediate and late steps of viral gene expression (37). VACV morphogenesis is a complex process that starts within the virus factories, or virosomes, in parallel with the late stage of gene expression. Crescent-shaped virus membranes evolve into immature spherical particles (IV) that subsequently progress to form brick-shaped mature virions (MV) (reviewed in reference 8). Virus assembly and maturation are complex processes requiring telomere resolution of newly replicated DNA (20, 36, 40), genome encapsidation (6, 22, 50), and the proteolytic processing of major structural proteins (38, 51). For the past decade, several reports have analyzed in detail these numerous steps of VACV morphogenesis, unraveling the role of distinct virus late proteins in the progression of viral particle formation (reviewed in reference 8).Cantagalo virus (CTGV) is a strain of VACV isolated from pustular lesions on cows in Brazil (10). Similar outbreaks of vaccinia-like viruses have been reported frequently over the past 8 years (14, 39, 48). Interestingly, the majority of these vaccinia viruses circulating in the wild in Brazil bear a striking similarity to the Brazilian vaccine strain used for systematic vaccination during the eradication campaign, which was produced in Rio de Janeiro (19) and called strain IOC (10). This similarity raises the interesting possibility that the circulating vaccinia viruses represent feral derivatives of IOC or of a closely related ancestor. Little is known about the sensitivity of these novel vaccinia viruses to antiviral compounds. In the absence of an active smallpox vaccination campaign, the spread of these vaccinia viruses in the wild, the prevalence of cowpox infections in Europe and elsewhere (39), and the occurrence of complications from smallpox vaccination (52) make the need for effective antipoxvirus treatment a worldwide concern.Cidofovir (CDV), an acyclic pyrimidine phosphonate analogue, has shown a potent antiviral effect on several poxvirus infections (4, 15, 44, 46). Recently, we have reported the efficacy of CDV in inhibiting the replication of the Brazilian VACV strains CTGV and IOC (26). The mechanism of action of CDV on reactions catalyzed by the VACV DNA polymerase has been studied in vitro. CDV is not a chain-terminating analogue but drastically slows chain extension and inhibits the 3′-5′ exonuclease proofreading activity of the enzyme (34). In addition, templates containing CDV cause inhibition of DNA elongation (33). Differences between the effects of CDV on human cytomegalovirus (55) and VACV enzymes have been observed, but overall it has been widely accepted that CDV acts by inhibiting the process of virus DNA replication. Moreover, most CDV-resistant VACV strains contain mutations in the catalytic domain or in the 3′-5′ exonuclease domain of the DNA polymerase (2, 5, 28, 45).Despite the consensus regarding mechanisms of action, the effects of CDV on the stages of the VACV replicative cycle have never been analyzed. We report here that although CDV led to approximately 90% inhibition of VACV progeny production, we observed only 30% inhibition of DNA replication and normal levels of postreplicative virus gene expression. However, the encapsidation of DNA into virus particles and the proteolytic processing of the major core proteins were inhibited in CDV-treated cells, leading to an impairment of virus morphogenesis. These effects on virus assembly are an indirect result of a primary effect of CDV on VACV DNA synthesis. Atomic force microscopy (AFM) analysis revealed that virus DNA isolated from the cytoplasm of CDV-treated cells formed aggregates of highly entangled and intertwined DNA molecules that were not observed in cytoplasmic viral DNA isolated from untreated cells. In addition, these DNA aggregates were not detected in encapsidated virus genomes isolated from particles purified from untreated or CDV-treated cells. Our data suggest that incorporation of CDV into VACV DNA during the replication process may lead to aberrant DNA structures, which are less able to be packaged into virus particles.     

Replicating Damaged DNA in Eukaryotes

DNA damage is one of many possible perturbations that challenge the mechanisms that preserve genetic stability during the copying of the eukaryotic genome in S phase. This short review provides, in the first part, a general introduction to the topic and an overview of checkpoint responses. In the second part, the mechanisms of error-free tolerance in response to fork-arresting DNA damage will be discussed in some detail.Before eukaryotic cells divide, the successful completion of DNA replication during S phase is essential to preserve genomic integrity from one generation to the next. During this process, the replication apparatus traverses in the form of bidirectionally moving forks to synthesize new daughter strands. Cells use several means to ensure faithful copying of the parental strands—first, by means of regulatory mechanisms a correctly coordinated replication apparatus is established, and second, a high degree of fidelity during DNA synthesis is maintained by replicative polymerases (Kunkel and Bebenek 2000; Reha-Krantz 2010). However, under several stressful circumstances, endogenously or exogenously induced, the replication apparatus can stall (Tourriere and Pasero 2007). Mostly, structural deformations in the form of lesions or special template-specific features arrest the replication process, activate checkpoint pathways and set in motion repair or tolerance mechanisms to counter the stalling (Branzei and Foiani 2009; Zegerman and Diffley 2009). Basic replication mechanism, its regulatory pathways and means to tolerate DNA damage are largely conserved across eukaryotic species (Branzei and Foiani 2010; Yao and O’Donnell 2010). Understanding the mechanisms involved may enable therapeutic intervention to several human conditions arising from an incomplete replication or from the inability to tolerate perturbations (Ciccia et al. 2009; Preston et al. 2010; Abbas et al. 2013). Enhanced replication stress has also been commonly identified in precancerous lesions, and the inactivation of checkpoint responses coping with this presumably oncogene-induced condition is considered necessary to establish the fully malignant phenotype (Bartkova et al. 2005; Negrini et al. 2010).It is not possible to treat this topic in a comprehensive manner in the allotted space; the reader is referred to excellent recent reviews for more details (Branzei and Foiani 2010; Jones and Petermann 2012). We will attempt to provide an overview of the various strategies that a eukaryotic cell invokes to avoid problems caused by replication stress related to DNA damage and, if problems arise, to tolerate damage without endangering the entire process of genome duplication. In this context, we will only give a brief outline of checkpoint responses that are discussed in more detail in Sirbu and Cortez (2013) and Marechal and Zou (2013). Also, a detailed discussion of translesion synthesis can be reviewed in Sale (2013).     

Dormant Origins,the Licensing Checkpoint,and the Response to Replicative Stresses

Only ∼10% of replication origins that are licensed by loading minichromosome maintenance 2-7 (MCM2-7) complexes are normally used, with the majority remaining dormant. If replication fork progression is inhibited, nearby dormant origins initiate to ensure that all of the chromosomal DNA is replicated. At the same time, DNA damage-response kinases are activated, which preferentially suppress the assembly of new replication factories. This diverts initiation events away from completely new areas of the genome toward regions experiencing replicative stress. Mice hypomorphic for MCM2-7, which activate fewer dormant origins in response to replication inhibition, are cancer-prone and are genetically unstable. The licensing checkpoint delays entry into S phase if an insufficient number of origins have been licensed. In contrast, humans with Meier-Gorlin syndrome have mutations in pre-RC proteins and show defects in cell proliferation that may be a consequence of chronic activation of the licensing checkpoint.Replicating the large amount of DNA in eukaryotic cells is a complex task, requiring the activation of hundreds or thousands of origins spread throughout the genome. To maintain genetic stability, it is essential that during S phase genomic DNA is precisely duplicated, with no sections of DNA left unreplicated and no section of DNA replicated more than once. To prevent re-replication, cells divide the process of DNA replication into two non-overlapping phases. Prior to S phase, origins are licensed by the binding of minichromosome maintenance 2-7 (MCM2-7) double hexamers (Gillespie et al. 2001; Blow and Dutta 2005; Arias and Walter 2007). During S phase, these are activated as the core of the CMG (Cdc45-MCM-GINS) replicative helicase (Moyer et al. 2006; Ilves et al. 2010). Prior to the onset of S phase, licensing proteins are down-regulated or inhibited, so that no more origins can be licensed (Wohlschlegel et al. 2000; Tada et al. 2001; Li et al. 2003; Li and Blow 2005). One consequence of using this mechanism for preventing re-replication of DNA is that it is imperative that enough origins are licensed prior to S-phase entry, so that no regions of the genome remain unreplicated, even if some replication forks stall or some origins fail to initiate (Blow et al. 2011). Metazoan cells employ a licensing checkpoint to monitor that sufficient origins are licensed, inhibiting S-phase entry until this is established (Shreeram et al. 2002; Blow and Gillespie 2008).Here we review recent research showing how cells ensure complete genome duplication by licensing more replication origins in G1 than are normally used during S phase. The otherwise dormant replication origins become important for ensuring the completion of DNA replication if replication forks stall or are inhibited during S phase. We also review research showing how the licensing checkpoint ensures that a large enough number of origins are licensed before cells embark on S phase.     

Regulating DNA Replication in Plants

Chromosomal DNA replication in plants has requirements and constraints similar to those in other eukaryotes. However, some aspects are plant-specific. Studies of DNA replication control in plants, which have unique developmental strategies, can offer unparalleled opportunities of comparing regulatory processes with yeast and, particularly, metazoa to identify common trends and basic rules. In addition to the comparative molecular and biochemical studies, genomic studies in plants that started with Arabidopsis thaliana in the year 2000 have now expanded to several dozens of species. This, together with the applicability of genomic approaches and the availability of a large collection of mutants, underscores the enormous potential to study DNA replication control in a whole developing organism. Recent advances in this field with particular focus on the DNA replication proteins, the nature of replication origins and their epigenetic landscape, and the control of endoreplication will be reviewed.Faithful genome duplication during the S phase of the cell cycle uses strategies largely conserved in all eukaryotes (DePamphilis and Bell 2011) and is pivotal to preserve genome integrity. Genome duplication in dividing plant cells has the same requirements and constraints than in animal cells, including the strict rule of occurring once and only once every cell cycle. The initial discoveries of several basic biological processes were performed in studies with plant cells, e.g., transposons, telomeres, RNA interference, to cite a few. DNA replication in eukaryotes is not an exception. Pioneering work in the mid-1950s showed the semiconservative nature of chromosomal DNA replication in the common bean Vicia faba (Taylor et al. 1957). Since then, plant DNA replication studies have focused primarily on defining temporal patterns of DNA replication at the chromosomal level along the S phase. DNA fiber autoradiography was first used in plants to determine replicon size at the single molecule level (Nitta and Nagata 1976; Van’t Hof 1976) and genome organization in early and late replicon families (Van’t Hof et al. 1978; Van’t Hof and Bjerknes 1981; reviewed in Bryant 2010; Costas et al. 2011a).In this work, we will focus on: (1) plant DNA replication proteins (see also Supplemental Table 3 online); (2) plant DNA replication origins and their epigenetic landscape; (3) novel licensing mechanisms; (4) the relevance of DNA replication proteins in the control of the endoreplication cycle during plant development, and, finally; (5) a brief overview of duplication of plant DNA viruses.     

Heavily Isotype-Dependent Protective Activities of Human Antibodies against Vaccinia Virus Extracellular Virion Antigen B5

Antibodies against the extracellular virion (EV or EEV) form of vaccinia virus are an important component of protective immunity in animal models and likely contribute to the protection of immunized humans against poxviruses. Using fully human monoclonal antibodies (MAbs), we now have shown that the protective attributes of the human anti-B5 antibody response to the smallpox vaccine (vaccinia virus) are heavily dependent on effector functions. By switching Fc domains of a single MAb, we have definitively shown that neutralization in vitro—and protection in vivo in a mouse model—by the human anti-B5 immunoglobulin G MAbs is isotype dependent, thereby demonstrating that efficient protection by these antibodies is not simply dependent on binding an appropriate vaccinia virion antigen with high affinity but in fact requires antibody effector function. The complement components C3 and C1q, but not C5, were required for neutralization. We also have demonstrated that human MAbs against B5 can potently direct complement-dependent cytotoxicity of vaccinia virus-infected cells. Each of these results was then extended to the polyclonal human antibody response to the smallpox vaccine. A model is proposed to explain the mechanism of EV neutralization. Altogether these findings enhance our understanding of the central protective activities of smallpox vaccine-elicited antibodies in immunized humans.The smallpox vaccine, live vaccinia virus (VACV), is frequently considered the gold standard of human vaccines and has been enormously effective in preventing smallpox disease. The smallpox vaccine led to the worldwide eradication of the disease via massive vaccination campaigns in the 1960s and 1970s, one of the greatest successes of modern medicine (30). However, despite the efficacy of the smallpox vaccine, the mechanisms of protection remain unclear. Understanding those mechanisms is key for developing immunologically sound vaccinology principles that can be applied to the design of future vaccines for other infectious diseases (3, 101).Clinical studies of fatal human cases of smallpox disease (variola virus infection) have shown that neutralizing antibody titers were either low or absent in patient serum (24, 68). In contrast, neutralizing antibody titers for the VACV intracellular mature virion (MV or IMV) were correlated with protection of vaccinees against smallpox (68). VACV immune globulin (VIG) (human polyclonal antibodies) is a promising treatment against smallpox (47), since it was able to reduce the number of smallpox cases ∼80% among variola-exposed individuals in four case-controlled clinical studies (43, 47, 52, 53, 69). In animal studies, neutralizing antibodies are crucial for protecting primates and mice against pathogenic poxviruses (3, 7, 17, 21, 27, 35, 61, 66, 85).The specificities and the functions of protective antipoxvirus antibodies have been areas of intensive research, and the mechanics of poxvirus neutralization have been debated for years. There are several interesting features and problems associated with the antibody response to variola virus and related poxviruses, including the large size of the viral particles and the various abundances of many distinct surface proteins (18, 75, 91, 93). Furthermore, poxviruses have two distinct virion forms, intracellular MV and extracellular enveloped virions (EV or EEV), each with a unique biology. Most importantly, MV and EV virions share no surface proteins (18, 93), and therefore, there is no single neutralizing antibody that can neutralize both virion forms. As such, an understanding of virion structure is required to develop knowledge regarding the targets of protective antibodies.Neutralizing antibodies confer protection mainly through the recognition of antigens on the surface of a virus. A number of groups have discovered neutralizing antibody targets of poxviruses in animals and humans (3). The relative roles of antibodies against MV and EV in protective immunity still remain somewhat unclear. There are compelling data that antibodies against MV (21, 35, 39, 66, 85, 90, 91) or EV (7, 16, 17, 36, 66, 91) are sufficient for protection, and a combination of antibodies against both targets is most protective (66). It remains controversial whether antibodies to one virion form are more important than those to the other (3, 61, 66). The most abundant viral particles are MV, which accumulate in infected cells and are released as cells die (75). Neutralization of MV is relatively well characterized (3, 8, 21, 35). EV, while less abundant, are critical for viral spread and virulence in vivo (93, 108). Neutralization of EV has remained more enigmatic (3).B5R (also known as B5 or WR187), one of five known EV-specific proteins, is highly conserved among different strains of VACV and in other orthopoxviruses (28, 49). B5 was identified as a protective antigen by Galmiche et al., and the available evidence indicated that the protection was mediated by anti-B5 antibodies (36). Since then, a series of studies have examined B5 as a potential recombinant vaccine antigen or as a target of therapeutic monoclonal antibodies (MAbs) (1, 2, 7, 17, 40, 46, 66, 91, 110). It is known that humans immunized with the smallpox vaccine make antibodies against B5 (5, 22, 62, 82). It is also known that animals receiving the smallpox vaccine generate antibodies against B5 (7, 20, 27, 70). Furthermore, previous neutralization assays have indicated that antibodies generated against B5 are primarily responsible for neutralization of VACV EV (5, 83). Recently Chen at al. generated chimpanzee-human fusion MAbs against B5 and showed that the MAbs can protect mice from lethal challenge with virulent VACV (17). We recently reported, in connection with a study using murine monoclonal antibodies, that neutralization of EV is highly complement dependent and the ability of anti-B5 MAbs to protect in vivo correlated with their ability to neutralize EV in a complement-dependent manner (7).The focus of the study described here was to elucidate the mechanisms of EV neutralization, focusing on the human antibody response to B5. Our overall goal is to understand underlying immunobiological and virological parameters that determine the emergence of protective antiviral immune responses in humans.     

Endocytosis of Viruses and Bacteria

Of the many pathogens that infect humans and animals, a large number use cells of the host organism as protected sites for replication. To reach the relevant intracellular compartments, they take advantage of the endocytosis machinery and exploit the network of endocytic organelles for penetration into the cytosol or as sites of replication. In this review, we discuss the endocytic entry processes used by viruses and bacteria and compare the strategies used by these dissimilar classes of pathogens.Many of the most widespread and devastating diseases in humans and livestock are caused by viruses and bacteria that enter cells for replication. Being obligate intracellular parasites, viruses have no choice. They must transport their genome to the cytosol or nucleus of infected cells to multiply and generate progeny. Bacteria and eukaryotic parasites do have other options; most of them can replicate on their own. However, some have evolved to take advantage of the protected environment in the cytosol or in cytoplasmic vacuoles of animal cells as a niche favorable for growth and multiplication. In both cases (viruses and intracellular bacteria), the outcome is often destructive for the host cell and host organism. The mortality and morbidity caused by infectious diseases worldwide provide a strong rationale for research into pathogen–host cell interactions and for pursuing the detailed mechanisms of transmission and dissemination. The study of viruses and bacteria can, moreover, provide invaluable insights into fundamental aspects of cell biology.Here, we focus on the mechanisms by which viral and bacterial pathogens exploit the endocytosis machinery for host cell entry and replication. Among recent reviews on this topic, dedicated uniquely to either mammalian viruses or bacterial pathogens, we recommend the following: Cossart and Sansonetti (2004); Pizarro-Cerda and Cossart (2006); Kumar and Valdivia (2009); Cossart and Roy (2010); Mercer et al. (2010b); Grove and Marsh (2011); Kubo et al. (2012); Vazquez-Calvo et al. (2012a); Sun et al. (2013).The term “endocytosis” is used herein in its widest sense, that is, to cover all processes whereby fluid, solutes, ligands, and components of the plasma membrane as well as particles (including pathogenic agents) are internalized by cells through the invagination of the plasma membrane and the scission of membrane vesicles or vacuoles. This differs from current practice in the bacterial pathogenesis field, where the term “endocytosis” is generally reserved for the internalization of molecules or small objects, whereas the uptake of bacteria into nonprofessional phagocytes is called “internalization” or “bacterial-induced phagocytosis.” In addition, the term “phagocytosis” is reserved for internalization of bacteria by professional phagocytes (macrophages, polymorphonuclear leucocytes, dendritic cells, and amoebae), a process that generally but not always leads to the destruction of the ingested bacteria (Swanson et al. 1999; May and Machesky 2001; Henry et al. 2004; Zhang et al. 2010). With a few exceptions, we will not discuss phagocytosis of bacteria or the endocytosis of protozoan parasites such as Toxoplasma and Plasmodium (Robibaro et al. 2001).     

Modified Vaccinia Virus Ankara Triggers Chemotaxis of Monocytes and Early Respiratory Immigration of Leukocytes by Induction of CCL2 Expression

Orthopoxviruses commonly enter into humans and animals via the respiratory tract. Herein, we show that immigration of leukocytes into the lung is triggered via intranasal infection of mice with modified vaccinia virus Ankara (MVA) and not with the vaccinia virus (VACV) Elstree, Wyeth, or Western Reserve (WR) strain. Immigrating cells were identified as monocytes, neutrophils, and CD4⁺ lymphocytes by flow cytometry and could be detected 24 h and 48 h postinfection. Using an in vitro chemotaxis assay, we confirmed that infection with MVA induces the expression of a soluble chemotactic factor for monocytes, identified as CCL2 (monocyte chemotactic protein-1 [MCP-1]). In contrast to infection with several other VACV strains, MVA induced the expression of CCL2, CCL3, CCL4, and CXCL10 in the human monocytic cell line THP-1 as well as in primary human monocytes. Thus, MVA, and not the VACV Elstree, Wyeth, or WR strain, consistently triggered the expression of a panel of chemokines, including CCL2, in the murine lung, correlating considerably with the immigration of leukocytes. Using CCL2-deficient mice, we demonstrate that CCL2 plays a key role in MVA-triggered respiratory immigration of leukocytes. Moreover, UV irradiation of MVA prevented CCL2 expression in vitro and in vivo as well as respiratory immigration of leukocytes, demonstrating the requirement for an activated molecular viral life cycle. We propose that MVA-triggered chemokine expression causes early immigration of leukocytes to the site of infection, a feature that is important for rapid immunization and its safety and efficiency as a viral vector.The World Health Organization (WHO) announced the worldwide eradication of smallpox at the end of the 1970s. Nevertheless, the threat of an outbreak of smallpox or a smallpox-like disease, either by natural means or via bioterrorism, exists to this day. This danger was illustrated in 2003 by the mini-epidemic of monkeypox in the U.S. Midwest (33). Thus, a vaccine against smallpox is, even today, essential. Although vaccination against smallpox using vaccinia virus (VACV) was quite successful, the incidence of severe side effects prompted the WHO to discontinue the use of the vaccine. Therefore, there is currently still a need for an effective and safe vaccine against smallpox.Among the orthopoxviruses, VACV is frequently used to study poxvirus infection, since it displays many properties of variola virus, the etiologic agent of smallpox, including the capability of modulating and suppressing the immune system by means of expressing several immunoregulatory proteins (44). The assumed natural primary infection site of variola virus is considered to be the respiratory tract (5). Here, the virus encounters lung epithelial cells, conventional dendritic cells, and macrophages. More than three decades ago, viral antigen was detected in alveolar macrophages after a sublethal infection of rabbits with inhaled VACV by using immunofluorescence assays (3). Additionally, under the electron microscope, the cells obtained from repeated washing of rabbit lungs demonstrated that VACV infects and replicates exclusively in macrophages (18). Macrophages, the most abundant hematopoietic cell type in the lung, play a key role in antiviral immune defense through the phagocytosis of infectious particles and the production of reactive oxygen species as well as leukotrienes and inflammatory cytokines (14).The coordinated migration, differentiation, and activation of dendritic cells as well as lymphocytes are required for the efficient elimination of microbes, including viruses, from the lung (15). Activated alveolar macrophages, in particular, enhance the cellular immune response by triggering the immigration of several leukocyte types into the lung owing to the production of chemokines (35). The importance of alveolar macrophages in limiting the replication of a recombinant VACV strain Western Reserve (WR) was recently demonstrated with mice (38). It is likely that the stimulation of cytokine production contributed to the elimination of the virus. In summary, the infection of resident alveolar macrophages with orthopoxviruses and the subsequent upregulation of chemokine expression attract several different leukocyte types, representing a critical event in antiviral defense.A highly attenuated orthopoxvirus strain, modified VACV Ankara (MVA), is being considered as a candidate for the production of a vaccine against smallpox and as a viral vector in gene therapeutic protocols (9). MVA, administered intranasally (i.n.) or intramuscularly as a short-term immunization, has been proven to be protective against a lethal challenge with the virulent VACV strain WR in a mouse model. In contrast, the VACV strain Elstree failed to protect mice when administered intramuscularly 48 h before challenge but was effective when the lethal challenge occurred 14 days postimmunization. Interestingly, an elevated concentration of various types of leukocytes, including monocytes, granulocytes, and T lymphocytes, was present in the lung 48 h after i.n. immunization with MVA (46). This respiratory immigration of leukocytes was most likely triggered by a chemoattractant factor produced by resident lung cells due to infection with MVA. In order to elucidate the mechanism underlying the migration of leukocytes, pilot experiments were performed to test whether MVA triggers the expression of a soluble factor capable of attracting leukocytes, especially monocytes.     

Clinical and Molecular Features of POLG-Related Mitochondrial Disease

The inability to replicate mitochondrial genomes (mtDNA) by the mitochondrial DNA polymerase (pol γ) leads to a subset of mitochondrial diseases. Many mutations in POLG, the gene that encodes pol γ, have been associated with mitochondrial diseases such as myocerebrohepatopathy spectrum (MCHS) disorders, Alpers-Huttenlocher syndrome, myoclonic epilepsy myopathy sensory ataxia (MEMSA), ataxia neuropathy spectrum (ANS), and progressive external ophthalmoplegia (PEO). This chapter explores five important topics in POLG-related disease: (1) clinical symptoms that identify and distinguish POLG-related diseases, (2) molecular characterization of defects in polymerase activity by POLG disease variants, (3) the importance of holoenzyme formation in disease presentation, (4) the role of pol γ exonuclease activity and mutagenesis in disease and aging, and (5) novel approaches to therapy and avoidance of toxicity based on primary research in pol γ replication.While many polymerases are responsible for replicating billions of nucleotides in the nucleus, DNA polymerase γ replicates thousands of copies of the 16-kilobase mitochondrial genome (mtDNA) in each human cell. The holoenzyme of DNA polymerase γ (pol γ) consists of a catalytic subunit (encoded by POLG at chromosomal locus 15q25) and a dimeric form of its accessory subunit (encoded by POLG2 at chromosomal locus 17q24.1) (Longley et al. 1998b; Lim et al. 1999). MtDNA encodes 13 proteins that are essential for the electron transport chain that provides most of the ATP in the cell. Therefore, mtDNA replication is essential for life as demonstrated by the embryonic lethality of POLG knockout mice (Hance et al. 2005). Studies over the last decade have identified over 200 mutations in POLG that are associated with certain mitochondrial diseases (http://tools.niehs.nih.gov/polg/) (Longley et al. 2005: Copeland 2008; Longley et al. 2010; Walter et al. 2010; Stumpf and Copeland 2011; Tang et al. 2011). POLG-related disorders are currently defined by at least five major phenotypes of neurodegenerative disease that include: (1) Alpers-Huttenlocher syndrome (AHS), (2) childhood myocerebrohepatopathy spectrum (MCHS), (3) myoclonic epilepsy myopathy sensory ataxia (MEMSA), (4) the ataxia neuropathy spectrum (ANS), and (5) progressive external ophthalmoplegia (PEO) with or without sensory ataxic neuropathy and dysarthria (SANDO) (Wong et al. 2008; Cohen et al. 2010; Saneto and Naviaux 2010; Saneto et al. 2010). These diseases associate with mutations that have been shown to decrease the activity of the polymerase, the fidelity of replication, and the formation of the holoenzyme. This work reviews the important conclusions regarding POLG-related diseases and discusses some strategies for disease treatment options.     

Helicase Activation and Establishment of Replication Forks at Chromosomal Origins of Replication

Many replication proteins assemble on the pre-RC-formed replication origins and constitute the pre-initiation complex (pre-IC). This complex formation facilitates the conversion of Mcm2–7 in the pre-RC to an active DNA helicase, the Cdc45–Mcm–GINS (CMG) complex. Two protein kinases, cyclin-dependent kinase (CDK) and Dbf4-dependent kinase (DDK), work to complete the formation of the pre-IC. Each kinase is responsible for a distinct step of the process in yeast; Cdc45 associates with origins in a DDK-dependent manner, whereas the association of GINS with origins depends on CDK. These associations with origins also require specific initiation proteins: Sld3 for Cdc45; and Dpb11, Sld2, and Sld3 for GINS. Functional homologs of these proteins exist in metazoa, although pre-IC formation cannot be separated by requirement of DDK and CDK because of experimental limitations. Once the replicative helicase is activated, the origin DNA is unwound, and bidirectional replication forks are established.The main events at the initiation step of DNA replication are the unwinding of double-stranded DNA and subsequent recruitment of DNA polymerases, to start DNA synthesis. Eukaryotic cells require an active DNA helicase to unwind the origin DNA. The core components of the replicative helicase, Mcm2–7, are loaded as a head-to-head double hexamer connected via their amino-terminal rings (Evrin et al. 2009; Remus et al. 2009; Gambus et al. 2011) onto Orc-associated origins, to form the pre-RC in late M and G₁ phases (see Bell and Kaguni 2013). However, Mcm2–7 alone does not show DNA helicase activity at replication origins. After the formation of the pre-RC, other replication factors assemble on origins, and the pre-initiation complex (pre-IC) is formed. The pre-IC is defined as a complex formed just before the initiation of DNA replication (Zou and Stillman 1998); in yeast, it contains at least seven additional factors: Cdc45, GINS, Dpb11, Sld2, Sld3, Cdc45, and DNA polymerase ε (Pol ε) (Muramatsu et al. 2010). The formation of the pre-IC is a prerequisite for the activation of the Mcm2–7 helicase; two additional factors, Cdc45 and GINS, associate with Mcm2–7 and form a tight complex, the Cdc45–Mcm–GINS (CMG) complex (Gambus et al. 2006; Moyer et al. 2006). This reaction requires components of the pre-IC and two protein kinases, cyclin-dependent kinase (CDK) and Dbf4-dependent kinase (DDK) (for reviews, see Labib 2010; Masai et al. 2010; Tanaka and Araki 2010). In this article, we summarize and discuss the manner via which the pre-IC is formed in yeasts and metazoa. Although there are some discrepancies, the process of formation of the pre-IC is conserved fairly well in these organisms.     

Postreplicative Mismatch Repair

The mismatch repair (MMR) system detects non-Watson–Crick base pairs and strand misalignments arising during DNA replication and mediates their removal by catalyzing excision of the mispair-containing tract of nascent DNA and its error-free resynthesis. In this way, MMR improves the fidelity of replication by several orders of magnitude. It also addresses mispairs and strand misalignments arising during recombination and prevents synapses between nonidentical DNA sequences. Unsurprisingly, MMR malfunction brings about genomic instability that leads to cancer in mammals. But MMR proteins have recently been implicated also in other processes of DNA metabolism, such as DNA damage signaling, antibody diversification, and repair of interstrand cross-links and oxidative DNA damage, in which their functions remain to be elucidated. This article reviews the progress in our understanding of the mechanism of replication error repair made during the past decade.The mismatch repair (MMR) system is one of the key guardians of genomic integrity. Its malfunction leads to a substantial increase in spontaneous mutagenesis, illegitimate recombination, and cancer in mammals. MMR improves the fidelity of DNA replication by several orders of magnitude by excising sections of the nascent strand containing mispaired nucleotides. It is likely that MMR has evolved to carry out this function in order to ensure that daughter cells inherit an exact replica of the parental genome. But MMR also controls the fidelity of recombination by removing mispairs from heteroduplexes arising between donor and recipient strands and possibly even rejecting synapses between sequences that are too diverged. Indeed, the existence of MMR was first invoked in the 1960s to explain the unanticipated segregation of genetic markers in fungi and bacteria (for a comprehensive overview of the field, see chapter 12 in Friedberg et al. 1995). During the intervening 50 years, our understanding of MMR has made enormous progress; the main protagonists, as well as many “extras” that participate in this complex process, have been identified, initially in a series of genetic and biochemical experiments and later by sequence homology searches that were made possible by the high degree of evolutionary conservation of MMR. Analysis of the primary sequences of these polypeptides then helped to uncover their enzymatic activities that were confirmed by biochemical and structural studies. In vitro MMR assays using cell extracts and recombinant DNA substrates carrying single mismatches at defined positions led to the discovery of criteria required for efficient, strand-directional MMR. Finally, the Escherichia coli and the minimal human MMR systems could be reconstituted from purified recombinant proteins (Dzantiev et al. 2004). Despite this wealth of knowledge, however, we still lack detailed understanding of the molecular transactions that lead to successful repair of replication errors, and our notion of the role(s) of MMR proteins during recombination is highly speculative.Since the discovery of a link between its malfunction and cancer (for recent reviews, see Wimmer and Etzler 2008; Hewish et al. 2010), MMR has attracted a great deal of attention, and recent progress in our understanding of this pathway has been the subject of several reviews (Stojic et al. 2004; Kunkel and Erie 2005; Iyer et al. 2006; Jiricny 2006; Hsieh and Yamane 2008; Li 2008; George and Alani 2012; Peña-Diaz and Jiricny 2012). This article therefore provides only a brief overview of the MMR process and focuses primarily on the most recent insights into this complex pathway of DNA metabolism.     

DNA Replication Origins

The onset of genomic DNA synthesis requires precise interactions of specialized initiator proteins with DNA at sites where the replication machinery can be loaded. These sites, defined as replication origins, are found at a few unique locations in all of the prokaryotic chromosomes examined so far. However, replication origins are dispersed among tens of thousands of loci in metazoan chromosomes, thereby raising questions regarding the role of specific nucleotide sequences and chromatin environment in origin selection and the mechanisms used by initiators to recognize replication origins. Close examination of bacterial and archaeal replication origins reveals an array of DNA sequence motifs that position individual initiator protein molecules and promote initiator oligomerization on origin DNA. Conversely, the need for specific recognition sequences in eukaryotic replication origins is relaxed. In fact, the primary rule for origin selection appears to be flexibility, a feature that is modulated either by structural elements or by epigenetic mechanisms at least partly linked to the organization of the genome for gene expression.Timely duplication of the genome is an essential step in the reproduction of any cell, and it is not surprising that chromosomal DNA synthesis is tightly regulated by mechanisms that determine precisely where and when new replication forks are assembled. The first model for a DNA synthesis regulatory circuit was described about 50 years ago (Jacob et al. 1963), based on the idea that an early, key step in building new replication forks was the binding of a chromosomally encoded initiator protein to specialized DNA regions, termed replication origins (Fig. 1). The number of replication origins in a genome is, for the most part, dependent on chromosome size. Bacterial and archaeal genomes, which usually consist of a small circular chromosome, frequently have a single replication origin (Barry and Bell 2006; Gao and Zhang 2007). In contrast, eukaryotic genomes contain significantly more origins, ranging from 400 in yeast to 30,000–50,000 in humans (Cvetic and Walter 2005; Méchali 2010), because timely duplication of their larger linear chromosomes requires establishment of replication forks at multiple locations. The interaction of origin DNA and initiator proteins (Fig. 1) ultimately results in the assembly of prereplicative complexes (pre-RCs), whose role is to load and activate the DNA helicases necessary to unwind DNA before replication (Remus and Diffley 2009; Kawakami and Katayama 2010). Following helicase-catalyzed DNA unwinding, replisomal proteins become associated with the single-stranded DNA, and new replication forks proceed bidirectionally along the genome until every region is duplicated (for review, see O’Donnell 2006; Masai et al. 2010).Open in a separate windowFigure 1.Revised versions of the replicon model for all domains of life. For cells of each domain type, trans-acting initiators recognize replication origins to assemble prereplicative complexes required to unwind the DNA and load DNA helicase. Eukaryotic initiators are preassembled into hexameric origin recognition complexes (ORCs) before interacting with DNA. In prokaryotes, single initiators (archaeal Orc1/Cdc6 or bacterial DnaA) bind to recognition sites and assemble into complexes on DNA. In all cases, the DNA helicases (MCMs or DnaB) are recruited to the origin and loaded onto single DNA strands. In bacteria, DNA-bending proteins, such as Fis or IHF, may modulate the assembly of pre-RC by bending the origin DNA. Two activities of DnaA are described in the figure. The larger version binds to recognition sites, and the smaller version represents DnaA required to assist DnaC in loading DnaB helicase on single-stranded DNA.Initiator proteins from all forms of life share structural similarities, including membership in the AAA⁺ family of proteins (ATPases associated with various cellular activities) (Duderstadt and Berger 2008; Wigley 2009) that are activated by ATP binding and inactivated by ATP hydrolysis (Duderstadt and Berger 2008; Duncker et al. 2009; Kawakami and Katayama 2010). Despite these similarities, initiators assemble into prereplicative complexes in two fundamentally different ways (Fig. 2). In prokaryotes, initiator monomers interact with the origin at multiple repeated DNA sequence motifs, and the arrangement of these motifs (see below) can direct assembly of oligomers that mediate strand separation (Erzberger et al. 2006; Rozgaja et al. 2011). In eukaryotes, a hexameric origin recognition complex (ORC) binds to replication origins and then recruit additional factors (as Cdc6 and Cdt1) that will themselves recruit the hexameric MCM2-7 DNA helicase to form a prereplicative complex (for review, see Diffley 2011). This process occurs during mitosis and along G₁ and is called “DNA replication licensing,” a crucial regulation of eukaryotic DNA replication (for review, see Blow and Gillespie 2008). Importantly, this complex is still inactive, and only a subset of these preassembled origins will be activated in S phase. This process is, therefore, fundamentally different from initiation of replication in bacteria. Moreover, because sequence specificity appears more relaxed in large eukaryotic genomes, prokaryotic mechanisms that regulate initiator–DNA site occupation must be replaced by alternative mechanisms, such as structural elements or the use of epigenetic factors.Open in a separate windowFigure 2.Functional elements in some well-studied prokaryotic replication origins. (A) Bacterial oriCs. The DNA elements described in the text are (arrows) DnaA recognition boxes or (boxes) DNA unwinding elements (DUEs). When recognition site affinities are known, colored arrows designate high- (K_d > 100 nm) and low- (K_d < 100 nm) affinity sites. (B) Archaeal oriCs. Arrows and boxes designate DNA elements as in A, but the initiator protein is Orc1/Cdc6 rather than DnaA. (Thick arrows) Long origin recognition boxes (ORBs); (thin arrows) shorter versions (miniORBs). Both ORBs and miniORBs are identified in Pyrococcus. DUEs are not yet well defined for Helicobacter or Sulfolobus genera and are not labeled in this figure.Here, we describe replication origins on prokaryotic and eukaryotic genomes below, with a particular focus on the attributes responsible for orderly initiator interactions and origin selection specificity, as well as on the shift from origin sequence-dependent regulation to epigenetic regulation. You are also referred to other related articles in this collection and several recent reviews covering the topics of DNA replication initiation in more detail (Méchali 2010; Beattie and Bell 2011; Blow et al. 2011; Bryant and Aves 2011; Ding and MacAlpine 2011; Dorn and Cook 2011; Kaguni 2011; Leonard and Grimwade 2011; Sequeira-Mendes and Gomez 2012).