Genetic Instability and Herpes Simplex Virus

Although inhibitory mechanisms that safeguard cells against DNA damages occur in all cells, genetic instability is widely present throughout living world. Some well known viruses cause alterations in host genome, leading to the formation of malignant transformations. Throughout this review we discuss some forms of instabilities caused by herpes simplex virus. Herpes simplex virus may induce chromosomal instabilities by various mechanisms, such as by formation of syncytium or ICP0-induced degradation of constitutive centromere proteins as well as may induce accumulation of point mutations by means of oxidative stress in neurons. We believe that further investigation of the ability of herpes simplex virus to cause genetic instability may help us to increase our understanding about the nature of this phenomenon.


INTRODUCTION TO GENETIC INSTABILITY
During billion years of evolution cells have evolved the mechanisms that safeguard their own genome against exogenous and endogenous sources of DNA damage and ensure accurate passage of genetic information onto daughter cells. Although cells have mechanisms (intricate machinery of repair, damage tolerance, and checkpoint pathways) preventing mutations and DNA rearrangements, genetic instability is widely present throughout the living world, from bacteria and single cell organisms to multicellular beings. In spite of the

Review article
harmful influence for the cell and subsequently the whole organism, genetic instability contributes to adaptation at the molecular level and propagates genetic diversity. Genetic instability also involved in the generation of variability in the developmental processes, such as immunoglobulin diversification [1]. Nonetheless, genetic instability is usually associated with pathological upsets, and in humans it is associated with premature ageing [2], with inherited diseases (such as mental disorders and retardations) and with various types of cancer.
Genetic instability is a fundamental hallmark of almost all human cancers. It refers to a range of genetic alterations from chromosomal rearrangements to point mutations that are similar for both exogenous and endogenous mutagens and occur in all types of cancer. There are various forms of genetic instability. Chromosomal instability refers to changes in chromosome number that lead to chromosome loss or augmentation, and it is usually caused by failures in chromosome segregation or spindle checkpoint (Table1) [3]. Micro-and minisatellite instability leads to shrinkages or expansions of short DNA repeat sequences and can occur by homologous recombination, by mismatch repair errors and by replicative fork slippage [4]. Most hereditary alterations in the human genome encompass point mutations, including micro-insertions, micro-deletions and nucleotide substitutions, and those instabilities are mainly associated with replication errors, impairment of mismatch repair [5] and base excision repair [6]. Production of many copies of gene or genes Genetic instability has been reviewed extensively elsewhere and many works have been dedicated to complete examination of various types of genetic instability and their role in cancer rise and further progression [7]. In this review we discuss how viruses (particularly herpes simplex virus) lead to various types of genetic instability during their acute or chronic infections.

VIRUS-INDUCED GENETIC INSTABILITY
The discovery of Epstein-Barr virus in cells from Burkitt's lymphoma in 1964 [8], hepatitis B virus in human sera positive for hepatitis B surface antigen in 1970 [9] and human papillomavirus 16 and 18 from human cervical cancer specimens in 1983 and 1984 respectively [10,11], restored interest in the roles of viruses in genetic instability and human cancer. After 50 years of exciting discoveries, it has become extremely apparent that several viruses have been etiologically linked to specific forms of cancer and many other viruses encode proteins that are implicated in genetic instability. Viruses are one of the main cancer risk factors in humans, and several cancers are directly associated with viral infections ( Table 2) [12].  [13]), and it can result in chromosomal deletions and transpositions of viral sequences from one chromosome to another [14]. In other words, hepatitis B virus integration leads to genetic instability [15] and this event may precede tumor development [16]. On the other hand human papillomavirus E6 protein targets Bak (proapoptotic Bcl-2 family member) for degradation. Bak plays an important role in signaling apoptosis in response to UV irradiation. Also, E6 protein associates with and dysregulates cellular regulatory protein complexes, especially p53 and pRB that control cellular proliferation, differentiation and apoptosis. Cells that are infected by human papillomaviruses are less prone to undergo apoptosis after UV induced DNA damages [17], have a decreased ability to maintain genomic integrity [18] and contain extensive chromosome aberrations.
In contrast to the well studied DNA tumorigenic viruses, the role of RNA viruses in genetic instability and carcinogenesis is poorly investigated. At present, only retroviruses have been associated with genetic instability. It is well known that after infection the retroviral RNA genome is reverse-transcribed by the virally encoded reverse transcriptase into a doublestranded DNA copy which then integrates into the host chromosome. As a consequence of integration, retroviruses reorganize cellular genetic material (which is a classical example of genetic instability) and control host genes expression. Though the biological impact is still poorly understood, extensive studies of retroviruses led to the discovery of proto-oncogenes that have crucial roles in the control of cell growth and differentiation [19,20]. The discovery of proto-oncogenes has become a turning point in our understanding of mechanisms of carcinogenesis.

HERPES SIMPLEX VIRUS AND GENETIC INSTABILITY
Herpes simplex virus (HSV) belongs to the family of Herpesviridae. HSV has two serotypes, HSV-1 and HSV-2, whose formal designation according to the rules of the International Committee on Taxonomy of Viruses are human herpesviruses 1 and 2 (through this review we use informal designation). Although their size and complexity served as obstacles to intensive research for many years, now HSV is among the most intensively studied of all viruses. The attractiveness of HSV is a result of distinct biologic properties that is possesses, particularly the ability to cause a variety of infections, remain latent in their host and reactivate to cause lesions near the sites of infection.
The genome of HSV is double-stranded, linear DNA, which becomes circular in the absence of protein synthesis after entry to the nuclei of infected cells [21]. The nuclei of cells are the only place where HSV replication takes place. Knipe and colleagues [22] showed that effective viral infection follows from the remodeling of the nucleus of HSV-infected cells because remodeling provides the structural basis for viral DNA replication and gene expression [23]. Major alterations of nuclear structure may lead to genetic instability in host cells. Below we discuss some forms of instabilities caused by lytic or chronic HSV infection.

Abnormal Chromosome Set due to the Formation of Syncytium
The syncytium, as a distinct phenomenon, is usually referred to the genetic instability due to abnormal chromosome set in a single multinucleate cell [24]. The formation of multinucleate cells, or syncytium, results from destruction of cell walls and fusion of single nuclear cells into a single cell mass with two or more nuclei [24]. The fusion of cells seems to be the main mechanism that is responsible for the formation of syncytium both in vivo [25] and in vitro [26,27].
Viruses, such as HSV, that replicate in the nucleus could benefit from the presence of additional nuclei within a syncytium. The ability of HSV to cause the formation of syncytium was shown by Roizman in 1962 [28]. At present, it has been published that a few numbers of viral glycoproteins and cell receptors are involved in HSV-induced cell fusion, but a precise mechanism that leads to fusion remains unclear. It is believed that HSV-induced cell fusion is a complex process that involves the concerted and subsequent actions of multiple viral glycoproteins and membrane proteins. The glycoproteins gD, gB, gH and gL form a functional complex that is required for both virus entry [29] and virus-induced cell fusion [30]. The binding of gD to its cognate host receptors, herpesvirus entry mediator (HVEM) or nectin-1, probably triggers conformational and possibly sequential changes to gH/gL heterodimer and then gB to promote membrane fusion [31,32,33]. Tiwari and colleagues [34,35] showed that the formation of syncytium heavily depends not only on HVEM or nectin-1 interaction with glycoproteins but also on expression of 3-O-sulfated heparin sulfate, a well known receptor for HSV attachment [36] and penetration [37], on targets cells. Although HSV-induced formation of syncytium plays an important role in the cytopathology of HSV, there is a huge gap in our knowledge about accurate mechanisms of syncytium formation, and further studies may fill the gap. In any case polykaryocyte formation is an important means of viral spread (cell-to-cell spread by polykaryocyte formation) and a clue for HSV-1 keratitis [35].

Abnormal Chromosome Set: Polyploidy
Polyploidy, a condition in which a normally diploid cell acquires one or more additional sets of chromosomes, occurs frequently in nature. Polyploid cells can arise by a variety of mechanisms, including cytokinesis failure [38], mitotic slippage [39], virus-induced cells and nuclear fusion [40] etc. The influence of HSV on cell cycle control leading to deviation of cell cycle and polyploid cell formation was discussed many years ago in some articles [41]. The hypothesis arose among members of the HSV community that a few viral proteins like ICP4 (infected cell protein 4), ICP8 or DNA polymerase may be involved in mitotic arrest and subsequently in polyploid cell formation. Further studies examined the influence of another viral protein, ICP0, whose leading role in mitotic arrest have been demonstrated by two different research groups [42,43]. They showed that ICP0 can destruct CENP-C thereby arresting cells in G2/M phase. According to recent studies ICP0 disassembles microtubules of infected cells which may also lead to cell arrest [44]. In both cases, HSV uses a pathway that is fundamentally different from the inactivation of p53 by the HPV E6 protein for ICP0mediated G2/M arrest, utilizing Chk2, a protein kinase that is usually activated in response to DNA damages [45].
In addition to ICP0-mediated cell arrest we can suggest two alternative mechanisms that may be implicated in polyploid cell formation. Love and Wildy in 1963 reported that the first detectable abnormality in HSV-infected HeLa cells was an enlargement of RNA containing cellular organelle, nucleolini, followed by their extrusion from the nucleolus into the nucleoplasm [46]. The role of nucleolini in cell cycle was discovered by Alliegro and colleagues who showed that normal spindle formation is tightly correlated with location of nucleolini in nucleolus [47]. These findings suggest that the direct influence of HSV on location of nucleolini may result in deviation of cell cycle and polyploidy formation.
The second proposed mechanism based on our studies (unpublished) is HSV-induced nuclei fusion in syncytium. We observed the formation of giant nuclei in SK-N-MC cells after a short time of HSV infection (10-12 hours). The giant nuclei were characterized by defective chromatin structure, absence of nucleoli and up to 90c (c-value refers to the amount of DNA contained within a haploid nucleus) DNA content. The formation of giant nuclei after such a short time of infection can be the consequence of virus-induced nuclei fusion. We can speculate that some viral glycoproteins are involved in nuclear fusion in the same way as they are responsible for the fusion of the viral envelope with the outer nuclear membrane [48]. However, this phenomenon requires further investigation.

Abnormal Chromosome Set: Aneuploidy
Aneuploidy is one sort of chromosome abnormality characterized by missing a chromosome or having extra copies. A frequent characteristic of cancer cells, aneuploidy generally occurs during cell division when the chromosomes do not separate correctly between the two newly forming daughter cells [49]. Chromosomal rupture around the centromeric region and a poorly controlled mitotic spindle are main mechanisms leading to aneuploidy [50].
HSV infection of cultured cells induces the formation of aneuploidy by two different ways. As previously mentioned, ICP0 plays an important role in HSV-induced chromosomal instability due to destruction of essential component of centromere. ICP0 is a RING finger nuclear protein with characterized E3 ubiquitin ligase activity [51]. As soon as ICP0 enters the nucleus, it provisionally localizes to centromeres and induces the proteasomal degradation of centromere proteins [42,52,53]. Therefore, ICP0-induced degradation of constitutive centromere proteins is likely to modify the structure of the central core region, thereby preventing the assembly of the kinetochore. Chromosomes frequently fail to align at a compact metaphase plate when CENP-C is targeted by ICP0 [42], leading to lost of accurate chromosomal segregation and aneuploidy.
The amplification of only some parts of the genome usually results in formation of genetic instability, particularly aneuploidy [54]. Although HSV suppresses the ability of the host to replicate [55], it may induce amplification of small-sized parts of the host genome. Some experiments show that the replication machinery of HSV can be involved in the amplification of other viral genomes like papillomavirus or simian virus 40 [56,57]. The importance of these findings is that HSV may amplify part of the DNA that does not belong to the host genome. Other studies demonstrate that viral polymerase holoenzyme (UL30/UL42) as well as helicase-primase heterotrimer (UL5/UL8/UL52), origin binding protein (UL9) and singlestranded DNA binding protein (UL29) are implicated in the amplification of host genome [58,59].
Galloway and McDougall [60] showed that HSV is able to cause mutations-both point mutations and gene rearrangements. HSV is also able to induce gene amplification, particularly of sequences harboring an origin of replication such as SV40 or papillomaviruses. Other experiments have shown that HSV can activate the expression of endogenous retroviruses.

Chromosome Structural Abnormalities
One of the consequences of a viral infection of mammalian cells is chromosome structural damage. Many DNA and RNA viruses induce a wide range of aberrations in vitro and in vivo [61]. The ability of HSV to cause chromosome structural abnormalities was reported many times during the 1960s [62,63,64]. For example, Hampar and Elison demonstrated that HSV infection forms multiple chromosomal aberrations in Chinese hamster cells, especially deletions that occur at a frequency of 80-100 per cent in all infected cells [65]. Different types of translocations and non-homologue recombination are also detected during HSV infection, including translocations looking like robertsonian type, as almost all infected cells have a new chromosome which would not be identified, and is morphologically different from cell to cell [66,67]. HSV may induce uncoiling of chromosomes near the lq12-21 and to a lesser extent the pericentric regions of chromosomes 1, 9 and 16 [68].
How does HSV induce such a variety of chromosome structural abnormalities? The accurate mechanisms involved in the formation of chromosome abnormalities in general are still poorly investigated. At least three theories -telomere attrition, defects in double-strand break (DSB) repair and fragile sites -have been suggested as structural abnormalities [50]. Errors in mitotic chromosome segregation may generate DNA breaks via the formation of structures called micronuclei. It has been shown that ICP0 induces centromeric disruption and appearance of micronuclei [69]. The centromeric protein CENP-C was lost from centromeres during virus infection in an ICP0-and proteasome-dependent manner, causing substantial ultrastructural changes in the kinetochore. This results in not only cellular arrest in the G2/M phase (shown above), but also an unusual cytokinesis with many micronuclei [42].
Improper DSB repair may lead to chromosomal rearrangements, such as deletions or translocations, detected during HSV infection, because of continuous de novo generation of breaks [70,71]. HSV-infected cells exhibit high frequency of improper DSB repair, which means that the virus may influence host repair mechanisms, leading to the accumulation of chromosomal rearrangements [72,73]. On the other hand, HSV can induce DSBs by means of the reactivation of some cellular proteins, such as endonuclease G. Endonuclease G is normally localized in mitochondria and released from them during apoptosis, inducing DNA fragmentation [74]. HSV requires endonuclease G in abundance for successful infection, and therefore it is recruited from mitochondria and translocated in nucleus, implicating in initiation of genomic inversion of HSV and formation of DSB in host DNA [75].

Does HSV Induce Gene Mutations?
After the pioneering work of Schlehofer and zur Hausen [76], reporting on the induction of mutations in the host genome by HSV-1, many scientists from the HSV community tried to identify the mutagenic role of the virus on host genes. In spite of extensive investigation, only few studies identified the mutagenic influence of HSV on host genes [77,78]. Conversely, there are striking evidences that virus growth is impaired in cells that lack key components of the DNA damage response and are prone to accumulate point mutations [72,79]. Restriction on viral growth is explained by the fact that HSV exploits cellular response mechanisms to DNA damage for its own optimal growth, particularly for conducting uracil-initiated base excision repair [80,81,82]. However, we believe that HSV infection may induce accretion of point mutations in the host genome due to a delay of apoptosis. It is well known that HSV and other viruses prevent apoptotic cell death from proceeding which allows them to produce new progeny during infection cycle. As HSV penetrate into the nucleus, viral proteins, such as ICP 4, ICP 27 and US3 are instantly synthesized and involved in regulation of apoptosis, postponing cell dead [83,84,85].

Latent Infection and Genetic Instability
A latent infection is a phase of viral life cycle in which, after initial infection, virus production ceases. During a latent infection the viral genome is not fully eradicated and undergoes dramatic changes, forming episomes or insertions into the host genome. Infection of neurons by HSV, in particular those in the trigeminal and dorsal root ganglia, usually results in lifelong latency of the virus in the host which is one of the most challenging features of HSV biology [86]. An HSV latent infection is characterized as episomal latency because viral DNA persists in the nucleus, in a circular episomal form, associated with nucleosomes. Other nuclear domains including promyelocytic leukemia nuclear bodies and centromeres are functionally involved in the control of HSV-1 latency, and represent a key level of host/virus interaction [87].
While most of HSV genes are repressed in a latent infection, the latency-associated transcript (LAT) is intensively expressed. This results in several RNA species contributing to the establishment of latency. Recent findings showed that LAT not only regulates the establishment, maintenance and reactivation of virus from a latent state, but also inhibits the two major apoptotic pathways in neurons, caspase 8-and caspase 9-induced apoptosis [88][89][90]. LAT also inhibits the activation of caspase 3 that is implicated in apoptosis [91]. In LAT expressed neurons was detected a high level of protein kinase B (AKT), which serves as a key factor in many types of cancer [92]. Activation of AKT was shown to overcome cell cycle arrest in either phase G1 or G2 and may promote the survival and proliferation of cells [93,94]. Unblocked cells, due to ineffective checkpoint control may serve as an anchorage for mutations in DNA. In addition, Plo and Lopez reported that AKT represses homologous recombination induced by DSB, resulting in the accumulation of DSB in cells [95]. The correlation between LAT expression and high level of AKT needs detailed investigation in the near future as well as a link between LAT and genetic instability.
There is little evidence that the long-term persistence of HSV may directly have an influence on host genome stability, leading to the various types of rearrangements. As shown by Chenet-Monte and Heilbronn some viral proteins can be involved in the chromosomal aberrations and DNA amplification in cells during both forms of infection [96,97]. More recent results indicated that latent HSV infection in the murine nervous system is usually associated with focal chronic inflammation and with oxidative damage which can be expressed by the formation of chromosomal rearrangements [98,99,100].

CONCLUSION
As mentioned previously, cells have evolved mechanisms that are preventing them from various exogenous and endogenous sources of DNA damage. Although these mechanisms occur in all cells, the problem of genetic stability seems to be unsolved enigma. While some forms of instabilities contribute to the adaptation of cells on molecular level, others make harmful influence for cells and even for whole organisms, leading to the diseases such as cancer.
The studies of virus-induced genetic instability have provided critical insights into key mechanisms of mutagenesis. Some viruses play essential roles in the initiation as well as progression of genetic instability, leading to the formation and maintenance of transformed phenotype. During the past decades we accumulated evidences that confirm the leading role of some viruses in genetic instability and carcinogenesis. On the other hand, there are many other viruses that have always been with us and their role in genetic rearrangements remains unexplored and needs for further investigation. Herpes simplex virus is thought to be one of the best candidates due to its wide distribution among population and long term persistency. Analysis demonstrated that HSV infection induced a general increase of the level of mutations. This type of response is thought to be compatible with the specific behavior of the virus. The further studies promise many more discoveries that may help to uncover new biological effects of HSV.