BioFactors 14 (2001) 205–210 205IOS Press

Mini-review

Glutathione peroxidase and viral replication:Implications for viral evolution andchemoprevention

Alan M. Diamonda,∗, Ya Jun Hua and David B. MansurbaDepartment of Human Nutrition and Dietetics, University of Illinois, Chicago, IL 60612, USAbRadiation Oncology Center, Mallinckrodt Institute of Radiology, Washington University, St. Louis,MO 63110, USA

Received 5 October 2000

Accepted 29 January 2001

Abstract. It is likely that several of the biological effects of selenium are due to its effects on selenoprotein activity. While theeffects of the anti-oxidant selenoprotein glutathione peroxidase (GPx) on inhibiting HIV activation have been well documented,it is clear that increased expression of this enzyme can stimulate the replication and subsequent appearance of cytopathic effectsassociated with an acutely spreading HIV infection. The effects of GPx on both phases of the viral life cycle are likely mediatedvia its influence on signaling molecules that use reactive oxygen species, and similar influences on signaling pathways mayaccount for some of the anti-cancer effects of selenium. Similarly, selenium can alter mutagenesis rates in both viral genomesand the DNA of mammalian cells exposed to carcinogens. Comparisons between the effects of selenium and selenoproteins onviral infections and carcinogenesis may yield new insights into the mechanisms of action of this element.

1. Introduction

Selenium is an essential trace element, whose levels of consumption have been associated with a widevariety of human health-related consequences. While initially regarded for its toxicity at high levels ofexposure, selenium-deficiency is also associated with several clinical phenomena [13]. Of more recentinterest, levels of dietary selenium in the nutritional range are thought to influence the occurrence andseverity of several diseases, including cancer, heart disease, and infectious diseases. The mechanismsby which selenium influences the progression of these diseases remains unknown. Selenium in proteinis in the form of the amino acid selenocysteine, which is co-translationally inserted during translationof selenoprotein mRNAs and typically resides at the active site of the selenoprotein [8]. Several dozenselenoproteins are present in mammalian cells and only about a third of these have been characterizedin detail [8]. Of those, approximately half have been implicated as anti-oxidants, the best studied beingthe members of the glutathione peroxidase (GPx) family of selenoproteins [5]. In this manuscript, theeffects of GPx and selenium are discussed for both viral replication and evolution, and contrasted to dataavailable on their role in preventing cancer.

∗Corresponding author: Alan M. Diamond, Department of Human Nutrition and Dietetics, 1919 W. Taylor Street, Chicago,IL, 60612, USA. Tel.: +1 312 996 2083; Fax: +1 312 413 0319; E-mail: adiamond@uic.edu.

0951-6433/01/$8.00 2001 – IOS Press. All rights reserved

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2. Glutathione peroxidase activity can influence HIV replication

As is typical for members of the retroviruses, the HIV life cycle can be considered to have two phases.Initially, infection is initiated by the attachment of the viral particle to a receptor on the membrane ofthe target cell, followed by the entry of the RNA genome into the cytoplasm. Primed by a molecule oftRNA encoded by the host, the retroviral RNA genome is converted into a DNA copy by the activity ofthe virally encoded enzyme reverse transcriptase. The DNA copy then integrates into the host genomewhere it remains and functions as any other host gene, this being referred to as the proviral state. Thesecond phase of the life cycle occurs as the result of transcription of the provirus, which results in RNAmolecules that can serve as both mRNAs for the production of viral proteins and full length transcriptsthat eventually are packaged into new virions.

The influence of oxidative stress and reactive oxygen species (ROS) on the HIV life cycle has receivedconsiderable attention. HIV activation, the increase in the generation of new virus particles from an HIV-infected cell, can be stimulated by ROS. This is due, at least in part, to the presence of ROS-responsivetranscription regulatory regions located in the viral long terminal repeats [15]. Following ROS exposure,transcription of the provirus is stimulated by activated transcription factors such as NFκB and AP-1,stimulating the production of new infectious viral particles and initiating new rounds of viral replication.As would be anticipated, anti-oxidants have been shown to suppress the activation of HIV replicationby ROS. Several molecules with anti-oxidant activity have been shown to be effective in this regard,including glutathione, glutathione ester and N-acetyl-cysteine [15]. Similarly, anti-oxidant enzymeshave also been shown to suppress HIV activation, including catalase and glutathione peroxidases [19].

In addition to being able to suppress the activation of HIV from the proviral state, other data implicatedthis selenium-containing enzyme in the progression of an HIV infection. Levels of the cytosolic form ofglutathione peroxidase were found to be reduced in both symptomatic and asymptomatic HIV-infectedindividuals [6,12] and chronically infected human T cells [18]. These observations stimulated studiesdesigned to directly evaluate whether elevated GPx levels, achieved by introduction of a classicalGPx (GSHPx-1) expression construct, could influence the progression of an acutely spreading HIVinfection [17]. A retroviral-based construct containing the cDNA for GSHPx-1 expressed from a CMVpromoter was introduced into human SupT1, individual clones containing the construct were isolatedby limiting dilution and total GPx activity was assessed by standard coupled spectrophotometric assay.The GPx activity of cells infected with vector-alone was similar to that observed in native cells, whilesignificant increments in activity between 2–4 fold were observed in cell lines obtained following theintroduction of the GSHPx-1 expression construct. These lines showing elevated GPx activity, aswell as cells infected with vector alone, were infected with HIV-1 at a low M.O.I. (0.002) and thecultures were followed for two weeks. Surprisingly, cell lines with elevated GPx levels exhibited adramatic decline in viability and syncytia formation 5 days post-infection, while the control infectedcells maintained viability until day 14. The accelerated appearance of cytopathic effects associated withthe HIV infection was associated with the enhancement of viral replication as evidenced by the increasedappearance of the HIV-1 proteins p24 and reverse transcriptase in the supernatant of infected cultures.In these same studies, it was also shown that the converse was true, that reducing anti-oxidant levelscould attenuate viral spread. This was achieved using the compound BSO, a competitive inhibitor ofγ-glutamylcysteine synthetase, the rate limiting enzyme in glutathione synthesis. Non-toxic levels ofBSO were able to reduce viral replication both in SupT1 cells, and peripheral blood lymphocytes [17].These results are consistent with those showing that low levels of H2O2 (> 0.05 mM) were able to inhibitHIV-1 replication in CD4+ HeLa cells [16].

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While the mechanism by which increased GSHPx-1 expression stimulates HIV-1 replication remainsunproven, it seems likely that it involves the suppression of the host cells apoptotic response to viralinfection. Over-expression of GSHPx-1 has been shown to prevent apoptosis of human T cells followinginterleukin withdrawal [9] while the mitochondrial phospholipid hydroperoxide glutathione peroxide(PHGPx) has been shown to suppress apoptosis via the mitochondrial death pathway [14]. In addition,several anti-apoptotic genes, including bcl-2, adenovirus E1B 19K, and poxvirus Crm A have been shownto similarly result in the stimulation of an acutely spreading HIV-1 infection, as has been the chemicalapoptosis inhibitor z-VAD-fmk ([17], for references).

The experiments described above present the possibility that GSHPx-1 could have contradictory effectson the ultimate replication of retroviruses such as HIV. Early in the replication cycle, increased GSHPx-1expression could stimulate the spread of an acute viral infection by reducing the ability of infectedcells to kill themselves and in that way limit viral spread. Later in the life cycle, following the reversetranscription of the viral genome and integration into the infected cell’s genome, anti-oxidants suchas GSHPx-1 could potentially limit viral spread from chronically infected cells by suppressing geneexpression from ROS-responsive transcriptional regulatory sequences. Because of the complexity of theconsequences of GSHPx-1 activity on the entire replicative life cycle of HIV, we have urged caution inthe use of selenium and anti-oxidants in general in the treatment of AIDS or persons who are infectedwith HIV [17].

3. Glutathione peroxidases in viral genomes

If indeed GSHPx-1 expression is capable of circumventing a defensive host mechanism at the timeof infection, it is not surprising that viruses have been shown to have acquired homologues of thisselenoprotein during the course of their evolution. The best described example of this became evidentwhen the entire genome sequence of Molluscum contagiosum virus (MCV), a human poxvirus regardedat the etiologic agent associated with skin cancers in children and immunosuppressed adults, wasdetermined and shown to contain a homologue of GSHPx-1 [20]. It was shown that this gene includesthe sequence element in its 3-untranslated region (3’-UTR) required for the reading of in-frame UGAcodons as selenocysteine (referred to as a SECIS element), as do all other mammalian selenoproteingenes [8]. The SECIS element was located approximately 40 nucleotides downstream of the open readingframe of the MCV gene, and detected due to structural features, including a characteristic stem-loopstructure as well as a conserved 4 base paired region within the stem. That this SECIS element wasindeed functional was established by substituting it for the corresponding SECIS element present in the3’-UTR of type I iodothyronine deoidinase where it supported the appropriate recognition of the in-frameUGA codon of that proteins mRNA as selenocysteine. Furthermore, it was shown that the MCV GPxcontains selenium and, when over-expressed in human keratinocytes, protected those cells from the toxicconsequences of exposure to either H2O2 or ultraviolet irradiation [21]. Similarly, the recent presentationof the genome sequence of the pathogenic Fowlpox virus (FPV) revealed a GPx homologue with 44%identity to its human counterpart [2]. Collectively, these data indicate that members of the poxviruseshave acquired GPx homologues during their evolution. Whether the acquisition of this gene by thisfamily of viruses provides a benefit at the time of infection by making the suicide cellular response lesseffective, or by protecting the host cell from oxidative damage, or both remains to be determined.

In addition to the data described above indicating the presence of GPx-homologues in the poxvirues,Taylor and his colleagues have presented data based on molecular modeling to suggest that severalhuman pathogenic viruses, including HIV and Hepatitis C, contain “selenium-dependent GPx modules”

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within their genomes [23]. Recently, extensive modeling analysis expanding on earlier work evoking atranslational frameshift required to generate an HIV-encoded truncated GPx homologue, supported thepossibility of an HIV-1 encoded GPx [24]. In this same manuscript, the authors were able to express theseHIV-1 sequences in either human breast carcinoma cells or canine kidney cells, and achieve measurableincreases in GPx activity [24]. However, the relatively small increase in GPx activity obtained andthe need to provide a SECIS element from an established selenoprotein gene (rat 5’-deiodinase gene)still leaves some question as to whether the detected sequences encode a functional GPx gene. Thecharacterization of an HIV-encoded SECIS element and the detection of the predicted protein product inHIV-infected cells will be required to resolve this issue.

4. GPx, signal transduction and cancer

As described above, GPx activity can prevent HIV activation by suppressing expression from ROS-responsive transcriptional elements present in the viral LTR. These transcriptional elements include thosefor AP-1 and NFκB, molecules with defined roles in growth regulation, stress response and apoptosis.In addition, the mechanism by which GPx and other anti-oxidants suppress apoptosis is not yet defined,and it may include influencing signaling pathways that respond to ROS levels to promote cell death. IfGPx can influence signaling pathways associated with viral infections, it is therefore also possible thatthe anti-carcinogenic effects of selenium are also mediated, at least in part, via the effect of GPx andperhaps other anti-oxidant selenoproteins on signaling pathways associated with cell proliferation. Theinhibition of NFκB activation as a direct result of GSHPx-1 over-expression has been reported in T47Dcell transfectants [11].

There is increasing evidence that reactive oxygen species (ROS) are involved in signal transductionpathways and that such signaling is important in carcinogenesis [1]. Importantly, it has been recentlyshown that cell cycle progression in Ras transformed NIH 3T3 fibroblasts is mediated through ROSsignaling [10]. In this study, Ras-oncogene transformed fibroblasts produced a 2-fold increase in ROSlevels and this was inhibited by dominant negative forms of Ras. Interestingly, synthetic anti-oxidantswere shown to decrease the content of ROS and also inhibit DNA synthesis. Investigators using theNIH3T3 in vitro model have also demonstrated increased ROS following either Ras or Rac transfectionor stimulation with cytokines such as PDGF, EGF, TNF, or IL-1 [22]. In addition, results from our labindicate that the GPx-mimetic compound ebselen can inhibit transformation and that this is associatedwith inhibition of c-Jun NH2 – terminal kinase (JNK) activation (unpublished data). Thus, emergingevidence implicates ROS in a broad range of signal transduction responses, and specifically in thesignaling pathways downstream from Ras resulting in transformation. Since Ras and its downstreamtargets are estimated to be involved in many human cancers, these findings have implications for ourunderstanding of the cancer-suppressive mechanisms of anti-oxidants. While the existing paradigmhas been that anti-oxidants are potentially chemopreventive by limiting oxidative damage to DNA andthereby preventing mutation, anti-oxidants may also reduce cancer incidence by the inhibition of effectorpathways from activated oncogenes that utilize ROS.

5. Selenium-mediated effects on mutagenesis

Another possible commonality between the effects of selenium and selenoproteins on viral propagationand cancer risk may be found with regard to the effects of selenium on mutagenesis. Beck et al.

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showed that the genome of a benign, non-virulent coxsackievirus B3 (CVB3/0) recovered from micefed a selenium-deficient diet had undergone six nucleotide differences that resulted in this strain beingconverted to a virulent one capable of causing heart damage [4]. Subsequently, these results were extendedto show that the benign strain could be converted to the virulent one by passage of the virus through amouse genetically engineered to completely lack GSHPx-1 [3]. These results demonstrated increasedmutagenesis of the viral genome under conditions of selenium deficiency and implicate GSHPx-1 in thiseffect. Similarly, selenium has been shown to prevent mutations induced by ionizing radiation [7]. UsingChinese Hamster ovary cells, the effects of selenium on radiation-induced mutagenesis was quantifiedby measuring mutation frequencies at the hypoxanthine-guanine phosphoribosyl transferase (hprt) locus.Using this assay, it was shown that 30 nM selenium, provided to the culture medium 3 days prior toradiation, significantly reduced the mutation frequency at the hprt locus. Furthermore, this dose ofselenium resulted in a 5-fold increase in GPx activity in the treated cells. It is therefore possible thatsimilar mechanisms are involved with the increased mutation rate observed in selenium deficiency thatresults in a virulent coxsackievirus and the reduced mutation rate observed in irradiated mammaliantissue culture cells under conditions of selenium supplementation.

6. Conclusion

The role of selenium and selenoproteins in biological processes is a subject of intense study, and themechanisms involved are being elucidated. Influences on signaling pathways and mutagenesis appear tobe possible common themes in their effects on both viral infectivity and carcinogenesis. By examiningthe common effects of selenium in these different systems, new insights on the mechanisms of actionmight be considered and eventually manipulated to maximize the health benefits of this element.

Acknowledgements

This work was supported by Grant # R01 CA81153 to AMD.

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