herpes simplex virus 1 ( hsv1 ) is an -herpesvirus and the etiological cause of several diseases . hsv1 establishes a life - long latent infection in the sensory nerve ganglia of the nerves innervating the primary site of infection ( arvin et  al . , 2007 ) . the host immune response toward hsv1 entails both adaptive responses and innate responses , which includes both cell - intrinsic and cell - extrinsic responses ( arvin et  al . , 2007 ) . the cell - intrinsic response involves cell - autonomous sensing of viral infection and subsequent production of type i interferons that induce interferon - stimulated genes , including the potent antiviral protein tetherin ( levy et  al . , 2011 ) . tetherin acts as a tether of budding viruses , adhering them to the infected cell surface and limiting viral spread ( neil , 2013 ) . the cell - extrinsic innate immune response involves recruitment of cellular immune responders , including natural killer ( nk ) cells . nk cells are innate lymphocytes that kill tumor and virally infected cells ( vivier et  al . , 2011 ) and are considered members of the innate lymphoid cell ( ilc ) family , classified under the ilc1 family ( artis and spits , 2015 ) . the nk response to target cells is dictated by a balance of signals delivered from activating and inhibitory receptors . the inhibitory nk cell receptors recognize mainly major histocompatibility complex ( mhc ) class i proteins ( koch et  al . , 2013 ) , whereas the activating receptors recognize tumor- , pathogen- , and stress - induced ligands and self - ligands ( long et  al . , 2013 ) . a principal activating nk receptor is nkg2d , which recognizes several stress - induced ligands : mhc class i polypeptide - related sequence a and b ( mica and micb ) and the ul16 binding protein 1 - 6 ( ulbp1 - 6 ) ( lanier , 2015 ) , variably expressed by tumor cells and upregulated by cellular stresses , like those that occur during viral infections ( iannello and raulet , 2013 ) . additionally , nk cells express several other activating receptors , such as 2b4 , which recognizes cd48 , a cognate ligand / receptor expressed by other immune cells ( kim et  al . , 2014b ) . other activating nk receptors , such as the natural cytotoxicity receptors ( ncrs ) nkp30 , nkp44 , and nkp46 , bind mostly viral hemagglutinins and unknown cellular ligands ( koch et  al . , 2013 ) . micrornas ( mirnas or mirs ) are short non - coding rnas that bind target mrnas and repress their translation ( guo et  al . , 2010 ) . generally , the effect of mirnas on target protein expression is relatively mild ( baek et  al . , 2008 ) . notably , viruses , especially of the herpesvirus family , encode for mirnas that can modulate host gene expression ( skalsky and cullen , 2010 ) . indeed , we have previously demonstrated viral mirna - based immune - evasive mechanisms ( stern - ginossar et  al . , 2007 , nachmani et  al . , 2010 , nachmani et  al . , 2009 , bauman et  al . , 2011 ) . hsv1 encodes 27 known mirnas that are variably expressed during the viral lifecycle ( cui et  al . , 2006 , umbach et  al . , 2009 , the functions of the hsv1 mirnas are largely unknown , and it is unknown whether they affect the counter - hsv1 immune response . most cell surface proteins are membrane - integral ; however , some are covalently attached to glycosylphosphatidylinositol ( gpi ) lipid anchors . the gpi anchoring pathway is a multistep process that involves more than 25 proteins and in which the gpi - transamidase ( gpit ) complex covalently attaches the substrate pro - protein c terminus to the gpi moiety ( kinoshita , 2014 ) . gpit consists of five known proteins : pigk , pigs , pigt , pigu , and gpaa1 , of which pigk is widely accepted as the catalytic subunit ( kinoshita , 2014 ) . importantly , pigt and pigk share a disulfide bond , and pigt is thought to be involved in gpit complex stabilization ( kinoshita , 2014 ) . additionally , according to the predicted 3d model , pigt forms a -propeller gate that regulates access into the catalytic site of gpit ( eisenhaber et  al . , 2003 ) . here we demonstrate that hsv1 mir h8 targets pigt to avoid nk cell attack and the effects of tetherin . previously , we described viral mirna - based mechanisms utilized by herpesviruses to downregulate the expression of micb , an nkg2d ligand , to evade nk cell recognition and elimination ( stern - ginossar et  al . , 2007 , nachmani et  al . , 2009 , nachmani et  al . , 2010 ) . for this , we transduced the b cell line bjab , which endogenously expresses five nkg2d ligands  mica , micb , and ulbp13as well as many other known and unknown activating nk receptor ligands with lentiviral vectors encoding for artificial hairpins that are processed into mature hsv1 mirnas , as described previously ( stern - ginossar et  al . , 2007 ) . we found that 19 of the hsv1-encoded mirnas tested had no effect on the expression of the nkg2d stress - induced ligands ( figure  s1 ) . however , mir h8 did significantly reduce the expression level of two nkg2d ligands , ulbp2 and ulbp3 , and the expression of the 2b4 ligand cd48 ( figure  1a , quantified in figure  1b ) . notably , the mir h8 effect was very pronounced ( figure  1a ) , unlike the expected effects ( baek et  al . , 2008 , bauman et  al . , 2011 , stern - ginossar et  al . , 2007 , nachmani et  al . , 2009 , nachmani et  al . , 2010 ) . we verified expression of mir h8 in the transduced cells ( figure  1c ) . several other activating nk receptors , including the ncrs nkp44 and nkp46 , and the short - tailed killer - cell immunoglobulin - like receptors ( kirs ) bind to yet unknown ligands . to investigate whether mir h8 might affect the expression of these ligands , we utilized fusion proteins consisting of the extracellular portions of the different activating nk receptors fused to the fc portion of the human immunoglobulin 1 ( igg1 ) . expression of mir h8 did not affect the expression of any of these unknown activating nk cell ligands ( figure  1d ) . we also transduced bjab cells with the full - length precursor mir of h8 ( pre - h8 ) . using this modality the results obtained using pre - h8 were practically identical to the results obtained using the mir h8 from the artificial hairpin ( figure  1e ) . for this , we utilized the rna hybridization prediction program rna - hybrid ( rehmsmeier et  al . , 2004 ) . initially we examined the transcripts of ulbp2 , ulbp3 , and cd48 for a binding site for mir h8 , but no such binding sites were predicted ( data not shown ) . next , because ulbp2 , ulbp3 , and cd48 are all gpi - anchored proteins , we examined the transcripts of the proteins involved in gpi anchoring for predicted binding by mir h8 . one of these , pigt of the gpit complex , had a predicted binding site for mir h8 ( figure  1f , top ) . this site was in the 3 utr of pigt ; it displayed full seed ( bases 27 of the mir ) complementarity and had additional supplemental binding sites in the mir 3. to validate this bioinformatic prediction , we utilized a dual - luciferase assay , as described previously ( nachmani et  al . as can be seen in figure  1 g , when the 3 utr of pigt was expressed downstream of the luciferase , normalized luciferase activity was dramatically reduced in the presence of mir h8 . however , mutating 2 base pairs ( bp ) in the seed binding site for mir h8 ( figure  1f ) reversed most of the mir h8 effect ( figure  1 g ) . thus , mir next , to ensure that endogenous pigt is targeted by mir h8 , we stained bjab cells expressing mir h8 or a control mir with an anti - pigt antibody and quantified pigt expression using fluorescence - activated cell sorting ( facs ) analysis . as can be seen in figure  2a , quantified in figure  2b , pigt levels were reduced 40% in mir h8-expressing cells . pigt mrna levels remained unchanged ( figure  2c ) , suggesting that mir h8 induces translational inhibition . to validate that the effect of mir h8 on the expression of the activating nk ligands is indeed pigt - dependent , we targeted pigt directly using short hairpin rna ( shrna ) . pigt expression was reduced , as assessed by facs staining ( figure  2d , quantified in figure  2e ) and by qrt - pcr ( figure  2f ) . moreover , the expression of ulbp2 , ulbp3 , and cd48 was reduced to levels similar to the effect elicited of mir h8 ( figure  2 g ) . the functionality of the reductions observed in the levels of ulbp2 , ulbp3 , and cd48 was tested using nk cytotoxicity assays . first we co - incubated our mir h8 and control cells with the nk cell line yts - eco , known to kill target cells exclusively via 2b4-cd48 interactions ( chuang et  al . , 2000 ) . cells expressing mir h8 were killed significantly less than control mirna - expressing cells ( figure  3a ) . moreover , when mir h8 and control cells were incubated with primary bulk nk cells , the same effect was observed . this reduction in nk cell killing was also seen when the target cells expressed shpigt but not shscramble ( figure  3b ) . furthermore , when mir h8-expressing cells were rescued using exogenous pigt , the mir h8 effect was reversed ( figure  3c ) . next we tested whether mir h8 s effect on pigt might affect the expression of the gpi anchored protein tetherin ( also known as bst2/cd317 ) . for this , we stained our hsv1 mir h8- and control mirna - expressing cells with an anti - tetherin antibody and observed that expression of mir h8 significantly reduced the expression levels of tetherin ( figure  4a ; quantified in figure  4b ) . importantly , pre - h8 caused the same marked reduction in tetherin expression ( figure  4c ) , as did shpigt ( figure  4d ) . next , to examine the functionality of the decrease in tetherin expression , we infected mir h8- and control empty vector - expressing cells with hsv1-rfp . 72  hr after infection , the supernatants were used to titrate the viral load in vero cells . as expected , mir h8-expressing cells with low tetherin expression were significantly more susceptible to hsv1 infection and produced more viral progeny than control vector - expressing cells . indeed , shpigt elicited the same effect and so did shtetherin , but to a stronger degree ( figure  4e ) . additionally , reconstitution of pigt in mir h8-expressing cells ( h8-pigt ) reversed this effect compared with mir h8-expressing cells transfected with an empty vector ( h8-ds ) ( figure  4f ) . in this setting , the cells expressing shtetherin were also the cells that displayed the highest release of virus , and the controls , expressing a control mir with the empty vector ( control mir - ds ) and an shscramble , displayed the lowest viral release . when relative viral release was measured ( explained in experimental procedures ) , cells expressing mir h8 showed a higher proportion of viral release , as did the shtetherin- and shpigt - expressing cells compared with their appropriate controls ( figure  4 g ) . finally , to validate that the mir h8 effect on hsv1 spread is mediated via pigt and tetherin , we expressed tetherin exogenously in a549 cells and transduced these cells with mir h8 . as can be seen in figure  4h , mir h8 also enhanced viral release in this setting . thus , the effect of hsv1 mir h8 on pigt expression and the downstream effects on gpi - anchored proteins facilitate viral immune evasion at several different checkpoints . here we demonstrate that mir h8 significantly reduces the membrane expression of the nkg2d ligands ulbp2 and ulbp3 as well as the levels of cd48 , a ligand for 2b4 . we demonstrate that mir h8 targets the 3 utr of pigt of the gpit complex seed dependently . next we show that these effects are biologically significant and reduce nk cell killing of mir h8-expressing cells . finally , we show that the dominant cell - intrinsic antiviral protein tetherin , which is gpi - anchored , is also affected by mir h8 expression . tetherin is a cell - intrinsic , interferon - inducible viral restriction factor ( neil , 2013 ) . the importance of tetherin in the anti - hsv1 response is emphasized by the viral countermeasures employed to subvert its activity . two recent papers found that hsv1 counteracts tetherin via its viral host shutoff gene as well as by its glycoprotein m ( blondeau et  al . however , the effect of mir h8 via pigt on tetherin levels was prominent enough to be of biological significance in titration experiments even when these mechanisms were at play . interestingly , the effect of mir h8 on pigt levels and gpi anchoring did not encompass all gpi - anchored proteins . namely , ulbp1 ( which is also gpi - anchored ) levels did not change . this can be explained by preferential loading of different pro - proteins into the remaining gpit complexes if the predicted role of pigt as the  gatekeeper  ( eisenhaber et  al . , indeed , partial activity of the gpit can be expected because it was reported previously that a complete lack of pigt is required for complete blocking of gpi anchoring ( krawitz et  al . , 2013 ) , and hypomorphic activity of pigt resulting in partial loss of gpi - anchored protein presentation has been observed ( kvarnung et  al . , 2013 ) . thus , our results that demonstrate a decrease in pigt levels of approximately 40% are consistent with previous findings . recent research characterized the expression dynamics of hsv1 mirnas ( du et  al . , 2015 ) , and this finding is important when taken in conjunction with the results presented here because the role of mir h8 is somewhat clarified . the function of immune cells , particularly cd8 + t  cells , in maintaining viral latency and inhibiting hsv1 reactivation has been well established , and the role of nk cells in controlling hsv1 infection is also well documented ( egan et  al . , 2013 , moraru et  al . , 2012 ) . additionally , during hsv1 reactivation , the infected cells undergo various stresses stemming from the de novo expression of viral transcripts and proteins . taken together , these findings point to the importance of immune evasion during early hsv1 reactivation before the main viral protein - based mechanisms are fully active . for this , utilization of viral mirnas is ideal because mirnas are considered non - immunogenic ( boss and renne , 2011 ) . we do not know why mir h8 is expressed only during reactivation ; its effects could be utilized by the virus to subvert tetherin during lytic infection as well . one possible explanation could be that , during reactivation , hsv1 needs to replicate and egress from a few selected neurons to establish widespread infection , and the effects mir h8 might help the virus overcome this initial limitation . on the other hand , during most of the lytic infection , a  much larger mass of cells is infected , and thus a much higher viral load is enabled simply by virtue of infected cell mass . the abovementioned reactivation exclusivity of mir h8 expression presents a distinct problem : all experiments that aim to ascertain the effect of mir h8 in a bona fide infection will require a setting in which hsv1 is reactivated . to date , however , all existing experimental models of hsv1 reactivation involve research animals ( du et  al . , 2015 , ramakrishna et  al . , 2015 , yao et  al . , 2014 , kobayashi et  al . , 2012 , kim et  al . , 2014a , roehm et  al . , 2011 ) , and because mir h8 targets the human pigt transcript exclusively ( figures s2 and s3 ) , performing such experiments in animals would be non - informative . thus , an hsv1 latency reactivation model in human cells is needed in combination with specific mir h8 knockout ( ko ) viruses or an anti - mir h8 antagomir to determine the physiological importance of our findings . importantly , however , the expression levels of mir h8 in our system were comparable with the levels observed during reactivation in mouse ganglions ( figure  1c in this paper and figure  3 by du et  al . , 2015 ) thus , even though we did  not demonstrate the importance of mir h8 in a bona fide hsv1 reactivation setting , our experiments using the ectopic expression of mir h8 closely resemble the settings observed during hsv1 reactivation . in conclusion , in this paper we discover that hsv1 mir h8 targets pigt of the gpi anchoring pathway , enabling hsv1 to vitiate the host immune response , inhibiting elimination by nk cells and curbing the cell - intrinsic capacity to limit viral spreading . thus , by using a single microrna to target a basic cellular pathway , hsv1 is able to subvert the host immune response at several key points . the importance of understanding hsv1 biology and life cycle has recently been reiterated as its possible roles in alzheimer s disease ( itzhaki , 2014 ) and even cancer ( yun et  al . rna artificial hairpins ( table s1 ) , orthologs of pre - mirna , were generated and cloned into lentiviral vectors . transduction efficiency was assessed by gfp , and only cell populations with > 90% efficiency were used for experiments . a detailed description of the primers and procedures used for cell culture and cloning is included in the supplemental experimental procedures . the cytotoxic activity of nk cells against various targets was assessed in 5-hr s release assays as described previously ( mandelboim et  al . , 1996 ) . nk cells were isolated from peripheral blood using the human nk cell isolation kit and the cell - sep kit ( stemcell technologies ) according to the manufacturer s instructions . 2   10 bjab cells expressing mir h8 or an empty sin - gfp vector were infected with hsv-1 ok14 at a multiplicity of infection ( moi ) of 0.2 for 72  hr . supernatants were collected and used to infect vero cells , and , 48 hours post infection ( hpi ) , viral plaques were counted . for relative viral release assays , cells were infected with hsv1 as above for 24  hr . the cell pellet was then frozen and thawed three times to release viral progeny , and the supernatant and cell pellet fractions were used for titration in vero cells as above . hsv1 release proportions  = supernatant virus/(supernatant virus  + cell - associated virus ) . the hsv-1 ok14 was a gift from oren kobiler and generated by cotransfecting bamhi - digested phsv1(17+)lox - mrfpvp26 and purified hsv-1(17 + ) dna ( o. kobiler and l.w . enquist , personal communication ) . the phsv1(17+)lox - mrfpvp26 was a kind gift from drs . katinka dhner and beate sodeik ( nagel et  al . , 2012 , nagel et  al . , cells were incubated on ice for 1  hr with the primary antibody at a concentration of 0.2  g / well . appropriate secondary antibodies were applied for 30  min on ice at a concentration of 0.75  g / well . unless otherwise noted , cells expressing hsv1 mir - h15 were used as a control for the mir h8-expressing cells . for a detailed account and a list of antibodies used , see the supplemental experimental procedures . details of the qrt - pcr procedure and a list of primers are included in the supplemental experimental procedures and table s2 . the generation of the firefly luciferase constructs was as described previously ( stern - ginossar et  al . , 2007 ) . for more details , please consult the supplemental experimental procedures and table s3 . for statistical significance
summaryherpes simplex virus 1 ( hsv1 ) is a ubiquitous human pathogen that utilizes variable mechanisms to evade immune surveillance . 
 the glycosylphosphatidylinositol ( gpi ) anchoring pathway is a multistep process in which a myriad of different proteins are covalently attached to a gpi moiety to be presented on the cell surface . among the different gpi - anchored proteins there are many with immunological importance . 
 we present evidence that the hsv1-encoded mir h8 directly targets pigt , a member of the protein complex that covalently attaches proteins to gpi in the final step of gpi anchoring . 
 this results in a membrane down - modulation of several different immune - related , gpi - anchored proteins , including ligands for natural killer - activating receptors and the prominent viral restriction factor tetherin . 
 thus , we suggest that by utilizing just one of dozens of mirnas encoded by hsv1 , the virus can counteract the host immune response at several key points .