Leishmania mexicana cysteine peptidases (CPs) have been identified as important parasite virulence factors. More recently, a natural inhibitor of CPs (ICP) from L. mexicana has been characterized, and ICP mutants have been created. Infection of BALB/c mice with ICP null mutants or ICP reexpressing mutants resulted in nonhealing, progressively growing lesions albeit slightly attenuated compared with the growth of lesions produced by wild-type parasites. In contrast, BALB/c mice infected with mutants overexpressing ICP were able to significantly control lesion growth or heal. While BALB/c mice infected with wild-type parasites, ICP null mutants, or ICP reexpressing mutants produced significant antibody responses, including immunoglobulin E (IgE), no Th1 response, as indicated by antigen-induced splenocyte gamma interferon (IFN-γ) production, could be demonstrated. In contrast, BALB/c mice infected with mutants overexpressing ICP produced significantly less antibody, particularly IgE, as well as significantly reduced splenocyte interleukin-4 and enhanced IFN-γ production. BALB/c mice were able to resolve infection following infection with one ICP overexpressing clone, which was subsequently used for vaccination studies with BALB/c mice. However, no protection was afforded these mice when they were challenged with wild-type parasites. Nevertheless, two other mouse strains susceptible to L. mexicana, C3H and C57BL/6, vaccinated with overexpressing ICP mutants were able to control challenge infection associated with an enhanced Th1 response. This study confirms that L. mexicana CPs are virulence factors and that ICPs have therapeutic potential.
Plasmodium parasites express a potent inhibitor of cysteine proteases (ICP) throughout their life cycle. To analyze the role of ICP in different life cycle stages, we generated a stage-specific knockout of the Plasmodium berghei ICP (PbICP). Excision of the pbicb gene occurred in infective sporozoites and resulted in impaired sporozoite invasion of hepatocytes, despite residual PbICP protein being detectable in sporozoites. The vast majority of these parasites invading a cultured hepatocyte cell line did not develop to mature liver stages, but the few that successfully developed hepatic merozoites were able to initiate a blood stage infection in mice. These blood stage parasites, now completely lacking PbICP, exhibited an attenuated phenotype but were able to infect mosquitoes and develop to the oocyst stage. However, PbICP-negative sporozoites liberated from oocysts exhibited defective motility and invaded mosquito salivary glands in low numbers. They were also unable to invade hepatocytes, confirming that control of cysteine protease activity is of critical importance for sporozoites. Importantly, transfection of PbICP-knockout parasites with a pbicp-gfp construct fully reversed these defects. Taken together, in P. berghei this inhibitor of the ICP family is essential for sporozoite motility but also appears to play a role during parasite development in hepatocytes and erythrocytes.
Coordinated protease activity is essential to parasite survival. Throughout its life cycle, the Plasmodium parasite expresses a potent cysteine protease inhibitor that has the potential to inhibit parasite as well as host cell cysteine proteases. We have generated a stage-specific knockout of this inhibitor and were able to analyze its function in all life cycle stages. Interestingly, although constitutively expressed, the inhibitor primarily appears to play an important role in sporozoite gliding, liver stage development and egress from hepatocytes whereas blood stage parasites lacking the inhibitor exhibited only mild attenuation. Parasite sexual stage development was not affected and development continued normally within the mosquito. However, sporozoites lacking the inhibitor show a strong phenotype; they are completely blocked in motility and thus cannot transmigrate or invade cells. Complementation of knockout parasites by exogenous expression of the inhibitor completely restored parasite virulence.
The biological role of a natural inhibitor of cysteine peptidases (designated ICP) of Leishmania has been investigated by genetic manipulation of the parasite. Null mutants grew normally in vitro, were as infective to macrophages in vitro as wild-type parasites, but had reduced infectivity to mice. Mutants re-expressing ICP from a single gene gave partial restoration of virulence in vivo, whereas mutants over-expressing ICP secreted the inhibitor and showed markedly reduced virulence in mice. Promastigotes of the null mutants had similar cysteine peptidase activities as the wild-type parasites, suggesting that ICP is not required for the expression or processing of the enzymes. The only proteins found to bind to ICP in promastigote cell lysates were fully processed forms of CPA and CPB, showing that ICP does not bind in abundance either to zymogens of the cysteine peptidases or other leishmanial proteins. However, only a small proportion of ICP co-localised with CPA and CPB in the promastigote (in the endoplasmic reticulum and Golgi) and the majority of ICP resided in vesicles that are apparently distinct from endosomes and the multivesicular tubule (MVT)-lysosome. These data suggest that ICP has a role other than modulation of the activity of the parasite's own cysteine peptidases and their normal trafficking to the MVT-lysosome via the flagellar pocket. The finding that ICP partially co-localised with an endocytosed cysteine peptidase leads us to postulate that ICP has a role in protection of the parasite against the hydrolytic environment of the sandfly gut and/or the parasitophorous vacuole of host macrophages.
cysteine peptidase inhibitor; ICP; chagasin; lysosomes; endosomes; Leishmania
ICP is a chagasin-family natural tight binding inhibitor of Clan CA, family C1 cysteine peptidases (CPs). We investigated the role of ICP in Trypanosoma brucei by generating bloodstream form ICP-deficient mutants (Δicp). A threefold increase in CP activity was detected in lysates of Δicp, which was restored to the levels in wild type parasites by re-expression of the gene in the null mutant. Δicp displayed slower growth in culture and increased resistance to a trypanocidal synthetic CP inhibitor. More efficient exchange of the variant surface glycoprotein (VSG) to procyclin during differentiation from bloodstream to procyclic form was observed in Δicp, a phenotype that was reversed in the presence of synthetic CP inhibitors. Furthermore, we showed that degradation of anti-VSG IgG is abolished when parasites are pretreated with synthetic CP inhibitors, and that parasites lacking ICP degrade IgG more efficiently than wild type. In addition, Δicp reached higher parasitemia than wild type parasites in infected mice, suggesting that ICP modulates parasite infectivity. Taken together, these data suggest that CPs of T. brucei bloodstream form play a role in surface coat exchange during differentiation, in the degradation of internalized IgG and in parasite infectivity, and that their function is regulated by ICP.
Plasmodium falciparum (Pf) blood stages express falstatin, an inhibitor of cysteine proteases (ICP), which is implicated in regulating proteolysis during red blood cell infection. Recent data using the Plasmodium berghei rodent malaria model suggested an additional role for ICP in the infection of hepatocytes by sporozoites and during liver stage development. Here we further characterize the role of ICP in vivo during infection with Plasmodium yoelii (Py) and Pf. We found that Py-ICP was refractory to targeted gene deletion indicating an essential function during asexual blood stage replication, but significant down-regulation of ICP using a regulated system did not impact blood stage growth. Py-ICP localized to vesicles within the asexual blood stage parasite cytoplasm, the parasitophorous vacuole, and was exported to dynamic exomembrane structures in the infected erythrocyte. In sporozoites, expression was observed in rhoptries, in addition to intracellular vesicles distinct from TRAP containing micronemes. During liver stage development, Py-ICP was confined to the parasite compartment until the final phase of liver stage development when, after parasitophorous vacuole membrane breakdown, it was released into the infected hepatocyte. Finally, we identified the cysteine protease yoelipain-2 as a binding partner of Py-ICP during blood stage infection. These data show that ICP may be important in regulating proteolytic processes during blood stage development, and is likely playing a role in liver stage-hepatocyte interactions at the time of exoerythrocytic merozoite release.
The herpes simplex virus type 1 (HSV-1) alpha or immediate-early proteins ICP4 (IE175), ICP0 (IE110), and ICP27 (IE63) are trans-acting proteins which affect HSV-1 gene expression. We previously showed that ICP27 in combination with ICP4 and ICP0 could act as a repressor or an activator in transfection assays, depending on the target gene (R. E. Sekulovich, K. Leary, and R. M. Sandri-Goldin, J. Virol. 62:4510-4522, 1988). To investigate the regions of the ICP27 protein which specify these functions, we constructed a series of in-frame insertion and deletion mutants in the ICP27 gene. These mutants were analyzed in transient expression assays for the ability to repress or to activate two different target genes. The target plasmids used consisted of the promoter regions from the HSV-1 beta or early gene which encodes thymidine kinase and from the beta-gamma or leaky late gene. VP5, which encodes the major capsid protein, each fused to the chloramphenicol acetyltransferase gene. Our previous studies showed that induction of pTK-CAT expression by ICP4 and ICP0 was repressed by ICP27, whereas the stimulation of pVP5-CAT expression seen with ICP4 and ICP0 was significantly increased when ICP27 was also added. In this study, a series of transfection assays was performed with each of the ICP27 mutant plasmids in combination with plasmids containing the ICP4 and ICP0 genes with each target. The results of these experiments showed that mutants containing insertions or deletions in the region from amino acids 262 to 406 in the carboxy-terminal half of the protein were unable to stimulate expression of pVP5-CAT but were able to repress induction of pTK-CAT activity by ICP4 and ICP0. Mutants in the carboxy-terminal 78 amino acids lost both activities; that is, these mutants did not show repression of pTK-CAT activity or stimulation of pVP5-CAT activity, whereas mutants in the hydrophilic amino-terminal half of ICP27 were able to perform both functions. These results show that the carboxy-terminal half of ICP27 is important for the activation and repression functions. Furthermore, the carboxy-terminal 62 amino acids are required for the repressor activity, because mutants with this region intact were able to repress. Analysis of the DNA sequence showed that there are a number of cysteine and histidine residues encoded by this region which have some similarity to zinc finger metal-binding regions found in other eucaryotic regulatory proteins. These results suggest that the structural integrity of this region is important for the function of ICP27.
Malaria is transmitted when motile sporozoites are injected into the dermis by an infected female Anopheles mosquito. Inside the mosquito vector, sporozoites egress from midgut-associated oocysts and eventually penetrate the acinar cells of salivary glands. Parasite-encoded factors with exclusive vital roles in the insect vector can be studied by classical reverse genetics. Here, we characterized the in vivo roles of Plasmodium berghei falstatin/ICP (inhibitor of cysteine proteases). This protein was previously suggested to act as a protease inhibitor during erythrocyte invasion. We show by targeted gene disruption that loss of ICP function does not affect growth inside the mammalian host but causes a complete defect in sporozoite transmission. Sporogony occurred normally in icp(−) parasites, but hemocoel sporozoites showed a defect in continuous gliding motility and infectivity for salivary glands, which are prerequisites for sporozoite transmission to the mammalian host. Absence of ICP correlates with enhanced cleavage of circumsporozoite protein, in agreement with a role as a protease regulator. We conclude that ICP is essential for only the final stages of sporozoite maturation inside the mosquito vector. This study is the first genetic evidence that an ICP is necessary for the productive motility of a eukaryotic parasitic cell.
Cysteine proteases and their inhibitors are considered ideal drug targets for the treatment of a wide range of diseases, including cancer and parasitic infections. In protozoan parasites, including Leishmania, Trypanosoma, and Plasmodium, cysteine proteases play important roles in life cycle progression. A mouse malaria model provides an unprecedented opportunity to study the roles of a parasite-encoded inhibitor of cysteine proteases (ICP) over the entire parasite life cycle. By precise gene deletion, we found no evidence that ICP influences disease progression or parasite virulence. Instead, we discovered that this factor is necessary for parasite movement and malaria transmission from mosquitoes to mammals. This finding in a fast-moving unicellular protozoan has important implications for malaria intervention strategies and the roles of ICPs in the regulation of eukaryotic cell migration.
Cysteine proteases play a crucial role in the development of the human malaria parasites Plasmodium falciparum and Plasmodium vivax. Our earlier studies demonstrated that these enzymes are equipped with specific domains for defined functions and further suggested the mechanism of activation of cysteine proteases. The activities of these proteases are regulated by a new class of endogenous inhibitors of cysteine proteases (ICPs). Structural studies of the ICPs of Trypanosoma cruzi (chagasin) and Plasmodium berghei (PbICP) indicated that three loops (termed BC, DE, and FG) are crucial for binding to target proteases. Falstatin, an ICP of P. falciparum, appears to play a crucial role in invasion of erythrocytes and hepatocytes. However, the mechanism of inhibition of cysteine proteases by falstatin has not been established. Our study suggests that falstatin is the first known ICP to function as a multimeric protein. Using site-directed mutagenesis, hemoglobin hydrolysis assays and peptide inhibition studies, we demonstrate that the BC loop, but not the DE or FG loops, inhibits cysteine proteases of P. falciparum and P. vivax via hydrogen bonds. These results suggest that the BC loop of falstatin acts as a hot-spot target for inhibiting malarial cysteine proteases. This finding suggests new strategies for the development of anti-malarial agents based on protease-inhibitor interactions.
Closely related African trypanosomes cause lethal diseases but display distinct host ranges. Specifically, Trypanosoma brucei brucei causes nagana in livestock but fails to infect humans, while Trypanosoma brucei gambiense and Trypanosoma brucei rhodesiense cause sleeping sickness in humans. T. b. brucei fails to infect humans because it is sensitive to innate immune complexes found in normal human serum known as trypanolytic factor (TLF) 1 and 2; the lytic component is apolipoprotein-L1 in both TLFs. TLF resistance mechanisms of T. b. gambiense and T. b. rhodesiense are now known to arise through either gain or loss-of-function, but our understanding of factors that render T. b. brucei susceptible to lysis by human serum remains incomplete. We conducted a genome-scale RNA interference (RNAi) library screen for reduced sensitivity to human serum. Among only four high-confidence ‘hits’ were all three genes previously shown to sensitize T. b. brucei to human serum, the haptoglobin-haemoglobin receptor (HpHbR), inhibitor of cysteine peptidase (ICP) and the lysosomal protein, p67, thereby demonstrating the pivotal roles these factors play. The fourth gene identified encodes a predicted protein with eleven trans-membrane domains. Using chemical and genetic approaches, we show that ICP sensitizes T. b. brucei to human serum by modulating the essential cathepsin, CATL, a lysosomal cysteine peptidase. A second cathepsin, CATB, likely to be dispensable for growth in in vitro culture, has little or no impact on human-serum sensitivity. Our findings reveal major and novel determinants of human-serum sensitivity in T. b. brucei. They also shed light on the lysosomal protein-protein interactions that render T. b. brucei exquisitely sensitive to lytic factors in human serum, and indicate that CATL, an important potential drug target, has the capacity to resist these factors.
The interplay among host innate immunity and resistance mechanisms in African trypanosomes has a major impact on the host range of these tsetse-fly transmitted parasites, defining their ability to cause disease in humans. A genome-scale RNAi screen identified a highly restricted set of four genes that sensitise trypanosomes to human serum: those encoding the haptoglobin-haemoglobin receptor, a predicted trans-membrane channel, a lysosomal membrane-protein and the cysteine peptidase inhibitor. An analysis of the cysteine peptidases revealed cathepsin-L as the protease regulated by the inhibitor – and with the capacity to render the parasite resistant to lysis by human serum. These findings emphasise the importance of parasite factors for the delivery and stability of host toxins. They also shed light on the control of proteolysis by parasites and potential unanticipated consequences of therapies that target the parasite proteases.
The herpes simplex virus type 1 immediate-early protein ICP0 enhances expression of a spectrum of viral genes alone and synergistically with ICP4. To test whether ICP0 and ICP4 interact physically, we performed far-Western blotting analysis of proteins from mock-, wild-type-, and ICP4 mutant virus-infected cells with in vitro-synthesized [35S]Met-labeled ICP0 and ICP4 as probes. The ICP4 and ICP0 polypeptides synthesized in vitro exhibited molecular weights similar to those of their counterparts in herpes simplex virus type 1-infected cells, and the in vitro-synthesized ICP4 was able to bind to a probe containing the ICP4 consensus binding site. Far-Western blotting experiments demonstrated that ICP0 interacts directly and specifically with ICP4 and with itself. To further define the interaction between ICP0 and ICP4, we generated a set of glutathione S-transferase (GST)-ICP0 fusion proteins that contain GST and either ICP0 N-terminal amino acids 1 to 244 or 1 to 394 or C-terminal amino acids 395 to 616 or 395 to 775. Using GST-ICP0 fusion protein affinity chromatography and in vitro-synthesized [35S]Met-labeled ICP0 and ICP4, ICP4 was shown to interact preferentially with the fusion protein containing ICP0 C-terminal amino acids 395 to 775, whereas ICP0 interacted efficiently with both the N-terminal GST-ICP0 fusion proteins and the C-terminal GST-ICP0 fusion proteins containing amino acids 395 to 775. Fusion protein affinity chromatography also demonstrated that the C-terminal 235 amino acid residues of ICP4 are important for efficient interaction with ICP0. Collectively, these results reveal a direct and specific physical interaction between ICP0 and ICP4.
Trypanosoma brucei is the etiological agent of Human African Trypanosomiasis, an endemic parasitic disease of sub-Saharan Africa. TbCatB and rhodesain are the sole Clan CA papain-like cysteine proteases produced by the parasite during infection of the mammalian host and are implicated in the progression of disease. Of considerable interest is the exploration of these two enzymes as targets for cysteine protease inhibitors that are effective against T. brucei.
Methods and Findings
We have determined, by X-ray crystallography, the first reported structure of TbCatB in complex with the cathepsin B selective inhibitor CA074. In addition we report the structure of rhodesain in complex with the vinyl-sulfone K11002.
The mature domain of our TbCat•CA074 structure contains unique features for a cathepsin B-like enzyme including an elongated N-terminus extending 16 residues past the predicted maturation cleavage site. N-terminal Edman sequencing reveals an even longer extension than is observed amongst the ordered portions of the crystal structure. The TbCat•CA074 structure confirms that the occluding loop, which is an essential part of the substrate-binding site, creates a larger prime side pocket in the active site cleft than is found in mammalian cathepsin B-small molecule structures. Our data further highlight enhanced flexibility in the occluding loop main chain and structural deviations from mammalian cathepsin B enzymes that may affect activity and inhibitor design. Comparisons with the rhodesain•K11002 structure highlight key differences that may impact the design of cysteine protease inhibitors as anti-trypanosomal drugs.
Proteases are ubiquitous in all forms of life and catalyze the enzymatic degradation of proteins. These enzymes regulate and coordinate a vast number of cellular processes and are therefore essential to many organisms. While serine proteases dominate in mammals, parasitic organisms commonly rely on cysteine proteases of the Clan CA family throughout their lifecycle. Clan CA cysteine proteases are therefore regarded as promising targets for the selective design of drugs to treat parasitic diseases, such as Human African Trypanosomiasis caused by Trypanosoma brucei. The genomes of kinetoplastids such as Trypanosoma spp. and Leishmania spp. encode two Clan CA C1 family cysteine proteases and in T. brucei these are represented by rhodesain and TbCatB. We have determined three-dimensional structures of these two enzymes as part of our ongoing efforts to synthesize more effective anti-trypanosomal drugs.
Herpes simplex virus type 1 immediate-early protein ICP0 is an E3 ubiquitin ligase of the RING finger class that degrades several cellular proteins during infection. This activity is essential for its functions in stimulating efficient lytic infection and productive reactivation from latency. ICP0 targets a number of proteins that are modified by the small ubiquitin-like SUMO family of proteins, and it includes a number of short sequences that are related to SUMO interaction motifs (SIMs). Therefore, ICP0 has characteristics that are related to those of cellular SUMO-targeted ubiquitin ligase enzymes. Here, we analyze the impact of mutation of a number of SIM-like sequences (SLSs) within ICP0 on HSV-1 replication and gene expression and their requirement for ICP0-mediated degradation of both sumoylated and unmodified promyelocytic leukemia (PML) and other sumoylated cellular proteins. One SLS in the central portion of the ICP0 sequence (SLS4) was found to be absolutely required for targeting cellular sumoylated species in general and sumoylated forms of PML other than those of PML isoform I. Mutation of a group of SLSs in the C-terminal quarter of ICP0 also reduced ICP0-mediated degradation of sumoylated PML in a cooperative manner. Although mutation of individual SLSs caused only modest decreases in viral replication, combined mutation of SLS4 with SLS sequences in the C-terminal quarter of the protein reduced plaque formation efficiency by up to two orders of magnitude. These results provide further evidence that the biological activities of ICP0 are connected with host cell sumoylation events.
IMPORTANCE Herpes simplex virus type 1 protein ICP0 plays important roles in regulating the initial stages of lytic infection and productive reactivation from latency. ICP0 mediates its effects through inducing the degradation of cellular proteins that have repressive effects on viral gene expression. An increasing number of cellular proteins are known to be sensitive to ICP0-mediated degradation; therefore, it is important to understand how ICP0 selects its substrates for degradation. This study identifies sequence motifs within ICP0 that are involved in targeting cellular proteins that are modified by the SUMO family of ubiquitin-like proteins and describes how mutation of combinations of these motifs causes a 100-fold defect in viral infectivity.
Elsewhere this laboratory reported that (i) ICP0 interacts with cyclin D3 but not D1 or D2. The 3 cyclins independently partially rescue ΔICP0 mutants. (ii) Interaction with cyclin D3 is required for the switch from nuclear to cytoplasmic accumulation of ICP0. (iii) In infected cells cdk4 is activated whereas cdk2 is not. Inhibition of cdk4 results in nuclear retention of ICP0. Overexpression of cyclin D3 reverses the effect of the inhibitor. Here we report the following. (i) cdk4 interacts with ICP0, ICP4, and possibly with ICP8. This interaction is required to recruit cdk4 initially to ND10 and later to the viral replication compartments. (ii) cdk4 inhibitor I reduced or delayed the transcription and ultimately translation of mRNAs of ICP4, ICP27, or ICP8 and to a lesser extent that of the ICP0 gene in wild-type virus-infected cells. (iii) Overexpression of cyclin D3 resulted in a more rapid transcription of these genes. In the presence of inhibitor, the rates of accumulation of the products of these genes resemble those of wild-type virus in the absence of inhibitor. (iv) Overexpression of cyclin D3 also results in mobilization of cdk6 in nuclei of infected cells. We conclude that ICP0 encodes a function that enhances the recruitment of cyclin D3 to ND10 structures to activate cdk4 and that ICP0 along with other viral proteins recruits cdk4 to ND10 structures and ultimately to replication compartments for enhanced expression of viral genes and viral DNA synthesis.
Plasmodium parasites must control cysteine protease activity that is critical for hepatocyte invasion by sporozoites, liver stage development, host cell survival and merozoite liberation. Here we show that exoerythrocytic P. berghei parasites express a potent cysteine protease inhibitor (PbICP, P. berghei inhibitor of cysteine proteases). We provide evidence that it has an important function in sporozoite invasion and is capable of blocking hepatocyte cell death. Pre-incubation with specific anti-PbICP antiserum significantly decreased the ability of sporozoites to infect hepatocytes and expression of PbICP in mammalian cells protects them against peroxide- and camptothecin-induced cell death. PbICP is secreted by sporozoites prior to and after hepatocyte invasion, localizes to the parasitophorous vacuole as well as to the parasite cytoplasm in the schizont stage and is released into the host cell cytoplasm at the end of the liver stage. Like its homolog falstatin/PfICP in P. falciparum, PbICP consists of a classical N-terminal signal peptide, a long N-terminal extension region and a chagasin-like C-terminal domain. In exoerythrocytic parasites, PbICP is posttranslationally processed, leading to liberation of the C-terminal chagasin-like domain. Biochemical analysis has revealed that both full-length PbICP and the truncated C-terminal domain are very potent inhibitors of cathepsin L-like host and parasite cysteine proteases. The results presented in this study suggest that the inhibitor plays an important role in sporozoite invasion of host cells and in parasite survival during liver stage development by inhibiting host cell proteases involved in programmed cell death.
Plasmodium sporozoites are transmitted by Anopheles mosquitoes to the vertebrate host. They migrate through the skin before entering blood vessels and being transported with the bloodstream to liver sinusoids. There the sporozoites transmigrate through Kupffer cells and several hepatocytes before they invade a final hepatocyte and develop into thousands of merozoites. These daughter parasites are transported inside host cell-derived vesicles (merosomes) back to the bloodstream where they are finally released and infect red blood cells. Most of these processes depend on the activity of proteases, which must be tightly controlled to avoid proteolytic destruction of the parasite. We have identified a potent cysteine protease inhibitor of the rodent parasite Plasmodium berghei, which is expressed throughout the life cycle of the parasite. The inhibitor appears to play a role in sporozoite invasion of host cells and in parasite survival during liver stage development by inhibiting host cell proteases involved in programmed cell death.
The herpes simplex virus 1 (HSV-1) infected cell proteins 0 and 4 (ICP0 and ICP4) are multifunctional proteins extensively posttranscriptionally processed by both cellular and viral enzymes. We examined by two-dimensional separations the posttranslational forms of ICP0 and ICP4 in HEp-2 cells and in human embryonic lung (HEL) fibroblasts infected with wild-type virus, mutant R325, lacking the sequences encoding the US1.5 protein and the overlapping carboxyl-terminal domain of ICP22, or R7914, in which the aspartic acid 199 of ICP0 was replaced by alanine. We report the following (i) Both ICP0 and ICP4 were sequentially posttranslationally modified at least until 12 h after infection. In HEL fibroblasts, the processing of ICP0 shifted from A+B forms at 4 h to D+G forms at 8 h and finally to G, E, and F forms at 12 h. The ICP4 progression was from the A′ form noted at 2 h to B′ and C′ forms noted at 4 h to the additional D′ and E′ forms noted at 12 h. The progression tended to be toward more highly charged forms of the proteins. (ii) Although the overall patterns were similar, the mobility of proteins made in HEp-2 cells differed from those made in HEL fibroblasts. (iii) The processing of ICP0 forms E and F was blocked in HEL fibroblasts infected with R325 or with wild-type virus and treated with roscovitine, a specific inhibitor of cell cycle-dependent kinases cdc2, cdk2, and cdk5. R325-infected HEp-2 cells lacked the D′ form of ICP4, and roscovitine blocked the appearance of the most highly charged E′ form of ICP4. (iv) A characteristic of ICP0 is that it is translocated into the cytoplasm of HEL fibroblasts between 5 and 9 h after infection. Addition of MG132 to the cultures late in infection resulted in rapid relocation of cytoplasmic ICP0 back into the nucleus. Exposure of HEL fibroblasts to MG132 late in infection resulted in the disappearance of the highly charged ICP0 G isoform. The G form of ICP0 was also absent in cells infected with R7914 mutant. In cells infected with this mutant, ICP0 is not translocated to the cytoplasm. (v) Last, cdc2 was active in infected cells, and this activity was inhibited by roscovitine. In contrast, the activity of cdk2 exhibited by immunoprecipitated protein was reduced and resistant to roscovitine and may represent a contaminating kinase activity. We conclude from these results that the ICP0 G isoform is the cytoplasmic form, that it may be phosphorylated by cdc2, consistent with evidence published earlier (S. J., Advani, R. R. Weichselbaum, and B. Roizman, Proc. Natl. Acad. Sci. USA 96:10996–11001, 2000), and that the processing is reversed upon relocation of the G isoform from the cytoplasm into the nucleus. The processing of ICP4 is also affected by R325 and roscovitine. The latter result suggests that ICP4 may also be a substrate of cdc2 late in infection. Last, additional modifications are superimposed by cell-type-specific enzymes.
Herpes simplex virus-1 immediate-early protein ICP0 activates viral genes during early stages of infection, affects cellular levels of multiple host proteins and is crucial for effective lytic infection. Being a RING-type E3 ligase prone to auto-ubiquitination, ICP0 relies on human deubiquitinating enzyme USP7 for protection against 26S proteasomal mediated degradation. USP7 is involved in apoptosis, epigenetics, cell proliferation and is targeted by several herpesviruses. Several USP7 partners, including ICP0, GMPS, and UHRF1, interact through its C-terminal domain (CTD), which contains five ubiquitin-like (Ubl) structures. Despite the fact that USP7 has emerged as a drug target for cancer therapy, structural details of USP7 regulation and the molecular mechanism of interaction at its CTD have remained elusive. Here, we mapped the binding site between an ICP0 peptide and USP7 and determined the crystal structure of the first three Ubl domains bound to the ICP0 peptide, which showed that ICP0 binds to a loop on Ubl2. Sequences similar to the USP7-binding site in ICP0 were identified in GMPS and UHRF1 and shown to bind USP7-CTD through Ubl2. In addition, co-immunoprecipitation assays in human cells comparing binding to USP7 with and without a Ubl2 mutation, confirmed the importance of the Ubl2 binding pocket for binding ICP0, GMPS and UHRF1. Therefore we have identified a novel mechanism of USP7 recognition that is used by both viral and cellular proteins. Our structural information was used to generate a model of near full-length USP7, showing the relative position of the ICP0/GMPS/UHRF1 binding pocket and the structural basis by which it could regulate enzymatic activity.
USP7 is a cellular protein that binds and stabilizes many proteins involved in multiple pathways that regulate oncogenesis and as such is recognized as a potential target for cancer therapy. In addition, USP7 is targeted by several viral proteins in order to promote cell survival and viral infection. One such protein is the ICP0 protein of herpes simplex virus 1, which must bind USP7 in order to manipulate the cell in ways that enable efficient viral infection. Here we use a structural approach to define the mechanism of the USP7-ICP0 peptide interaction, revealing a novel binding site on USP7. We then used this information to identify two cellular proteins, GMPS and UHRF1, that also bind USP7 through this binding site. Therefore we have identified a new mechanism by which both viral and cellular proteins can target USP7. This information will be useful for the development of strategies to block specific protein interactions with USP7.
Varicella-zoster virus is the etiological agent of chickenpox and zoster in humans and belongs to the Alphaherpesvirinae subfamily within the family Herpesviridae. Much of the current understanding of gene regulation in alphaherpesviruses has been derived from studies of the prototype herpes simplex virus (HSV). In HSV, two virus-encoded, trans-regulatory proteins, ICP4 and ICP27, are essential for the replicative cycle of the virus. ICP4 is important in modulating HSV genes of all three kinetic classes, whereas the trans-regulatory effects of ICP27 are primarily associated with the expression of late genes. Recent evidence indicates that the trans-regulatory effects of ICP27 involve posttranscriptional processing of target gene transcripts (R. M. Sandri-Golding and G. E. Mendoza, Genes Dev. 6:848-863, 1992). The ICP27 homolog in varicella-zoster virus is a 452-amino-acid polypeptide encoded by the open reading frame 4 (ORF4) gene. Contrary to what is found with ICP27, we show that the ORF4 polypeptide is a transcriptional activator of diverse target promoters and has a critical requirement for the presence of upstream elements within these promoters to mediate its transcriptional effects. Evidence is also presented to implicate a critical role for the cysteine-rich, C-terminal region of the ORF4 polypeptide in its trans-regulatory functions. Specifically, by oligonucleotide-directed site-specific mutagenesis, we demonstrate that of 10 cysteine residues in the ORF4 polypeptide, only C-421 and C-426 are essential for transactivator function and suggest that these cysteine residues may participate in critical protein-protein interactions rather than protein-nucleic acid interactions to mediate ORF4 inducibility.
Herpes simplex virus type 1 (HSV-1) protein ICP27 has many important functions during infection that are achieved through interactions with a number of cellular proteins. In its role as a viral RNA export protein, ICP27 interacts with TAP/NXF1, the cellular mRNA export receptor, and both the N and C termini of ICP27 must be intact for this interaction to take place. Here we show by bimolecular fluorescence complementation (BiFC) that ICP27 interacts directly with TAP/NXF1 during infection, and this interaction failed to occur with an ICP27 mutant bearing substitutions of serines for cysteines at positions 483 and 488 in the C-terminal zinc finger. Recently, we showed that ICP27 undergoes a head-to-tail intramolecular interaction, which could make the N- and C-terminal regions accessible for binding to TAP/NXF1. To determine the importance of intramolecular association of ICP27 to its interaction with TAP/NXF1, we performed BiFC-based fluorescence resonance energy transfer (FRET) by acceptor photobleaching. BiFC-based FRET showed that the interaction between ICP27 and TAP/NXF1 occurred in living cells upon head-to-tail intramolecular association of ICP27, further establishing that TAP/NXF1 interacts with both the N and C termini of ICP27.
ICP27 is a key regulatory protein during herpes simplex virus type 1 (HSV-1) infection. ICP27 interacts with a number of cellular proteins, and an important question asks how these interactions are regulated during infection. We showed previously that ICP27 undergoes a head-to-tail intramolecular interaction, and here we show that the cellular mRNA export receptor protein TAP/NXF1 interacts with ICP27 after its head-to-tail association. Several proteins that interact with ICP27 require that the N and C termini of ICP27 be intact. These results demonstrate that the head-to-tail interaction of ICP27 may regulate some of its protein interactions perhaps through alternating between open and closed configurations.
During the early stages of herpes simplex virus type 1 (HSV-1) infection, viral immediate-early regulatory protein ICP0 localizes to and disrupts cellular nuclear structures known as PML nuclear bodies or ND10. These activities correlate with the functions of ICP0 in stimulating lytic infection and reactivating quiescent HSV-1. The disruption of ND10 occurs because ICP0 induces the loss of the SUMO-1-modified forms of PML and the subsequent proteasome-mediated degradation of the PML protein. The functions of ICP0 are largely dependent on the integrity of its zinc-binding RING finger domain. Many RING finger proteins have been found to act as ubiquitin E3 ligase enzymes, stimulating the production of conjugated polyubiquitin chains in the presence of ubiquitin, the ubiquitin-activating enzyme E1, and the appropriate E2 ubiquitin-conjugating enzyme. Substrate proteins that become polyubiquitinated are then subject to degradation by proteasomes. We have previously shown that purified full-length ICP0 acts as an efficient E3 ligase in vitro, producing high-molecular-weight polyubiquitin chains in a RING finger-dependent but substrate-independent manner. In this paper we report on investigations into the factors governing the degradation of PML induced by ICP0 in a variety of in vivo and in vitro assays. We found that ICP0 expression increases the levels of ubiquitinated PML in transfected cells. However, ICP0 does not interact with or directly ubiquitinate either unmodified PML or SUMO-1-modified PML in vitro, suggesting either that additional factors are required for the ICP0-mediated ubiquitination of PML in vivo or that PML degradation is an indirect consequence of some other activity of ICP0 at ND10. Using a transfection-based approach and a family of deletion and point mutations of PML, we found that efficient ICP0-induced PML degradation requires sequences within the C-terminal part of PML and lysine residue 160, one of the principal targets for SUMO-1 modification of the protein.
The herpesvirus proteins HSV-1 ICP27 and HVS ORF57 promote viral mRNA export by utilizing the cellular mRNA export machinery. This function is triggered by binding to proteins of the transcription-export (TREX) complex, in particular to REF/Aly which directs viral mRNA to the TAP/NFX1 pathway and, subsequently, to the nuclear pore for export to the cytoplasm. Here we have determined the structure of the REF-ICP27 interaction interface at atomic-resolution and provided a detailed comparison of the binding interfaces between ICP27, ORF57 and REF using solution-state NMR. Despite the absence of any obvious sequence similarity, both viral proteins bind on the same site of the folded RRM domain of REF, via short but specific recognition sites. The regions of ICP27 and ORF57 involved in binding by REF have been mapped as residues 104–112 and 103–120, respectively. We have identified the pattern of residues critical for REF/Aly recognition, common to both ICP27 and ORF57. The importance of the key amino acid residues within these binding sites was confirmed by site-directed mutagenesis. The functional significance of the ORF57-REF/Aly interaction was also probed using an ex vivo cytoplasmic viral mRNA accumulation assay and this revealed that mutants that reduce the protein-protein interaction dramatically decrease the ability of ORF57 to mediate the nuclear export of intronless viral mRNA. Together these data precisely map amino acid residues responsible for the direct interactions between viral adaptors and cellular REF/Aly and provide the first molecular details of how herpes viruses access the cellular mRNA export pathway.
When invading host cells, herpes viruses highjack cellular components to allow them to replicate. It has been long recognized that each herpes virus has a specific signature adaptor protein which, among other functions, inserts viral mRNA into the cellular mRNA nuclear export pathway, enabling production of viral proteins by the host cell. This process has been extensively studied in vivo and in vitro, but despite many efforts, the molecular and structural mechanisms of key interactions between viral adaptors and cellular mRNA export factors have not been described. Here we present the first atomic-resolution structure of the key complex between the archetypal viral adaptor ICP27 (from Herpes simplex virus 1) and the cellular mRNA export factor REF, responsible for introducing viral mRNA into the cellular nuclear export pathway. We demonstrate that despite the absence of obvious sequence similarity, the adaptor protein ORF57 from a different herpes virus (Herpesvirus saimiri) binds REF in the same site and in a similar way. We have identified and studied amino acid residues responsible for REF recognition. Together the data provide the first molecular insight into how herpesviral signature proteins recognize cellular proteins, obtaining access to the cellular mRNA export machinery.
Herpes simplex virus 1 (HSV-1)-infected cell protein 0 (ICP0) is a multifunctional protein that plays a key role in overcoming numerous facets of host innate immunity. A key function of ICP0 that requires an intact RING finger domain is that of an ubiquitin E3 ligase: ICP0 interacts with at least three ubiquitin-conjugating enzymes of which one, UbcH5a, is required for degradation of PML and SP100. A preceding report showed that ICP0 is highly unstable at very early times after infection but becomes stable at later times. We report here that (i) the degradation of ICP0 is not infected cell specific, (ii) the degradation does not require the interaction of ICP0 with either UbcH5a, UbcH6, or UbcH9, (iii) ICP0 is degraded both early and late in cells infected with a mutant lacking the UL13 protein kinase, (iv) ICP0 encoded by wild-type virus or the ΔUL13 mutant is stable in cells transfected with a plasmid encoding UL13 before infection, (v) ICP0 carrying mutations in the RING finger domain is stable both early and late in infection, and, finally, (vi) in cells infected with both wild type and RING finger mutant only the wild-type ICP0 is rapidly degraded at early times. The results suggest that the stability of ICP0 is mediated by the UL13 protein kinase and that the target of proteolysis is a site at or near the RING domain of ICP0.
IMPORTANCE ICP0, a major regulatory protein of HSV-1, turns over rapidly early in infection but becomes stable at late times. We report that stabilization requires the presence of UL13 protein kinase and that an ICP0 with mutations in RING finger is stable. In mixed infections mutant ICP0 is stable, whereas the wild-type ICP0 is degraded. Our findings suggest that the lifestyle of HSV-1 requires an ICP0 that turns over rapidly if late proteins are absent.
The herpes simplex virus type 1 (HSV-1) alpha proteins ICP4, ICP0, and ICP27 are trans-acting proteins which affect HSV-1 gene expression. To investigate potential interactions between these alpha products and to determine the specificity of action of the alpha proteins in combination with each other compared with their activities individually, we performed a series of transient-expression assays. In these assays we used plasmids containing the alpha genes encoding ICP4, ICP0, and ICP27 either singly or in combination as effectors and HSV-1 genes of different kinetic classes and heterologous genes as targets. The HSV-1 targets consisted of promoter-regulatory domains from alpha (ICP0 and ICP27), beta (thymidine kinase and alkaline exonuclease), beta-gamma (glycoprotein D, glycoprotein B, and VP5), and gamma (glycoprotein C) genes, each fused to the chloramphenicol acetyltransferase (CAT) gene. The heterologous target genes consisted of the simian virus 40 early promoter with enhancer and the Rous sarcoma virus long terminal repeat promoter and enhancer each fused to the CAT gene. Target promoter activity was measured by the assay of CAT activity in extracts of transfected cells and by Northern (RNA) blot hybridization of CAT mRNA. The results of these experiments showed that ICP4 activated only HSV-1 target genes, whereas ICP0 activated all of the targets and ICP27 had little effect on any of the targets. ICP4 and ICP0 had a synergistic effect when inducing HSV-1 targets, but they did not have this effect on the heterologous targets pSV2-CAT or pRSV-CAT. In fact, lower levels of CAT activity and CAT mRNA were found in the presence of both effectors than with ICP0 alone. Most interestingly, although the effector plasmid containing the ICP27 gene had little effect on its own, two different and marked effects depending on the target were observed when ICP27 was combined with ICP4 or ICP0 or both. A trans-repression of the induction seen with ICP4 and ICP0 was found when ICP27 was present in the transfections with pSV2-CAT, pRSV-CAT, pICP0-CAT, pICP27-CAT, pTK-CAT, pgD-CAT, pgB-CAT, and pgC-CAT. This resulted in CAT activity levels which were similar to or lower than the basal level of expression of the target genes in the absence of effector plasmids. This trans-repression occurred over a wide range of concentrations of input ICP27 plasmid. In contrast to this repressive effect of ICP27, a trans-activation was seen when ICP4, ICP0, and ICP27 plasmids were combined in transfections with pAE-CAT and pVP5-CAT as targets. This trans-activation also occurred over a 10-fold range of input ICP27 plasmid. These results suggest that ICP27 can facilitate both down
Bovine herpesvirus 1 (BoHV-1) infected cell protein 0 (bICP0) is an important transcriptional regulatory protein that stimulates productive infection. In transient transfection assays, bICP0 also inhibits interferon dependent transcription. bICP0 can induce degradation of interferon stimulatory factor 3 (IRF3), a cellular transcription factor that is crucial for activating beta interferon (IFN-β) promoter activity. Recent studies also concluded that interactions between bICP0 and IRF7 inhibit trans-activation of IFN-β promoter activity. The C3HC4 zinc RING (really important new gene) finger located near the amino terminus of bICP0 is important for all known functions of bICP0. A recombinant virus that contains a single amino acid change in a well conserved cysteine residue of the C3HC4 zinc RING finger of bICP0 grows poorly in cultured cells, and does not reactivate from latency in cattle confirming that the C3HC4 zinc RING finger is crucial for viral growth and pathogenesis. A bICP0 deletion mutant does not induce plaques in permissive cells, but induces autophagy in a cell type dependent manner. In summary, the ability of bICP0 to stimulate productive infection, and repress IFN dependent transcription plays a crucial role in the BoHV-1 infection cycle.
bovine herpesvirus 1 (BoHV-1); bICP0; interferon; IRF3; IRF7; transcriptional regulation
ICP4 is expressed during the immediate-early phase of infection by herpes simplex virus (HSV) and activates transcription of viral genes during subsequent phases of productive infection. Several members of the alpha-herpesvirus family encode regulatory proteins that have extensive homology with ICP4 and exhibit a transactivation domain (TAD) at the N terminus. The portions of ICP4 required for nuclear localization, DNA binding, and dimerization have been defined, but a domain that is specifically required for transactivation has not been identified. We have defined a promoter-specific ICP4 TAD by analysis of the activity of GAL4-ICP4 fusion proteins cotransfected into HeLa cells with a luciferase reporter gene linked to a promoter with five GAL4 binding sites. The transactivation activity of GAL4-ICP4 hybrids is located entirely within the first 139 residues of ICP4 and is significantly less potent than the activity of GAL4-TAD hybrids derived from ICP4 homologs. ICP4 residues 97 to 109 are a critical component of this N-terminal TAD. Transient transfection assays performed with nonfusion forms of ICP4 and luciferase genes linked to the HSV glycoprotein D (gD) or thymidine kinase (tk) promoter revealed that ICP4 residues 97 to 109 are required for induction of the gD promoter but are not required for induction of the tk promoter. Comparative experiments with ICP4 homologs revealed that the pseudorabies virus TAD is a potent activator of the gD promoter and a weak activator of the tk promoter. Complementation assays revealed that loss of ICP4 residues 97 to 109 reduced the yield of virus from infected cells nearly 500-fold compared to wild-type ICP4. We conclude that ICP4 residues 97 to 109 are a core component of a promoter-specific transactivation domain that is required for efficient replication of herpes simplex virus.
ICP27 is a multifunctional protein that is required for herpes simplex virus 1 mRNA export. ICP27 interacts with the mRNA export receptor TAP/NXF1 and binds RNA through an RGG box motif. Unlike other RGG box proteins, ICP27 does not bind G-quartet structures but instead binds GC-rich sequences that are flexible in structure. To determine the contribution of arginines within the RGG box, we performed in vitro binding assays with N-terminal proteins encoding amino acids 1 to 160 of wild-type ICP27 or arginine-to-lysine substitution mutants. The R138,148,150K triple mutant bound weakly to sequences that were bound by the wild-type protein and single and double mutants. Furthermore, during infection with the R138,148,150K mutant, poly(A)+ RNA and newly transcribed RNA accumulated in the nucleus, indicating that viral RNA export was impaired. To determine if structural changes had occurred, nuclear magnetic resonance (NMR) analysis was performed on N-terminal proteins consisting of amino acids 1 to 160 from wild-type ICP27 and the R138,148,150K mutant. This region of ICP27 was found to be highly flexible, and there were no apparent differences in the spectra seen with wild-type ICP27 and the R138,148,150K mutant. Furthermore, NMR analysis with the wild-type protein bound to GC-rich sequences did not show any discernible folding. We conclude that arginines at positions 138, 148, and 150 within the RGG box of ICP27 are required for binding to GC-rich sequences and that the N-terminal portion of ICP27 is highly flexible in structure, which may account for its preference for binding flexible sequences.