Identification of hematoxylin-stainable protein in epidermal keratohyalin granules as phosphorylated cystatin alpha by protein kinase C.

The hematoxylin-stainable protein (HSP) in keratohyalin granules of the newborn rat epidermis was found to have the same amino acid composition and the same inhibitory and immunological properties as cystatin alpha. However, only its pI value (4.7) differed from that of cystatin alpha (5.3). Alkaline phosphatase treatment of HSP changed its pI value from 4.7 to 5.3. This pI change was inhibited by EDTA, an inhibitor of alkaline phosphatase. Furthermore, 32P from [gamma-32P]ATP was incorporated into recombinant cystatin alpha by a protein kinase C (PKC) preparation in the presence of phosphatidyl serine and Ca2+ ions as co-factors. The incorporation increased dose-dependently with the added cystatin alpha and was inhibited significantly by H-7, a specific inhibitor of PKC. SDS-PAGE autoradiography of the 32P-labeled proteins showed that 32P was incorporated into the cystatin alpha. This incorporation was not observed by the action of cAMP-dependent protein kinase. Therefore, it is highly possible that the HSP is a phosphorylated cystatin alpha and that the phosphorylation is catalyzed specifically by PKC.     

Viral therapy: prospects for protease inhibitors

Antiviral activities of known protease inhibitors were assayed in virus-infected cell cultures. Some members of the cystatin superfamily, in particular chicken cystatin, were able to block virus replication. In a binding assay, using purified components, chicken and human cystatin were able to bind poliovirus protease with affinities which were reflected in their relative antiviral potencies. Prospects for application of protease inhibitors in clinical viral infections are discussed.     

Inhibition of cysteine protease and growth of Staphylococcus aureus V8 and poliovirus by phosphorylated cystatin alpha conjugate of skin

The inhibitory properties of phosphorylated cystatin alpha (P-cystatin alpha) and a conjugated protein of the P-cystatin alpha with filaggrin linker segment peptide (FLSP) against the growth of Staphylococcus bacteria and poliovirus were investigated. Both the P-cystatin alpha and the conjugated protein (P-cystatin alpha-FLSP conjugate) as a model for the cornified envelope of skin inhibited the cysteine protease activity of Staphylococcus aureus V8. The protease activity was inhibited by normal cornified envelope of newborn rat skin, which contains P-cystatin alpha, and P-cystatin alpha in cornified envelope of newborn rat skin also suppressed the growth of S. aureus V8. When P-cystatin alpha or P-cystatin alpha-FLSP conjugate was added to cultured HeLa cells infected with poliovirus, 50-70% of the cell-death due to poliovirus infection was prevented. The poliovirus 3C protease activity in the infected HeLa cells was inhibited by P-cystatin alpha or P-cystatin alpha-FLSP conjugate. As a result, the processing of viral capsid peptides was suppressed. These findings suggest that P-cystatin alpha and P-cystatin alpha-FLSP conjugate could play the role of the barrier against microorganism infections due to inhibition of their cysteine protease activities.     

Bactericidal effect of rat cystatin S on an oral bacterium Porphyromonas gingivalis

We tested antibacterial and antiviral activities of rat cystatin S, a cysteine proteinase inhibitor, belonging to the family 2 cystatins against 18 different bacterial species and poliovirus type 1 (Sabin). Rat cystatin S specifically inhibited the growth of a human oral anaerobic bacterium Porphyromonas gingivalis due to a bactericidal effect.     

Human cystatin, a new protein inhibitor of cysteine proteinases

A new low-molecular weight protein inhibitor of cysteine proteinases, human cystatin, was isolated from sera of patients with autoimmune diseases. It inhibits papain, human cathepsin H and cathepsin B. According to its partially determined amino-acid sequence, human cystatin is highly homologous to egg white cystatin, but only distantly related to stefin, the cytosolic protein inhibitor of cysteine proteinases isolated from human polymorphonuclear granulocytes. Very probably human cystatin is identical with human gamma-trace, a microprotein of known sequence but hitherto unknown function.     

Intracellular topology and epitope shielding of poliovirus 3A protein

The poliovirus RNA replication complex comprises multiple viral and possibly cellular proteins assembled on the cytoplasmic surface of rearranged intracellular membranes. Viral proteins 3A and 3AB perform several functions during the poliovirus replicative cycle, including significant roles in rearranging membranes, anchoring the viral polymerase to these membranes, inhibiting host protein secretion, and possibly providing the 3B protein primer for RNA synthesis. During poliovirus infection, the immunofluorescence signal of an amino-terminal epitope of 3A-containing proteins is markedly shielded compared to 3A protein expressed in the absence of other poliovirus proteins. This is not due to luminal orientation of all or a subset of the 3A-containing polypeptides, as shown by immunofluorescence following differential permeabilization and proteolysis experiments. Shielding of the 3A epitope is more pronounced in cells infected with wild-type poliovirus than in cells with temperature-sensitive mutant virus that contains a mutation in the 3D polymerase coding region adjacent to the 3AB binding site. Therefore, it is likely that direct binding of the poliovirus RNA-dependent RNA polymerase occludes the amino terminus of 3A-containing polypeptides in the RNA replication complex.     

Proteolysis of the p220 component of the cap-binding protein complex is not sufficient for complete inhibition of host cell protein synthesis after poliovirus infection.

Infection of cells with poliovirus results in the complete shutoff of host protein synthesis. It is presumed that proteolysis of the p220 component of the cap-binding protein complex that is required for the translation of host mRNAs is responsible for the shutoff phenomenon. In this paper, we show that when cells are infected with poliovirus in the presence of guanidine or 3-methylquercetin, both inhibitors of poliovirus replication, complete cleavage of p220 occurs by 3.5 h postinfection. However, under these conditions only 55 to 77% of host protein synthesis is suppressed. Results obtained with extracts prepared from poliovirus-infected cells were similar to those obtained in vivo. These results suggest that complete inhibition of host protein synthesis after poliovirus infection requires at least one event in addition to proteolysis of p220. Thus, proteolysis of p220 is probably necessary but not sufficient for total suppression of host protein synthesis after poliovirus infection.     

Cystatin C, a human proteinase inhibitor, blocks replication of herpes simplex virus.

Cystatin C is a human cysteine proteinase inhibitor present in extracellular fluids. Cystatin C and a tripeptide derivative (Z-LVG-CHN2) that mimics its proteinase-binding center, were tested for possible antiviral activity against herpes simplex virus type 1 (HSV) and poliovirus type 1. Both recombinant cystatin C and Z-LVG-CHN2 displayed strong inhibitory effects on HSV replication, whereas no significant effect on poliovirus replication was seen. The molar concentration of cystatin C that gave total inhibition of HSV replication was lower than that of either Z-LVG-CHN2 or of acyclovir, the drug currently most used against HSV infections. These results suggest that cysteine proteinase inhibitors might play a physiological role as inhibitors of viral replication and that such proteinase inhibitors, or peptide derivatives that mimic their proteinase-binding centers, might be used as antiviral agents.     

Adenovirus inhibition of cellular protein synthesis involves inactivation of cap-binding protein.

Adenovirus (Ad) infection results in a marked inhibition of cellular protein synthesis that initiates during the late phase of the viral infectious cycle. We show that the mechanism used for suppression of cellular protein synthesis during cell cycle progression is exploited by Ad to repress host and enhance late viral mRNA translation. Discrimination between cellular and late Ad mRNAs and inhibition of host protein synthesis are shown to involve viral-mediated underphosphorylation of cap-binding protein (CBP) and subsequent inactivation of CBP complex, a large enzymatic complex required for cap-dependent mRNA translation. Late Ad mRNAs, like those of poliovirus, possess the unique ability to translate independent of a normal cap recognition process and do not require the activity of CBP complex. Inhibition of cellular translation by these two viruses is quite similar, except that whereas CBP complex is proteolytically degraded by poliovirus, it is functionally inactivated by Ad.     

A human virus protein, poliovirus protein 2BC, induces membrane proliferation and blocks the exocytic pathway in the yeast Saccharomyces cerevisiae.

Inducible synthesis of poliovirus protein 2BC in Saccharomyces cerevisiae arrests cell growth in the G2 phase of the cell cycle, while no effects are observed upon expression of poliovirus genes 2B or 2C, either individually or in combination. Expression of 2BC induces a number of morphological modifications in yeast cells, one of the most striking being the proliferation of small membranous vesicles that fill most of the cytoplasm. These vesicles are morphologically similar to the cytopathic vacuoles that proliferate during the infection of human cells by poliovirus. The transport and processing of several yeast proteins, including vacuolar carboxypeptidase Y, aminopeptidase I or yeast alpha-mating factor, is hampered upon expression of poliovirus 2BC, suggesting that transport of proteins through the Golgi apparatus is impaired by this viral protein. Finally, a number of 2BC variants were generated and the effects of their expression on yeast growth, cellular morphology and protein processing were analyzed. 2BC variants defective in the NTPase activity were still able to interfere with yeast growth and the exocytic system, while deletion of 30 amino acids at the N-terminus of 2BC impairs its function. These findings lend support to the idea that 2BC, but not 2B or 2C, is the protein responsible for vesicle proliferation in poliovirus-infected cells. In addition, the activity of a human virus protein in yeast cells opens new avenues to investigate the exact location at which poliovirus 2BC interferes with the vesicular system and to test the action of other animal virus proteins potentially involved in modifying the vesicular system in mammalian cells.     

Effect of interferon treatment on blockade of protein synthesis induced by poliovirus infection

Treatment of HeLa cells with lymphoblastoid interferon leads to a drastic inhibition of infective poliovirus. Even relatively high concentrations of human lymphoblastoid interferon HuIFN-alpha (Ly) (400 IU/ml) do not prevent destruction of the cell monolayer after most of the cells have been infected with poliovirus. Analysis of macromolecular synthesis in a single step growth cycle of poliovirus in interferon-treated cells detected no viral protein synthesis. In spite of this inhibition of viral translation, the shut-off of host protein synthesis in interferon-treated cells is apparent when they are infected both at low and high multiplicities. Although viral RNA synthesis is inhibited considerably in cells treated with interferon, a certain amount is detected, suggesting that some viral replication takes place. Analysis of membrane permeability after poliovirus infection shows a leakage to 86Rb+ ions and modification of membrane permeability to the translation inhibitor hygromycin B at the moment when the bulk of virus protein synthesis occurs. These changes are delayed and even prevented if cells are pretreated with interferon. A situation is described in which host protein synthesis is shut-down with no major changes in membrane permeability, as studied by the two tests mentioned above. Prevention of viral gene expression by inactivation with ultraviolet light of the input virus or by treatment with cycloheximide blocks the shut-off of protein synthesis. This does not occur in the presence of 3 mM guanidine. These observations are in agreement with the idea that some poliovirus protein synthesis takes place in interferon-treated cells and this early gene expression is necessary to block cellular protein synthesis.     

Synthesis of a (desSer1 Ile29 Leu89) chicken cystatin gene, expression in E. coli as fusion protein and its isolation

A synthetic gene coding for the cysteine proteinase inhibitor (desSer1 Ile29 Leu89) chicken cystatin was cloned and expressed in E. coli. The gene was assembled from 12 oligonucleotides and inserted into vector pUC 8. Expression as fusion protein was performed in a temperature-inducible E. coli system. The expression product was synthesized as 20% of total E. coli protein. The fusion protein was purified, the chicken cystatin homologue was split off with CNBr and the N-terminal sequence confirmed up to position 37. The properties of the purified material correspond to those of natural chicken cystatin. The recombinant cystatin variant binds anti-chicken cystatin IgG, is inhibitorily active and displays Ki values with papain and with cathepsin B similar to those determined for natural chicken cystatin.     

Adenovirus late protein synthesis is resistant to the inhibition of translation induced by poliovirus

Inhibition of host protein synthesis after poliovirus infection has been suggested to be a consequence of the proteolytic degradation of a p220 polypeptide necessary to translate capped mRNAs. However, the synthesis of several adenovirus late proteins on capped mRNAs was resistant to poliovirus inhibition. Thus, the hexon protein was still made 8 h after poliovirus superinfection. The synthesis of other adenovirus proteins such as the fiber was much more sensitive to poliovirus-induced inhibition than the hexon, either in the absence or in the presence of guanidine. Detailed densitometric analyses clearly showed the differential behavior of several adenovirus late mRNAs to poliovirus shut-off of translation. This is striking in view of the fact that a common leader sequence in the 5' termini is present in the adenovirus late mRNAs. The use of 3-methyl quercetin, an inhibitor of poliovirus RNA synthesis (Castrillo, J. L., Vanden Berghe, D., and Carrasco, L. (1986) Virology 152, 219-227), showed that translation of several capped adenovirus mRNAs took place in poliovirus-infected cells after the synthesis of host proteins had ceased. The poliovirus mRNA and the adenovirus mRNA coding for the hexon protein are very efficient mRNAs and have a leader sequence of more than 740 and 250 nucleotides, respectively, with very rich secondary structures making it difficult to predict how the scanning model will operate on these two mRNAs.     

The host protein required for in vitro replication of poliovirus is a protein kinase that phosphorylates eukaryotic initiation factor-2

The HeLa cell protein (host factor) required for in vitro replication of poliovirus has been identified as a 67,000 dalton phosphoprotein. The purified protein displays three activities in vitro: stimulation of poliovirus RNA synthesis in the presence of poliovirus replicase, apparent self-phosphorylation, and phosphorylation of the alpha-subunit of eukaryotic protein synthesis initiation factor 2 (eIF-2). All three activities can be removed or inhibited by an antibody to host factor. Partially purified preparations of reticulocyte eIF-2 contain a similar phosphoprotein and display host factor activity in the viral RNA synthesis assay in vitro. In vitro phosphorylation of the 67 kd protein can be stimulated by low concentrations of double-stranded RNA. Addition of phosphorylated host factor in an in vitro RNA synthesis assay significantly changes the kinetics of viral RNA synthesis, indicating that protein phosphorylation may play an important role in viral RNA replication.     

Interactions of viral protein 3CD and poly(rC) binding protein with the 5' untranslated region of the poliovirus genome

The poly(rC) binding protein (PCBP) is a cellular protein required for poliovirus replication. PCBP specifically interacts with two domains of the poliovirus 5' untranslated region (5'UTR), the 5' cloverleaf structure, and the stem-loop IV of the internal ribosome entry site (IRES). Using footprinting analysis and site-directed mutagenesis, we have mapped the RNA binding site for this cellular protein within the stem-loop IV domain. A C-rich sequence in a loop at the top of this large domain is required for PCBP binding and is crucial for viral translation. PCBP binds to stem-loop IV RNA with six-times-higher affinity than to the 5' cloverleaf structure. However, the binding of the viral protein 3CD (precursor of the viral protease 3C and the viral polymerase 3D) to the cloverleaf RNA dramatically increases the affinity of PCBP for this RNA element. The viral protein 3CD binds to the cloverleaf RNA but does not interact directly with stem-loop IV nor with other RNA elements of the viral IRES. Our results indicate that the interactions of PCBP with the poliovirus 5'UTR are modulated by the viral protein 3CD.     

87 kDa tomato cystatin exhibits properties of a defense protein and forms protein crystals in prosystemin overexpressing transgenic plants

Transgenic tomato plants (Lycopersicon esculentum) overexpressing the prosystemin transgene have been shown previously to accumulate a soluble 87 kDa cystatin constitutively. We report here that this protein can be found in a crystalline form which can be purified using a glycerol/sucrose gradient. Midgut homogenate of third-instar larvae of two coleopteran pest insects, Callosobruchus maculatus and Zabrotes subfasciatus, had their proteolytic activity content significantly inhibited by tomato cystatin (TC). In leaves of wild-type tomato plants, cystatin mRNA accumulated systemically in response to wounding, treatment with methyl jasmonate (MJ) and when supplied with systemin, corroborating the anti-herbivorous activity. Accumulation of cystatin mRNA occurred when plants were supplied with chitosan and oligogalacturonic acid fragments (OGA), suggesting an effect of TC against pathogens. Moreover, this protein reduced the growth of two fungi, Fusarium solani and Trichoderma viride in vitro. Taken together, the data reinforce a role for TC in defense response against pests or pathogens.     

Inhibition of cysteine proteinases by a protein inhibitor from potato

The inhibitory specificity of a protein from potato tubers that inhibits cysteine proteinases (potato cysteine proteinase inhibitor, PCPI) has been compared with that of chicken egg-white cystatin. Most proteinases that are inhibited by cystatin were also inhibited by PCPI, but the potato inhibitor inhibited stem bromelain and fruit bromelain, which are not inhibited by cystatin, and for which no protein inhibitor of comparable potency has previously been described. In contrast, papaya proteinase IV was unaffected by PCPI as it is by the cystatins, and the exopeptidase, dipeptidyl peptidase I, is inhibited by cystatins, but was unaffected by PCPI. The differences in inhibitory specificity between these proteins may well reflect differences between superfamilies of cysteine proteinase inhibitors.     

Rapamycin stimulates viral protein synthesis and augments the shutoff of host protein synthesis upon picornavirus infection.

The immunosuppressant drug rapamycin blocks progression of the cell cycle at G1 in mammalian cells and yeast. We recently showed that rapamycin inhibits both in vitro and in vivo cap-dependent, but not cap-independent, translation. This inhibition is causally related to reduced phosphorylation and consequent activation of 4E-BP1, a repressor of the function of the cap-binding protein, eIF4E. Two members of the picornavirus family, encephalomyocarditis virus and poliovirus, inhibit phosphorylation of 4E-BP1. Since translation of picornavirus mRNAs is cap independent, inhibition of phosphorylation of 4E-BP1 could contribute to the shutoff of host protein synthesis. Here, we show that rapamycin augments both the shutoff of host protein synthesis and the initial rate of synthesis of viral proteins in cells infected with encephalomyocarditis virus and poliovirus.     

Multimerization of poly(rC) binding protein 2 is required for translation initiation mediated by a viral IRES

The cellular protein, poly(rC) binding protein 2 (PCBP2), is known to function in picornavirus cap-independent translation. We have further examined the RNA binding properties and protein-protein interactions of PCBP2 necessary for translation. We have studied its putative multimerization properties utilizing the yeast two-hybrid assay and in vitro biochemical methods, including glutathione S-transferase (GST) pull-down assays and gel filtration. Through genetic analysis, the multimerization domain has been localized to the second K-homologous (KH) RNA binding domain of the protein between amino acids 125 and 158. To examine the function of multimerization in poliovirus translation, we utilized the truncated protein, DeltaKH1-PCBP2, which is capable of multimer formation, but does not bind poliovirus stem-loop IV RNA (an interaction required for translation). Utilizing RNA binding and in vitro translation assays, this protein was shown to act as a dominant negative, suggesting that PCBP2 multimerization functions in poliovirus translation and RNA binding. Additionally, PCBP2 containing a deletion in the multimerization domain (DeltaKH2-PCBP2) was not able to bind poliovirus stem-loop IV RNA and could not rescue translation in extracts that were depleted of endogenous PCBP2. Results from these experiments suggest that the multimerization of PCBP2 is required for efficient RNA binding and cap-independent translation of poliovirus RNA. By examining the functional interactions of the cellular protein PCBP2, we have discovered a novel determinant in the mechanism of picornavirus cap-independent translation.     

Requirement of poly(rC) binding protein 2 for translation of poliovirus RNA.

Poly(rC) binding protein 2 (PCBP2) is one of several cellular proteins that interact specifically with a major stem-loop domain in the poliovirus internal ribosome entry site. HeLa cell extracts subjected to stem-loop IV RNA affinity chromatography were depleted of all detectable PCBP2. Such extracts were unable to efficiently translate poliovirus RNA, although extracts recovered from control columns of matrix unlinked to RNA retained full translation activity. Both translation and production of infectious progeny virus were restored in the PCBP2-depleted extracts by addition of recombinant PCBP2, but not by PCBP1, which is a closely related member of the protein family. The data show that PCBP2 is an essential factor, which is required for efficient translation of poliovirus RNA in HeLa cells.