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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.

Cystatin C, a low Mr cysteine proteinase inhibitor was isolated from bovine parotid glands by a procedure which includes alkaline treatment of the homogenate, affinity chromatography, gel filtration and ion exchange chromatography. The purified inhibitor has a pl of 8.0 and Mr of 14500. The identity with bovine cystatin C from colostrum was confirmed by N-terminal sequence of the inhibitor and amino acid composition. Cystatin C rapidly (kass = 5.5 x 10(7) M-1s-1) and tightly inhibits papain (Ki = 0.02 nM), whereas its interaction with bovine cathepsin B is substantially weaker (Ki = 4.4 nM). Bovine cystatin C also shows a weak antiviral effect on poliovirus infected human Hela cells.

A full-length and C-terminally truncated version of human endogenous retrovirus (HERV)-K10 protease were expressed in Escherichia coli and purified to homogeneity. Both versions of the protease efficiently processed HERV-K10 Gag polyprotein substrate. HERV-K10 Gag was also cleaved by human immunodeficiency virus, type 1 (HIV-1) protease, although at different sites. To identify compounds that could inhibit protein processing dependent on the HERV-K10 protease, a series of cyclic ureas that had previously been shown to inhibit HIV-1 protease was tested. Several symmetric bisamides acted as very potent inhibitors of both the truncated and full-length form of HERV-K10 protease, in subnanomolar or nanomolar range, respectively. One of the cyclic ureas, SD146, can inhibit the processing of in vitro translated HERV-K10 Gag polyprotein substrate by HERV-K10 protease. In addition, in virus-like particles isolated from the teratocarcinoma cell line NCCIT, there is significant accumulation of Gag and Gag-Pol precursors upon treatment with SD146, suggesting the compound efficiently blocks HERV-K Gag processing in cells. This is the first report of an inhibitor able to block cell-associated processing of Gag polypeptides of an endogenous retrovirus.

Cleavage of non-viral proteins is rarely observed with the HIV-1 protease (HIV pr). One such cleavage event occurs with bcl-2, an important cytoprotective protein. The loss of bcl-2 has biological consequences, leading to enhanced HIV replication and programmed death of the host cell. A strategy is proposed to suppress HIV with non-cleavable mutants of bcl-2.

New, potent therapies for HIV disease are available, based on synthetic inhibitors of the viral protease, an essential viral enzyme. The results in clinical trials have been impressive with most treated individuals benefiting in terms of reduced quantity of detectable virus, enhanced numbers of CD4 lymphocytes and improvements in quality and duration of life. However, there are some remaining negatives associated with the new drugs, including high cost, side effects and appearance of drug-resistant strains of HIV. Problems and future prospects for use of protease inhibitors and alternate approaches in AIDS are discussed.

Expression of the human immunodeficiency virus type 1 (HIV) protease in cultured cells leads to apoptosis, preceded by cleavage of bcl-2, a key negative regulator of cell death. In contrast, a high level of bcl-2 protects cells in vitro and in vivo from the viral protease and prevents cell death following HIV infection of human lymphocytes, while reducing the yields of viral structural proteins, infectivity, and tumor necrosis factor alpha. We present a model for HIV replication in which the viral protease depletes the infected cells of bcl-2, leading to oxidative stress-dependent activation of NF kappa B, a cellular factor required for HIV transcription, and ultimately to cell death. Purified bcl-2 is cleaved by HIV protease between phenylalanine 112 and alanine 113. The results suggest a new option for HIV gene therapy; bcl-2 muteins that have noncleavable alterations surrounding the HIV protease cleavage site.

Human immunodeficiency viruses have infected millions of people world-wide, and infection almost invariably leads, over a several year time frame, to progression to AIDS with high mortality.

There are three approved antiviral drug therapies for individuals infected with human immunodeficiency viruses (HIV). They are all nucleosides, with the most widely used known as AZT, and appear to act by inhibiting essential reactions carried out by the viral reverse transcriptase. Unfortunately, in patients treated for some time with one of these drugs, viral progeny often occur which are much less susceptible to the antiviral action of the inhibitor. The resistant phenotypes are often associated with a characteristic set of changes in the viral RNA sequence coding for the reverse transcriptase (1). As a rule, high-level resistance is accompanied by multiple amino acid changes in the transcriptase. This is not a rare phenomenon in anti-microbial chemotherapies, and occurs with antiviral as well as antibacterial medicines, and can reduce or even eliminate any value of the therapy. If there is an alternative therapy, that can be attempted. Unfortunately, for viruses, there are usually no useful options, and so targeting a second viral target is highly desirable.

The retroviruses encode a processing protease which is essential for virus replication. The enzyme is an aspartic protease, and cleaves both the gag and gag-pol gene products to yield mature virus containing active reverse transcriptase. In the absence of active protease only non-infectious viral particles are produced, so the enzyme has become an important target for drug designers, with the aim of benefiting people infected with the human immunodeficiency viruses, HIV-1 and HIV-2, and related retroviruses, which are agents of other serious diseases in man and domestic animals.

Only a few viral diseases are presently treatable because of our limited knowledge of specific viral target molecules. An attractive class of viral molecules toward which chemotherapeutic agents could be aimed are proteases coded by some virus groups such as retro- or picornaviruses (poliomyelitis, common cold virus). The picornavirus enzymes were discovered first, and they have now been characterized by a combination of molecular-genetic and biochemical approaches. Several laboratories have expressed the picornaviral enzymes in heterologous systems and have reported proteolytic activity, as well as the high cleavage fidelity diagnostic of the viral proteases. After dealing with several technical difficulties often encountered in standard genetic engineering approaches, one viral protease is now available to us in quantity and is amendable to mutagenic procedures. The initial outcome of the mutagenesis studies has been the confirmation of our earlier work with inhibitors, which suggested a cysteine active-site class. There is a clustering of active-site residues which may be unique to these viruses. The requirement for an active-site cysteine-histidine pair in combination with detailed information on the viral cleavage sites has permitted design of selective inhibitors with attractive antiviral properties. Future goals include investigation of the structural basis for selective processing and application of the cleavage specificity to general problems in genetic engineering.

Infectious virions of human rhinovirus type 2 (HRV-2) migrate during electrophoresis to pH 6.4 in a sucrose-stabilized pH gradient. As already reported, acidification of HRV-2 produces two kinds of noninfectious components both of which have lost the smallest virion polypeptide, VP4: One has lost RNA in addition to VP4 and sediments at about 80 S, while the other retains RNA and sediments at 135 S. The 80 S particles migrate to pH 4.5 upon electrophoresis while the 135 S particles migrate to pH 4.2. Natural top component of HRV-2 is also separable into two subpopulations that are isoelectric at pH 6.3 and pH 4.5. Only the particles isoelectric at pH 6.3 react with virion (D) specific serum and attach to HeLa cells. The polypeptide compositions of both the attaching and nonattaching fractions of natural top component are the same, and therefore the differences in activity and isoelectric point may reflect the arrangement of the polypeptides in the capsids. After a preparation of purified infectious virus is focused to its isoelectric point some particles are found at pH 4.5. These particles are relatively noninfectious, yet contain RNA and a full complement of polypeptides, and they sediment at 140 S. They are unable to attach to host cells. These results argue against a direct role of VP4 or RNA in the attachment process and support the view that protein conformation changes occur during the inactivation of HRV-2 virions.

Mouse L cells grown in suspension culture when treated with L cell interferon have a greater electrophoretic mobility toward the anode than control cells. This change in electrophoretic mobility depends on the concentration of interferon in the medium and the duration of interferon interaction with the cells. It is concluded that the interferon-treated cells have a greater net negative charge on the cell surface than control cells and it is suggested that the cell surface is altered because of the interaction with interferon.

HeLa cells contain endoprotease activity, detected by a sensitive, solid phase assay. The endoprotease has the ability to cleave a variety of protein substrates and is trypsin-like in its sensitivity to inhibitors. The activity is in part associated with cellular ribosomes and polysomes. A variety of biological and physical-chemical treatments which alter ribosomes or protein synthesis also directly affect the ribosomal protease activity.

Zinc ion inhibits the posttranslational cleavages of human rhinovirus-1A, encephalomyocarditis virus, and poliovirus polypeptides. Each virus displayed a different susceptibility to zinc. However, in each case the cleavages of the capsid precursor and the cleavages analogous to the C → D → E conversion in encephalomyocarditis virus were most sensitive to zinc. Higher concentrations of zinc resulted in the buildup of even larger precursor polypeptides of a size between 106,000 and 214,000 daltons. The sizes of these polypeptides and the relative position of their gene loci on the viral RNA were determined. These data were used to place these polypeptides in the over-all scheme of viral protein processing.

Treatment of human diploid fibroblasts with fibroblast interferon for 8 hr inhibited replication of vesicular stomatitis virus. When the total cell protein of cells treated with interferon for 8 hr was compared to the total cell protein of untreated cells by two-dimensional gel electrophoresis, the interferon-treated cells were found to contain four proteins not found in untreated cells. Addition of actinomycin D to the cells concurrently with interferon inhibited the synthesis of the four proteins. We conclude that these proteins are induced by interferon and that they may be involved in the inhibition of virus replication.

Zonal electrophoresis resolves two aggregates of poliovirus type 2 cytoplasmic polypeptides. The more negatively charged aggregate contains mainly noncapsid viral-specific polypeptides (NCVP) 2 and x, whereas the other consists of the capsid polypeptides (VP) 0, 1, 2, and 3 (VP0, VP1, VP2, VP3). After treatment with sodium deoxycholate (DOC), the aggregates sediment at 5 to 6 S . Their electrophoretic mobilities are unaffected by DOC or RNase. The capsid polypeptide aggregate is similar in mobility to virions but can be converted to a faster electrophoretic form, resembling empty capsids, by heating. If infected HeLa cells are allowed to synthesize poliovirus polypeptides in the presence of iodoacetamide, no capsid polypeptides are produced, but rather NCVP1a (the precursor to capsid polypeptides) is accumulated, along with NCVP2 and NCVPx. When analyzed by electrophoresis and centrifugation, uncleaved NCVP1a migrates with the NCVP2-x aggregate. NCVP1a can be cleaved to capsid-like polypeptides in vitro by using extracts of infected cells, but not uninfected cells, indicating either a virus-specified protease or a cellular enzyme activated during infection. After cleavage of NCVP1a by infected cell extracts, the capsid polypeptides which are produced dissociate from the NCVP2-x complex.

Growth, purification and electrophoretic analysis of three rhinoviruses and an enterovirus are described. It is shown that, with regard to structural polypeptides, the three rhinoviruses differ from other picornaviruses, and from each other. However, all three rhinoviruses resemble one another in possessing structural polypeptides of molecular weights 33,000 and 30,000 in similar proportions.

The use of protease inhibitors causes the accumulation of very large polypeptides (polyprotein) in tissue culture cells infected with either poliovirus or echovirus 12. The effectiveness of the inhibitor varies, depending on the cell line chosen. In infected monkey kidney cells, polyprotein is not cleaved when a chymotrypsin inhibitor is added, but in infected HeLa cells a trypsin inhibitor is most effective. Therefore, at least a part of the proteolytic activity is supplied by the host cell. Extracted viral polyprotein can be cleaved in vitro by trypsin or chymotrypsin. As estimated by migration in sodium dodecyl sulfate gels and antigenicity, chymotrypsin cleavage of the poliovirus polyprotein yields fragments which are similar to the in vivo product. The polyprotein is not in soluble form but is attached to a fast-sedimenting, membrane-bound structure. Proteolytic activities in cell extracts were assayed using polyprotein as substrate, and infected and uninfected extracts produced qualitatively dissimilar cleavages.

Agrobacterium tumefaciens phage LV-1 and its DNA were studied by physical and chemical methods. The phage has a bipyramidal head attached by a “neck” to a flexible tail composed by helically arranged substructures. The buoyant density of the virus in CsCl is 1.505 g/ml. and its sedimentation coefficient ( s ) is 430 S. The phage DNA is double-stranded and linear, and the single strands are intact for the length of the molecule. The sedimentation coefficient was 34.5 S for the native DNA, and the contour length was 14.2 μm. The s value and the length are consistent with a linear molecule of molecular weight 31 × 10 6 daltons. The melting temperature, buoyant density in CsCl, and chromatography of the phage DNA indicate that the mole fraction of guanine-cytosine (G-C) is 57%. This compares to 61% G-C for the DNA of the host bacterium, A. tumefaciens , which is lysogenized by the virus.

The replication of picornaviruses requires the action of proteolytic enzymes on viral precursor proteins. The protein cleavages are often crucial to the virus replication process, so that a complete understanding of their role could lead to development of new types of antiviral agents. On a more fundamental level, the characterization of the proteolytic reactions in virus infections is leading to a fuller appreciation of regulation of function by limited proteolysis. The picornavirus is a model in which proteolytic modifications yield macromolecular complexes with altered conformational and antigenic properties, and altered affinities for nucleic acids and receptors on cell membranes. Obviously, there is a general significance of these phenomena in understanding similar events in cellular interactions and in the numerous proteolytic events occurring in the tissues and fluids of higher organisms. For recent reviews of protein cleavage in picornavirus replication, see ref. 1–3.

Publisher Summary This chapter explores the recent developments in the biochemistry of viruses. The goal of molecular viroligists is to understand the structure, expression, and control of viral genes. Probably, the most complete genome map has been determined for the small transforming papovavirus SV40. The goal of mapping a mutant is to determine what function is affected by the mutation, and where that function is located on the physical map. Consequently, the protein or protein region responsible for the function can be determined. The variations found in the genomes of the different strains of all the viruses examined, suggests that one might expect the structures of their gene products to vary considerably, also this could account for variations in disease severity, and suggests that antiviral chemicals may vary considerably in effectiveness. Viruses that contain RNA genome can be classed as positive and negative-stranded viruses depending on whether their genome RNA serves directly as mRNA (positive) or requires RNA transcription to synthesize mRNA molecules (negative). The positive-stranded RNA viruses are best represented by the picornaviruses, of which poliovirus is a model virus. The negative-stranded RNA viruses are larger and more complex than the positive-stranded viruses. They contain an RNA polymerase that transcribes their genome RNAs into functional mRNAs. Probably without exception among the animal viruses, enzymatic cleavage of viral proteins occurs in or on the surface of infected cells. The evidence collected for some of the representative viruses indicates that the cleavages are essential for the successful production of viral progeny. The growth of the virus in a high yielding cell gave infectious virions with virtually all the glycoprotein in a cleaved form. Cleavage allows the virus to proceed successfully through the early stages of infection to the point where the viral nucleic acid is released into the host cell.

The regulation of gene expression in the interferon system falls into two distinct sub-topics. First is the induction of the interferon structural genes in cells infected with a virus or treated with some other inducer, such as double-stranded RNA or lectins. The second involves the mechanism of interferon action, in which several distinct genes are induced in interferon-treated cells to be transcribed and translated as the cells become virus resistant or cease their usual division cycle. In neither the induction of interferon nor its action is much known about specific effects on chromatin structure, but it is presumed that perturbations do occur as in other examples of regulated gene expression. Besides providing yet another “model” system, the interferons offer the following features: very rapid alterations in gene expression initiated at the cell surface by a relatively few inducing molecules (only one molecule in some cell types), leading to profound biological sequelae (refractility to viruses and blockade of cell cycle movements). The interferons are particularly interesting because cell division often stops in treated cells, in comparison to other hormonally-regulated systems where division rates are enhanced. The property of interferons to make cell cultures quiescent thus avoids the problem of studying cascades of gene expression which occur in growth-stimulated cells.

Summary Chicken cystatin is a previously described small protein which has the property of inhibiting cysteine active site proteases. The protein, when added to cultured human cells, alters the intracellular proteolytic processing of pollovirus proteins, and causes a reduction in virus yield. It is suggested that the cystatin is able to penetrate to the cellular cytoplasm and inhibit the action of the pollovirus protease.

The picornaviruses and togaviruses are important pathogens in man and some other animal species. Human infections caused by picornaviruses are found in all geographical regions and climates. The prototype of the group are the polioviruses, causitive agents of poliomyelitis, a devastating disease of the central nervous system. Thirty or more years ago poliovirus epidemics caused paralytic disease in thousands of children and young adults, especially among the middle and upper class of Western countries. The terrifying nature of these epidemics helped launch high visibility, public-funded animal virology research in the U.S. and western Europe. Today the fruits of that support are visible; the picornaviruses are among the best understood of all viruses at the biochemical level. Togaviruses are usually arthropod borne but they are not limited to tropical regions. Their infections are often subclinical but sometimes lead to encephalitides and hemorrhagic fevers which rank among the most lethal of all virus diseases.

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 anitviral potencies. Prospects for application of protease inhibitors in clinical viral infections are discussed.

The site of guanidine action on poliovirus type 2 was investigated. Physical-chemical and biological characterizations of viral mutants with altered guanidine sensitivities indicate changes in capsid polypeptides. Differences between mutants include electrophoretic mobilities of virions and of capsid subunits, stability of virions to high temperature, and chromatographic behavior of individual capsid polypeptides. No alterations were detected in nonstructural proteins of guanidine mutants. Aside from guanidine sensitivity of RNA synthesis, the differences between mutants in replication were limited to early events, particularly uncoating rates. No evidence for an effect of guanidine on processed capsid polypeptides was obtained; however, the capsid precursor polypeptide of sensitive virus is altered by guanidine treatment. A model is proposed in which a precursor to poliovirus structural polypeptides participates in viral RNA synthesis.

Ribosomes have been shown to contain a proteolytic activity, characterized as an endopeptidase with serine in the active center. The enzyme has been given the name cathepsin R, following the recommendations of Barrett et al. (in a publication from the Cold Spring Harbor Laboratory, New York) for naming new proteinases. The present paper contains evidence that cathepsin R in rat liver ribosomes is present in a cryptic form. Upon dissociation of ribosomes to subunits (and to minor extent also by 0.5 M KC1 washes), the cryptic proteinase is released. Activation of the released cathepsin R is effected by equilibration with 2 M NaC1/0.05 M sodium acetate, pH 4.8. The molecular weight of free cathepsin R is 25 000-30 000.

Previous studies have shown that primary cleavages in nascent picornavirus precursors are accomplished by cellular proteases. This study has characterized the enzyme in infected cells that produces the capsid polypeptides by secondary cleavages of viral precursors. The kinetics of the production of protease activity correlate with the time course of virus protein synthesis, and the new enzyme has characteristic pH and temperature optima. Guanidine and cycloheximide, which are inhibitors of virus RNA and protein synthesis, prevent production of the protease. As determined by introduction of amino acid analogs into the protease or inhibition by a leucyl chloromethyl ketone, the enzyme is synthesized at a time of infection when host cell proteins are not produced, and the enzyme copurified with a 40,000-dalton virus polypeptide present in the cytoplasm of infected cells. Wild-type levels of protease activity are produced by viral mutants that are defective in coat protein synthesis. The conclusion is that a non-structural poliovirus gene product participates in protein cleavages that produce the viral coat proteins.

Human rhinovirus 1A virions are found at two densities after cesium chloride gradient centrifugation. Dialysis of the virus against ammonium formate converts all the virus into the more dense form. Virus, dialyzed against ammonium formate and left for several days at 6 C forms crystals. Viral infectivity associated with washed crystals is lost by treatment at either pH 5 or pH 10.3, however, the integrity of the crystals is retained at the acidic pH. The crystals, as characterized by light and electron microscopy, are hexagonally prismatic with tapered, pyramidal ends. Electron images of replicated surfaces and thin sections of crystals show ordered packing of virus with a center-to-center repeat of 28.5 nm.

The rate of attachment of type 2 virions to suspensions of HeLa cells is much greater than that of type 14, but the number of receptor sites per cell is similar for each type. The receptor sites may be partly saturated with excess virions; attachment is greatly reduced after about 10 4 particles have been taken up per cell. A lack of saturation of type 14 receptors by excess type 2 indicates that their receptor sites are separate on the cell surface. Excess of type 2 blocks attachment of type 1A, however, and excess of type 14 blocks type 51. Attachment of the human rhinoviruses is temperature-dependent with a Q 10 of 2.7. The eclipse reaction is also temperature-dependent. At 34.5 C, the irreversible eclipse of cell-associated rhinovirus type 2 requires only a few minutes, whereas the rate of eclipse of cell-associated type 14 is considerably slower. The eclipse product of type 2 rhinovirus has been recovered from infected cells. It sediments at about 90% of the rate of the infective virions and is missing virus polypeptide 4 (the smallest of the capsid polypeptides). Upon being subjected to CsCl gradient centrifugation, virus polypeptide 2 is also lost but the product still contains ribonucleic acid and bands at about 1.45 g/cc.

Preparations of purified virions of human rhinoviruses type 2 and type 14 and of an equine rhinovirus are each homogeneous by the criteria of electron microscopy and rate-zonal and isopycnic gradient centrifugation. The rhinoviruses differ from each other in intrinsic infectivity and buoyant density in CsCl, while their sedimentation rates in sucrose gradients are similar, about 4% less than poliovirus. Purification in CsCl gradients produces an artifactual increase of 7% in the rhinovirus sedimentation constant, which is reversed with time after removal of CsCl from the preparation. Rhinovirus types differ in part in their four size classes of major polypeptides: VP1, VP2, VP3, and VP4. Crude preparations also contain empty capsids which sediment at half the rate of the virions, contain VP1 and VP3 and polypeptides larger than any in the virions (VPO). There is another naturally occurring component, the slow component, which sediments 90% as fast as intact virions. The slow component gives rise to a dense component in CsCl gradients which is 0.04–0.05 g/ml more dense than intact virions. The dense component contains RNA, VP1, and VP3. On the basis of indirect evidence, only the smallest polypeptide, VP4, is missing from the slow component, and VP2 is lost during CsCl centrifugation. Components which resemble the slow and dense components can be produced from purified virions in vitro by careful acidification.

The purification of human fibroblast interferon has been simplified to a two-step procedure consisting of affinity chromatography on Blue Sepharose and sodium dodecyl sulfate polyacrlamide gel electrophoresis. A preliminary amino acid composition and the sequence of the 13 amino-terminal residues of homogeneous interferon prepared by this method is reported.