Gene Order of Encephalomyocarditis Virus as Determined by Studies with Pactamycin

Previous work has shown that translation of the encephalomyocarditis (EMC) viral ribonucleic acid (RNA) generates at least three primary products, polypeptides A, F, and C. The A and C polypeptides then undergo post-translational cleavages to complete the production of the stable viral polypeptides (delta, beta, gamma, alpha, G, I, F, H, and E). In this communication we show that A, F, and C are produced in equimolar amounts giving further support to the theory that the RNA of picornaviruses has only a single site for the initiation of protein synthesis. The biosynthesis of viral proteins in EMC virus-infected HeLa cells was studied in the presence of pactamycin at concentrations which preferentially inhibit the initiation of protein synthesis. The amount of each polypeptide formed during the residual period of protein synthesis observed after the addition of pactamycin was used as a criterion for ordering the genes on the viral RNA. The results obtained indicate that the primary gene products are ordered on the EMC viral RNA 5' --> 3' A-F-C and that the stable products are ordered delta-beta-gamma-alpha-G-I-F-H-E. Moreover, the intermediate chains B and epsilon map in the capsid region, whereas the intermediate chain D maps in the E region. This order is largely consistent with previously established relationships of the viral polypeptides and thus indicates that pactamycin is a valid tool for "genetic" mapping of polycistronic RNA molecules with single initiation sites.     

Polyprotein processing in picornavirus replication

The primary translation product of the picornavirus genome is a single large protein which is processed to the mature viral polypeptides by progressive, co- and post-translational cleavages. Replication of the picornaviruses is thus entirely dependent upon the proteolysis of viral precursor proteins. In poliovirus, two virus-encoded proteinases have been identified that catalyze all but the final cleavage of the viral polyprotein. The final processing event, maturation of the virion polypeptide VPO, appears to occur by an unusual autocatalytic serine proteinase-like mechanism. Proteolytic processing of viral precursor proteins is basically similar in all picornaviruses, but recently it has become clear that there are also important differences between these viruses. Understanding of the processing events in picornavirus replication may ultimately lead to the discovery of specific inhibitors of the viral enzymes that could prove clinically useful as anti-viral agents.     

Protein cleavage in virus-infected cells

A variety of proteins, including viral precursor polypeptides, were bound to a solid support and used in a sensitive assay for proteolytic enzymes in HeLa cells. A trypsin-like endoprotease, present on ribosomes of HeLa cells, loses activity after picornavirus infection. The decline follows synthesis and processing of a viral protein. Inhibition of cellfree activity of HeLa protease occurs when protein trypsin inhibitors or double-stranded RNA are added. After the mid-point of infection, protease activity with enhanced specificity for viral substrates is detected. The new protease has a pH optimum and heat stability different from endogenous host enzymes, and is synthesized following infection. A viral mutant was isolated which produces a temperature-sensitive protease. The results indicate that a poliovirus gene product participates enzymatically in the final cleavages of some polioviral proteins. A model for the regulation of poliovirus replication based on specific proteolysis is presented.     

Processing of the intracellular form of the west Nile virus capsid protein by the viral NS2B-NS3 protease: an in vitro study.

According to the existing model of flavivirus polyprotein processing, one of the cleavages in the amino-terminal part of the flavivirus polyprotein by host cell signalases results in formation of prM (precursor to one of the structural proteins, M) and the membrane-bound intracellular form of the viral capsid protein (Cint) retaining the prM signal sequence at its carboxy terminus. This hydrophobic anchor is subsequently removed by the viral protease, resulting in formation of the mature viral capsid protein found in virions (Cvir). We have prepared in vitro expression cassettes coding for both forms of the capsid protein, for the prM protein, for the C-prM precursor, and for the viral protease components of West Nile flavivirus and characterized their translation products. Using Cint and Cvir translation products as molecular markers, we have observed processing of the intracellular form of the West Nile capsid protein by the viral protease in vitro both upon cotranslation of the C-prM precursor and the viral protease-encoding cassette and by incubation of C-prM translation products with a detergent-solubilized extract of cells infected with a recombinant vaccinia virus expressing the active viral protease. The cleavage of Cint by the viral protease at the predicted dibasic site was verified by introduction of point mutations into the cleavage site and an adjacent region. These studies provide the first direct demonstration of processing of the intracellular form of the flavivirus capsid protein by the viral protease.     

Sequential Protein Synthesis Following Vaccinia Virus Infection

Inhibition of HeLa cell protein synthesis and the sequential synthesis of viral proteins were followed by pulse-labeling infected cells with (14)C-phenylalanine. Proteins were resolved by polyacrylamide gel electrophoresis. The viral origin of native proteins was confirmed by immunodiffusion. The inhibition of host protein synthesis and the synthesis of early viral proteins occur 1 to 3 hr after infection. This early sequence of events also occurs in the presence of 5-fluorodeoxyuridine, an inhibitor of deoxyribonucleic acid synthesis. Other viral proteins are synthesized at a later time. Those proteins which are not made in the absence of viral deoxyribonucleic acid synthesis can be further subdivided into intermediate and late classes. The intermediate protein is synthesized before the late proteins but does not appear to be a precursor of them. Many more viral polypeptides were resolved by polyacrylamide gel electrophoresis after solubilization of the entire cytoplasmic fraction with sodium dodecyl sulfate. Virion and nonvirion proteins were identified. Kinetic experiments suggested that certain structural proteins as well as certain nonstructural proteins are made early, whereas others of both classes are made primarily at later times.     

Molecular processing of adenovirus proteins

Late in adenovirus infection, a virus-encoded protease processes several viral structural proteins. The maturation cleavages are a prerequisite for full viral infectivity. The peptide fragment removed during processing is located at the amino end of the major core protein VII. The structure of the precursor peptide sequence was determined by both protein and nucleotide sequencing. Two processing events were elucidated. First, during protein biosynthesis, the initiator methionyl residue is removed and the penultimate seryl residue is acetylated. Second, the resulting NH2-terminal 23-residue fragment is removed during virus assembly. The specificity of the viral endoprotease was investigated by isolating and characterizing another viral proprotein precursor, Pro-VI. The propeptide of VI was also found to be extended at the amino end of the molecule. Comparison of the two propeptide sequences at the cleavage site revealed a consensus amino acid sequence of Gly-Gly-Ala. In addition, there is extensive similarity in the precursor sequences of both proteins. The analogous constitution of the precursor fragments in Pro-VI and Pro-VII suggests that a common mechanism is implicated in controlling the reorganization of VI and VII during virion assembly.     

Sites of synthesis of viral proteins in avian sarcoma virus-infected chicken cells.

We determined the sites of synthesis of avian sarcoma virus-specific proteins in infected chicken cells by immunoprecipitation of the products synthesized in vitro by free and membrane-bound polyribosomes; 85% of Pr76, the precursor of the viral internal structural proteins (group-specific antigens), was synthesized on free polyribosomes, and 15% was synthesized on membrane-bound polyribosomes. Pr92, the lycosylated precursor of the viral glycoproteins (gp85 and gp35), was synthesized exclusively on membrane-bound polyribomes, which is consistent with its role as a membrane protein. When we investigated the site of synthesis of pp60src, the product of the avian sarcoma virus src gene, we found that 90% was synthesized on free polyribosomes, whereas 10% was detected on membrane-bound polyribosomes. The implications of these results with respect to the subcellular location of pp60src are discussed.     

Translation and processing of mouse hepatitis virus virion RNA in a cell-free system.

The first event after infection with mouse hepatitis virus strain A59 (MHV-A59) is presumed to be the synthesis of an RNA-dependent RNA polymerase from the input genomic RNA. The synthesis and processing of this putative polymerase protein was studied in a cell-free translation system utilizing 60S RNA from MHV-A59 virions. The polypeptide products of this reaction included two major species of 220 and 28 kilodaltons. Kinetics experiments indicated that both p220 and p28 appeared after 60 min of incubation and that protein p28 was synthesized initially as the N-terminal portion of a larger precursor protein. When the cell-free translation products were labeled with N-formyl[35S]methionyl-tRNAi, p28 was the predominant radioactive product, confirming its N-terminal location within a precursor protein. Translation in the presence of the protease inhibitors leupeptin and ZnCl2 resulted in the disappearance of p28 and p220 and the appearance of a new protein, p250. This product, which approached the maximal size predicted for a protein synthesized from genomic RNA, was not routinely detected in the absence of inhibitors even under conditions which optimized the translation reaction for elongation of proteins. Subsequent chelation of ZnCl2 resulted in the partial cleavage of the precursor protein and the reappearance of p28. One-dimensional peptide mapping with Staphylococcus aureus V-8 protease confirmed the precursor-product relationship of p250 and p28. The results show that MHV virion RNA, like many other viral RNAs, is translated into a large polyprotein, which is cleaved soon after synthesis into smaller, presumably functional proteins. This is in marked contrast to the synthesis of other MHV proteins, in which minimal proteolytic processing occurs.     

Sequence of protein synthesis in cells infected by human cytomegalovirus: early and late virus-induced polypeptides.

At least 10 distinct early virus-induced polypeptides were synthesized within 0 to 6 h after infection of permissive cells with cytomegalovirus. These virus-induced polypeptides were synthesized before and independently of viral DNA replication. A majority of these early virus-induced polypeptides were also synthesized in nonpermissive cells, which do not permit viral DNA replication. The virus-induced polypeptides synthesized before viral DNA replication were hypothesized to be nonstructural proteins coded for by the cytomegalovirus genome. Their synthesis was found to be a sequential process, since three proteins preceded the synthesis of the others. Synthesis of all early cytomegalovirus-induced proteins was a transient process; the proteins reached their highest molar ratios before the onset of viral DNA replication. Late viral proteins were synthesized at the time of the onset of viral DNA replication, which was approximately 15 h after infection. Their synthesis was continuous and increased in molar ratios with the accumulation of newly synthesized viral DNA in the cells. The presence of the amino acid analog canavanine or azetadine during the early stage of infection suppressed viral DNA replication. The amount of viral DNA synthesis was directly correlated to the relative amount of late viral protein synthesis. Because synthesis of late viral proteins depended upon viral DNA replication, the proteins were not detected in permissive cells treated with an inhibitor of viral DNA synthesis or in nonpermissive cells that are restrictive for cytomegalovirus DNA replication.     

Human immunodeficiency virus type 1 (HIV-1) protein Vif inhibits the activity of HIV-1 protease in bacteria and in vitro.

Human immunodeficiency virus type 1 (HIV-1) Vif is required for productive infection of T lymphocytes and macrophages. Virions produced in the absence of Vif have abnormal core morphology and those produced in primary T cells carry immature core proteins and low levels of mature capsid (M. Simm, M. Shahabuddin, W. Chao, J. S. Allan, and D. J. Volsky, J. Virol. 69:4582-4586, 1995). To investigate whether Vif influences the activity of HIV-1 protease (PR), the viral enzyme which is responsible for processing Gag and Gag-Pol precursor polyproteins into mature virion components, we transformed bacteria to inducibly express truncated Gag-Pol fusion proteins and Vif. We examined the cleavage of polyproteins consisting of matrix to PR (Gag-PR), capsid to PR (CA-PR), and p6Pol to PR (p6Pol-PR) and evaluated HIV-1 protein processing at specific sites by Western blotting using antibodies against matrix, capsid, and PR proteins. We found that Vif modulates HIV-1 PR activity in bacteria mainly by preventing the release of mature MA and CA from Gag-PR, CA from CA-PR, and p6Pol from p6Pol-PR, with other cleavages being less affected. Using subconstructs of Vif, we mapped this activity to the N-terminal half of the molecule, thus identifying a new functional domain of Vif. Kinetic study of p6Pol-PR autocatalysis in the presence or absence of Vif revealed that Vif and N'Vif reduce the rate of PR-mediated proteolysis of this substrate. In an assay of in vitro proteolysis of a synthetic peptide substrate by purified recombinant PR we found that recombinant Vif and the N-terminal half of the molecule specifically inhibit PR activity at a molar ratio of the N-terminal half of Vif to PR of about 1. These results suggest a mechanism and site of action of Vif in HIV-1 replication and demonstrate novel regulation of a lentivirus PR by an autologous viral protein acting in trans.     

Evidence of ambiguous processing and selective degradation in the noncapsid proteins of rhinovirus 1A.

Pulse-chase kinetics and extensive pactamycin mapping studies show that the translation of rhinovirus 1A proceeds in the order: initiate-P1-S-P2-terminate, where P1 is the precursor to the capsid proteins, S is a stable primary gene product, and P2 is the precursor to a family of noncapsid products. Initial examination of the molar stoichiometry of the families of rhinoviral proteins in infected cells suggested that both the P1 and P2 regions were translated more frequently than the S region. However, we show that this apparent asymmetry in translation is an artifact arising from two phenomena: (i) ambiguous cleavage sites which result in two alternative products from the S region, having apparent molecular weights of 47,000 and 38,000, and (ii) several fates for the P2 precursors, including degradation of 35 to 45% of the P2 family to small unidentifiable products. Another artifact, a time-dependent shift in the pactamycin mapping position of polypeptide r-39, was traced to a selective inhibition of the rate of cleavage of its precursor (peak 76). The processing rate of the capsid precursor (peak 92) was not retarded by pactamycin.     

Analysis of deletion mutants indicates that the 2A polypeptide of hepatitis A virus participates in virion morphogenesis

Unlike all other picornaviruses, the primary cleavage of the hepatitis A virus (HAV) polyprotein occurs at the 2A/2B junction and is carried out by the only proteinase encoded by the virus, 3C(pro). The resulting P1-2A capsid protein precursor is subsequently cleaved by 3C(pro) to generate VP0, VP3, and VP1-2A, which associate as pentamers. An unidentified cellular proteinase acting at the VP1/2A junction releases the mature capsid protein VP1 from VP1-2A later in the morphogenesis process. Although these aspects of polyprotein processing are well characterized, the function of 2A is unknown. To study its role in the viral life cycle, we assessed the infectivity of synthetic, genome-length RNAs containing 11 different in-frame deletions in the 2A region. Deletions in the N-terminal 40% of 2A abolished infectivity, whereas deletions in the C-terminal 60% resulted in viruses with a small-focus replication phenotype. C-terminal deletions in 2A had no effect on RNA replication kinetics under one-step growth conditions, nor did they have an effect on capsid protein synthesis and 3C(pro)-mediated processing. However, C-terminal deletions in 2A altered the VP1/2A cleavage, resulting in accumulation of uncleaved VP1-2A precursor in virions and possibly accounting for a delay in the appearance of infectious particles with these mutants, as well as a fourfold decrease in specific infectivity of the virus particles. When the capsid proteins were expressed from recombinant vaccinia viruses, the N-terminal part of 2A was required for efficient cleavage of the P1-2A precursor by 3C(pro) and assembly of structural precursors into pentamers. These data indicate that the N-terminal domain of 2A must be present as a C-terminal extension of P1 for folding of the capsid protein precursor to allow efficient 3C(pro)-mediated cleavages and to promote pentamer assembly, after which cleavage at the VP1/2A junction releases the mature VP1 protein, a process that appears to be necessary to produce highly infectious particles.     

Deubiquitinating function of adenovirus proteinase

The invasion strategy of many viruses involves the synthesis of viral gene products that mimic the functions of the cellular proteins and thus interfere with the key cellular processes. Here we show that adenovirus infection is accompanied by an increased ubiquitin-cleaving (deubiquitinating) activity in the host cells. Affinity chromatography on ubiquitin aldehyde (Ubal), which was designed to identify the deubiquitinating proteases, revealed the presence of adenovirus L3 23K proteinase (Avp) in the eluate from adenovirus-infected cells. This proteinase is known to be necessary for the processing of viral precursor proteins during virion maturation. We show here that in vivo Avp deubiquitinates a number of cellular proteins. Analysis of the substrate specificity of Avp in vitro demonstrated that the protein deubiquitination by this enzyme could be as efficient as proteolytic processing of viral proteins. The structural model of the Ubal-Avp interaction revealed some similarity between S1-S4 substrate binding sites of Avp and ubiquitin hydrolases. These results may reflect the acquisition of an advantageous property by adenovirus and may indicate the importance of ubiquitin pathways in viral infection.     

Poliovirus mutant that contains a cold-sensitive defect in viral RNA synthesis.

By manipulating an infectious cDNA clone of poliovirus, we have introduced a single-codon insertion into the 3A region of the viral genome which has been proposed to encode a functional precursor of the virion-linked protein VPg. The resulting mutant was cold sensitive in monkey kidney cells. Viral RNA synthesis was poor at 32.5 degrees C, although no other function of the virus was obviously affected. The synthesis of both positive and negative strands was severely depressed. Temperature shift experiments suggest that a normal level of production of the affected function was required only during the early (exponential) phase of RNA synthesis. Analysis of viral polyprotein processing at the nonpermissive temperature revealed that some of the normal cleavages were not made, most likely as a consequence of the defect in RNA synthesis or as a result of the concomitant reduction in the level of virally encoded proteases.     

Adenovirus core protein synthesis in the absence of viral DNA synthesis late in infection.

The acid extraction of the adenovirus type 5 core proteins V, VII, and pVII (the precursor to VII) from infected cells and the subsequent electrophoresis on a 15% acrylamide-2.5 M urea-0.9 N acetic acid (pH 2.7) gel, revealed that peptide VII has a similar electrophoretic mobility to that of histone H1. The core proteins, which are coded by late adenovirus mRNA, continued to be synthesized late in infection when viral DNA synthesis was inhibited either by cytosine arabinoside in wild-type infections or by shifting adenovirus H5 ts 125-infected cells to the nonpermissive temperature (40 degree C). Only the initiation, not the continuation, of viral DNA replication was essential for core protein synthesis. The synthesis of viral core proteins continued for over 8 h after the cassation of DNA synthesis. This was in contrast to the rapid shutdown of cellular histone synthesis in the absence of cellular DNA synthesis.