Mature Capsid Protein Research Articles

The capsid gene of feline calicivirus contains linear B-cell epitopes in both variable and conserved regions.

In order to map linear B-cell (LBC) epitopes in the major capsid protein of feline calicivirus (FCV), an expression library containing random, short (100- to 200-bp) fragments of the FCV F9 capsid gene was constructed. Analysis of this library showed it to be representative of the region of the capsid gene that encodes the mature capsid protein. The library was screened by using polyclonal antisera from a cat that had been challenged experimentally with F9 to identify immunoreactive clones containing LBC epitopes. Twenty-six clones that reacted positively to feline antisera in immunoblots were identified. FCV-derived sequence from these clones mapped to a region of the capsid that spanned 126 amino acids and included variable regions C and E. An overlapping set of biotinylated peptides corresponding to this region was used to further map LBC epitopes by using F9 antisera. Four principal regions of reactivity were identified. Two fell within the hypervariable region at the 5' end of region E (amino acids [aa] 445 to 451 [antigenic site (ags) 2] and aa 451 to 457 [ags 3]). However, the other two were in conserved regions (aa 415 to 421 [ags 1; region D] and aa 475 to 479 [ags 4; central region E]). The reactivity of the peptide set with antisera from 11 other cats infected with a range of FCV isolates was also determined. Ten of 11 antisera reacted to conserved ags 4, suggesting that this region may be useful for future recombinant vaccine design.

Maturation of the hepatitis A virus capsid protein VP1 is not dependent on processing by the 3Cpro proteinase.

Most details of the processing of the hepatitis A virus (HAV) polyprotein are known. Unique among members of the family Picornaviridae, the primary cleavage of the HAV polyprotein is mediated by 3Cpro, the only proteinase known to be encoded by the virus, at the 2A/2B junction. All other cleavages of the polyprotein have been considered to be due to 3Cpro, although the precise location and mechanism responsible for the VP1/2A cleavage have been controversial. Here we present data that argue strongly against the involvement of the HAV 3Cpro proteinase in the maturation of VP1 from its VP1-2A precursor. Using a heterologous expression system based on recombinant vaccinia viruses directing the expression of full-length or truncated capsid protein precursors, we show that the C terminus of the mature VP1 capsid protein is located near residue 764 of the polyprotein. However, a proteolytically active HAV 3Cpro that was capable of directing both VP0/VP3 and VP3/VP1 cleavages in vaccinia virus-infected cells failed to process the VP1-2A precursor. Using site-directed mutagenesis of an infectious molecular clone of HAV, we modified potential VP1/2A cleavage sites that fit known 3Cpro recognition criteria and found that a substitution that ablates the presumed 3Cpro dipeptide recognition sequence at Glu764-Ser765 abolished neither infectivity nor normal VP1 maturation. Altered electrophoretic mobility of VP1 from a viable mutant virus with an Arg764 substitution indicated that this residue is present in VP1 and that the VP1/2A cleavage occurs downstream of this residue. These data indicate that maturation of the HAV VP1 capsid protein is not dependent on 3Cpro processing and may thus be uniquely dependent on a cellular proteinase.

Involvement of serine proteinase in infectious pancreatic necrosis virus capsid protein maturation and NS proteinase cleavage in CHSE‐214 cells

An investigation of virus-specific protein maturation in infectious pancreatic necrosis virus (IPNV) infected Chinook salmon embryo cells (CHSE-214) was undertaken. The precursor protein (pVP2-1) of the major mature capsid protein (VP2) was processed sequentially from pVP2-1 to pVP2-2 and VP2. Experiments using serine proteinase inhibitors showed that the maturation of the VP2 was blocked in the pVP2-1 post-translational cleavage steps. A protinin, a potent proteinase inhibitor, at 800 μg ml(-1) blocked pVP2-2 to VP2 and the cleavage of VP4 (28 kDa) to VP4-1 (25 kDa). Therefore, our data showed that the maturation of the capsid protein (VP2) and cleavage of VP4 (NS proteinase) can be blocked by serine proteinase inhibitors.

Cleavage of the feline calicivirus capsid precursor is mediated by a virus-encoded proteinase.

Feline calicivirus (FCV), a member of the Caliciviridae, produces its major structural protein as a precursor polyprotein from a subgenomic-sized mRNA. In this study, we show that the proteinase responsible for processing this precursor into the mature capsid protein is encoded by the viral genome at the 3'-terminal portion of open reading frame 1 (ORF1). Protein expression studies of either the entire or partial ORF1 indicate that the proteinase is active when expressed either in in vitro translation or in bacterial cells. Site-directed mutagenesis was used to characterize the proteinase Glu-Ala cleavage site in the capsid precursor, utilizing an in vitro cleavage assay in which mutant precursor proteins translated from cDNA clones were used as substrates for trans cleavage by the proteinase. In general, amino acid substitutions in the P1 position (Glu) of the cleavage site were less well tolerated by the proteinase than those in the P1' position (Ala). The precursor cleavage site mutations were introduced into an infectious cDNA clone of the FCV genome, and transfection of RNA derived from these clones into feline kidney cells showed that efficient cleavage of the capsid precursor by the virus-encoded proteinase is a critical determinant in the growth of the virus.

Genetic analysis of mengovirus protein 2A: its function in polyprotein processing and virus reproduction.

To examine the functional requirements of mengovirus 2A for virus reproduction, a series of mutants with overlapping deletions within the 2A region of mengovirus, and two chimeric constructs in which 2A is replaced either by Theiler's murine encephalomyelitis virus (TMEV) 2A or by coxsackie B3 virus (CBV3) 2Apro were generated. In vitro polyprotein synthesis showed that in both deletion mutants and the TMEV 2A chimeric construct, viral 3C protease (3Cpro)-mediated cleavage at the VP1-2A junction was disturbed, which resulted in decreased formation of mature capsid proteins and accumulation of the P1-2A precursor. 2Apro-mediated processing of the chimeric VP1-2Apro junction was highly efficient. Although the resulting L-P1 precursor was cleaved at the L-VP4 junction, further processing of the P1 precursor was abrogated. Two deletion mutant viruses and a TMEV 2A chimeric virus were obtained after transfection. The CBV 2Apro construct did not result in viable virus. Deletion mutant virus production was less than 3% compared to wild-type virus production, whereas chimeric virus production was reduced to 25%. Although inhibition of host-cell translation was identical in wild-type and mutant virus-infected cells, viral protein and RNA synthesis were reduced in cells infected with mutant virus, independently of the impaired P1-2A processing. It is concluded that mengovirus 2A may play a functional role in either virus translation or replication, and that the functional aspects of mengovirus and TMEV 2A cannot be exchanged. The results also confirm that the processing cascade of L-P1-2A occurs sequentially and is probably regulated by subsequent conformational transitions of the cleavage products after each proteolytic event. The sequential release of L and 2A may be essential in the context of their function in virus replication.

Human immunodeficiency virus type 1 Vif protein binds to the Pr55Gag precursor.

The Vif protein of human immunodeficiency virus type 1 is required for productive replication in peripheral blood lymphocytes. Previous reports suggest that vif-deleted viruses are limited in replication because of a defect in the late steps of the virus life cycle. One of the remaining questions is to determine whether the functional role of Vif involves a specific interaction with virus core proteins. In this study, we demonstrate a direct interaction between Vif and the Pr55Gag precursor in vitro as well as in infected cells. No interaction is observed between Vif and the mature capsid protein. The Pr55Gag-Vif interaction is detected (i) in the glutathione S-transferase system, with in vitro-translated proteins demonstrating a critical role of the NC p7 domain of the Gag precursor; (ii) with proteins expressed in infected cells; and (iii) by coimmunoprecipitation experiments. Deletion of the C-terminal 22 amino acids of Vif abolishes its interaction with the Pr55Gag precursor. Furthermore, point mutations in the C-terminal domain of Vif which have been previously shown to abolish virus infectivity and binding to cell membranes dramatically decrease the Gag-Vif interaction. These results suggest that the interaction between Vif and the pr55Gag precursor is a critical determinant of Vif function.

The major antigenic protein of infectious bursal disease virus, VP2, is an apoptotic inducer.

Infectious bursal disease virus (IBDV) is the causative agent of an economically important poultry disease. Vaccinia virus recombinants expressing the IBDV mature structural capsid proteins VP2 and VP3 were generated by using vectors for inducible gene expression. Characterization of these recombinant viruses demonstrated that expression of VP2 leads to induction of apoptosis in a variety of mammalian cell lines. Transfection of cell cultures with a expression vector containing the VP2 coding region under the control of the immediate-early promoter-enhancer region of human cytomegalovirus also triggers programmed cell death. The apoptotic effect of VP2 is efficiently counteracted by coexpression of the proto-oncogene bcl-2. The results presented demonstrate that VP2 is a bona fide apoptotic inducer. Evaluation of the significance of this finding for the virus life cycle must await further research.

A Conserved Internal Hydrophobic Domain Mediates the Stable Membrane Integration of the Dengue Virus Capsid Protein

The mature flavivirus capsid protein (virion C) is commonly thought to be free in the cytoplasm of infected cells and to form a nucleocapsid-like complex with genomic RNA in mature virus particles. There is little sequence conservation among flavivirus virion C proteins, but they are similar in size (e.g., 99 amino acids [aa] for the dengue-4 [DEN4] C) and in bearing a net positive charge. In addition, we noted that C contained a conserved internal hydrophobic segment (spanning aa 45–65 in the DEN4 C). Results ofin vivoexpression andin vitrotranslation of wt and mutant forms of the DEN4 virion C demonstrated that the conserved internal hydrophobic segment in the DEN C functioned as a membrane anchor domain. Signal peptide function of this segment was also suggested by its requirement for the entry of C into membranes. Virion C was integrated in membranes in a “hairpin” conformation; positively charged segments amino- and carboxy-terminal to the hydrophobic signal-anchor segment were accessible to protease digestion in the “cytoplasm.” The net positive charge in the amino-terminal extramembraneous portion of C (aa 1–44) was one determinant of the hairpin membrane orientation; a conserved positively charged residue within the hydrophobic segment (Arg-54 in the DEN4 C) was not.

Differential proteolytic processing leads to multiple forms of the CA protein in avian sarcoma and leukemia viruses

The CA (capsid) protein of avian sarcoma and leukemia viruses occurs in multiple species. Only one form has been previously characterized biochemically. We have now determined that the mature CA protein of avian sarcoma and leukemia viruses exists as three species with different C termini, ending in amino acid residues A-476, A-478, and M-479 of the Gag precursor, respectively. These structures were deduced from a combination of cyanogen bromide peptide mapping, sequence analysis of tryptic peptides, and electrospray mass spectrometry. The three forms of CA were detected in the same ratios in Rous sarcoma virus and avian myeloblastosis virus and therefore are likely to represent a common feature of members of this genus of avian retroviruses. The only previously reported CA species, CAM-479, accounts for only about 36% of the total CA protein, while CAA-476 and CAA-478 account for 55 and 9%, respectively. From the analysis of peptides cleaved in vitro by PR, the viral protease, we infer that the cleavage site between A-476 and A-477 not only is recognized by PR but is the preferred site. We were unable to determine if A-478/A-479 is a cleavage site for PR or alternatively if CAA-478 results from further processing of CAM-479 by a carboxypeptidase. To study the biological significance of residues A-477 to M-479, we constructed genetically altered viruses in which deletions removed either residues 477 to 479 or 477 to 488. The resulting virus particles appeared to assembly with normal efficiencies, but the latter mutant showed slowed proteolytic processing. Neither of the mutants was infectious.

A Study of the Dimerization of Rous Sarcoma Virus RNA in Vitro and in Vivo

The Rous sarcoma virus dimer linkage site (DLS) has been located by electron microscopy at position 511 ± 28 nucleotides. We have studied the dimerization of RNAs encompassing the first 634 nucleotides of Rous sarcoma virus and conclude that there are at least two dimerization signals. One is located between nucleotides 531 and 634 and may involve Watson-Crick pairing of an imperfect inverted repeat. The other signal is located between nucleotides 496 and 530. A tetraguanine sequence at nucleotides 523-526 is required for dimerization of this domain. The guanines are not involved in an identifiable Watson-Crick interaction or in guanine tetrad formation. Either dimerization domain can initiate the dimerization of RNA 1-634. It is possible that these domains are two parts of a single dimerization signal. Interstrand RNA contacts within the virion are not limited to the DLS but occur along the length of the genome. Nascent virions contain monomeric RNA which slowly associates to form an RNA dimer. The limiting step in dimerization is not proteolytic cleavage of the gag precursor because only the mature capsid protein p27 can be detected in these nascent virions.

Replication of an Osteopetrosis-Inducing Avian Leukosis Virus in Fibroblasts, Osteoblasts, and Osteopetrotic Bone

Avian leukosis virus (ALV)-induced osteopetrosis is caused by the abnormal growth and differentiation of osteoblasts. To evaluate the role of infection in osteopetrosis induction, the replication of an osteopetrosis-inducing virus (Br21) has been compared in osteopetrotic bone, calvarial-derived osteoblasts, and chick embryo fibroblasts. Much higher levels of infection occurred in diseased bone than in the cultures. Severe cases of osteopetrosis contained 10 times more viral DNA, 30 times more mature capsid protein, 5 to 10 times more Gag precursor protein, and 2 to 3 times more Env protein than the infected cultures. Virus replication in the cultured osteoblasts was similar to that in fibroblasts except for a distinctive asymmetric localization of Gag proteins. In osteopetrotic chickens, bones became atypically enlarged and sera contained elevated levels of osteoblast differentiation markers (alkaline phosphatase and osteocalcin). In cultures, infections did not affect the growth or differentiation of osteoblasts. Thus, the infected cultures lacked aspects of the bone environment that support both the high levels of infection and the aberrant function of osteoblasts characteristic of ALV-induced osteopetrosis.

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.

Characterization of the Foot-and-Mouth Disease Virus 3C Protease Expressed in Escherichia coli

We have constructed a clone encoding the foot-and-mouth disease virus (FMDV) 3C protease gene (p3C) using the polymerase chain reaction. The construct was engineered to contain initiation and termination codons and cloned into a plasmid under the control of the bacteriophage T7 promoter. The p3C gene was expressed both in an in vitro transcription-translation system and in vivo in an Escherichia coli system containing an inducible T7 RNA polymerase gene. In both systems the expressed products were of the appropriate molecular weight and immunologically reactive with bovine convalescent serum. E. coli -expressed 3C protein was mainly found in the insoluble fraction of cell lysates. The E. coli-expressed protease was assayed in an in vitro system with radiolabeled P1 capsid precursor protein and P2 precursor protein as substrates. E. coli-expressed 3C completely processed the P1 and P2 precursors into mature capsid and nonstructural proteins, respectively. The kinetics of processing of P1 by E. coli-expressed 3C revealed the following order of cleavage: VP3-VP1, VPO-VP3, VP1-2A.

Identification and sequence determination of the capsid protein gene of feline calicivirus

SummaryWe have determined 4380 bases of the sequence from a cDNA clone containing the 3′ end of feline calicivirus strain F9. We find four candidate open reading frames of which three are complete and comprise 245, 317 and 2012 nucleotides. The fourth continues toward the 5′ end. We have expressed the largest complete open reading frame inE. coli. Sera raised to this antigen react specifically with the capsid protein and its intracellular precursor molecule. N-terminal sequence analysis of purified, mature capsid protein confirms this assignment and has identified the position at which precursor is cleaved.

The hepatitis A virus polyprotein expressed by a recombinant vaccinia virus undergoes proteolytic processing and assembly into viruslike particles.

Hepatitis A virus (HAV) contains a single-stranded, plus-sense RNA genome with a single long open reading frame encoding a polyprotein of approximately 250 kDa. Viral structural proteins are generated by posttranslational proteolytic processing of this polyprotein. We constructed recombinant vaccinia viruses which expressed the HAV polyprotein (rV-ORF) and the P1 structural region (rV-P1). rV-ORF-infected cell lysates demonstrated that the polyprotein was cleaved into immunoreactive 29- and 33-kDa proteins which comigrated with HAV capsid proteins VP0 and VP1. The rV-P1 construct produced a 90-kDa protein which showed no evidence of posttranslational processing. Solid-phase radioimmunoassays with human polyclonal anti-HAV sera and with murine or human neutralizing monoclonal anti-HAV antibodies recognized the rV-ORF-infected cell lysates. Sucrose density gradients of rV-ORF-infected cell lysates contained peaks of HAV antigen with sedimentation coefficients of approximately 70S and 15S, similar to those of HAV empty capsids and pentamers. Immune electron microscopy also demonstrated the presence of viruslike particles in rV-ORF-infected cell lysates. Thus, the HAV polyprotein expressed by a recombinant vaccinia virus demonstrated posttranslational processing into mature capsid proteins which assembled into antigenic viruslike particles.

Limited expression of poliovirus by vaccinia virus recombinants due to inhibition of the vector by proteinase 2A

A recombinant vaccinia virus was constructed that expressed poliovirus coat precursor protein P1 fused to about two-thirds of the 2A proteinase. The truncated 2A segment could be cleaved away from the P1 region by coinfecting with poliovirus type 1, 2, or 3 or with human rhinovirus 14 but not with encephalomyocarditis virus. Further cleavage of the vector-derived P1 to yield mature poliovirus capsid proteins was not observed. Attempts to isolate vaccinia virus recombinants containing portions of the poliovirus genome that encompassed the complete gene for proteinase 2A were unsuccessful, unless expression of functional 2A was abolished by insertion of a frameshift mutation. We conclude that an activity of the 2A proteinase, probably its role in translational inhibition, prevented isolation of vaccinia virus recombinants that expressed 2A.

Partial purification and substrate analysis of bacterially expressed HIV protease by means of monoclonal antibody.

Retroviruses code for a specific protease which is essential for polyprotein precursor processing and viral infectivity. The HIV-specific protease has been predicted to be an aspartic protease which is located at the amino terminus of the pol gene. We have prepared several constructs for bacterial expression of the protease. Two of them span the whole protease region and result in its autocatalytic activation. Analysis of the dynamics of this activation indicates a two-step process which starts at the carboxy terminus and ends at the amino terminus of the protease. The activated protease is a molecule of 9 kd as evidenced by monoclonal antibody in immunoblot analysis. A construct in which the carboxy terminus of the protease is deleted results in a stable, enzymatically inactive 27-kd protein which proved useful as substrate since it contains one of the predicted cleavage sites. The stability of this protein indicates that the carboxy-terminal sequences of the protease are essential for its activity and its autocatalytic activation. The protease which is very hydrophobic was solubilized by acetone treatment and passaged over ultrogel and propylagarose columns for partial purification. It elutes as a dimer and tends to aggregate. It is inhibited by pepstatin A in agreement with its expected active site and its theoretical classification as aspartic protease. Cleavage of the gag precursor results in the mature capsid protein, p17. The protease does not, however, cleave the denatured 27-kd substrate or the denatured gag precursor. Therefore its specificity appears to be not solely sequence- but also conformation-dependent. This property needs to be taken into account for the development of protease inhibitors for therapy of AIDS.

Evidence that a phage T4 DNA packaging enzyme is a processed form of the major capsid gene product

A phage T4 DNA packaging enzyme appears to arise as a processed form of the major T4 capsid structural protein gp23. The enzyme activity and antigen are missing from all head gene mutants that block the morphogenetic proteolytic processing reactions of the head proteins in vivo. The enzyme antigen can be formed in vitro by T4 (gp21) specific processing of gp23 containing extracts. Enzyme antigen is found in active processed proheads but not in full heads. The enzyme and the major capsid protein show immunological cross-reactivity, produce common peptides upon proteolysis, and share an assembly-conformation-dependent ATP binding site. The packaging enzyme and the mature capsid protein (gp23∗) both appear to arise from processing of gp23, the former as a minor product of a specific gp23 structure in the pro-head, acting in DNA packaging as a DNA-dependent ATPase, and a headful-dependent terminase.

Nucleotide sequence at the 3′ terminus of pepper mottle virus genomic RNA: Evidence for an alternative mode of potyvirus capsid protein gene organization

The sequence of the 3′-terminal 1481 nucleotides of the pepper mottle virus (PeMV) genome has been determined. The sequence was determined by dideoxy nucleotide sequencing of complementary DNA which had been inserted into M13 bacteriophage cloning vectors and was confirmed by sequencing selected regions of PeMV RNA. A discrete open reading frame of 993 nucleotides, ending 333 nucleotides from the 3′-terminal polyadenylate tract, was identified that potentially encoded a 37,669-MW protein. The amino acids predicted at positions 64 through 84 of this putative polypeptide were identical to the amino-terminal 21 amino acids of the PeMV capsid protein ascertained by chemical sequencing. These combined nucleotide and amino acid sequence data suggest that the PeMV capsid protein is encoded by the 3′-most cistron on the genomic RNA and that it may be expressed as a precursor that is proteolytically processed to produce the mature capsid protein.

Processing of the encephalomyocarditis virus capsid precursor protein studied in rabbit reticulocyte lysates incubated with N-formyl-[35S]methionine-tRNAfMet.

Translation of encephalomyocarditis virus RNA in rabbit reticulocyte lysates in the presence of N-formyl-[(35)S]methionine-tRNA(f) (Met) revealed that a small polypeptide is cleaved from the N-terminus of the capsid protein precursor, preA, by virus-coded protease activity. Therefore, this N-terminal segment comprising the translation initiation site is not conserved in any of the mature capsid proteins.