Myristylation of poliovirus capsid precursor P1 is required for assembly of subviral particles.

The poliovirus capsid precursor polyprotein, P1, is cotranslationally modified by the addition of myristic acid. We have examined the importance of myristylation of the P1 capsid precursor during the poliovirus assembly process by using a recently described recombinant vaccinia virus expression system which allows the independent production of the poliovirus P1 protein and the poliovirus 3CD proteinase (D. C. Ansardi, D. C. Porter, and C. D. Morrow, J. Virol. 65:2088-2092, 1991). We constructed a site-directed mutation in the poliovirus cDNA encoding an alanine at the second amino acid position of P1 in place of the glycine residue required for the myristic acid addition and isolated a recombinant vaccinia virus (VVP1myr-) that expressed a nonmyristylated form of the P1 capsid precursor. The 3CD proteinase expressed by a coinfecting vaccinia virus, VVP3, proteolytically processed the nonmyristylated precursor P1 expressed by VVP1myr-. However, the processed capsid proteins, VP0, VP3, and VP1, did not assemble into 14S or 75S subviral particles, in contrast to the VP0, VP3, and VP1 proteins derived from the myristylated P1 precursor. When cells were coinfected with VVP1myr- and poliovirus type 1, the nonmyristylated P1 precursor expressed by VVP1myr- was processed by 3CD expressed by poliovirus, and the nonmyristylated VP0-VP3-VP1 (VP0-3-1) protomers were incorporated into capsid particles and virions which sedimented through a 30% sucrose cushion. Thus, the nonmyristylated P1 precursor and VP0-3-1 protomers were not excluded from sites of virion assembly, and the assembly defects observed for the nonmyristylated protomers were overcome in the presence of myristylated capsid protomers expressed by poliovirus. We conclude that myristylation of the poliovirus P1 capsid precursor plays an important role during poliovirus assembly by facilitating the appropriate interactions required between 5S protomer subunits to form stable 14S pentamers. The results of these studies demonstrate that the independent expression of the poliovirus P1 and 3CD proteins by using recombinant vaccinia viruses provides a unique experimental tool for analyzing the dynamics of the poliovirus assembly process.     

Interaction of liposomes with subviral particles of poliovirus type 2 and rhinovirus type 2.

Subviral particles ("A particles") were produced from rhinovirus type 2 by treatment with acid and from poliovirus type 2 by incubation at 37 degrees C in a low-ionic-strength buffer. A particles, but not virions, adsorbed to liposomes. It is proposed that these reactions may provide an in vitro model for two early steps of infection.     

Nucleotide binding by the poliovirus RNA polymerase.

Cross-linking of ribonucleoside triphosphates (NTPs) to specific binding sites on the poliovirus RNA-dependent RNA polymerase has been performed by ultraviolet irradiation and by reduction of oxidized nucleotide-protein complexes. The latter method approached a cross-linking efficiency of 1 NTP/molecule of enzyme. Nucleotide competition experiments suggested that the same binding site is occupied by all NTPs. Analysis of peptides produced by proteinase Glu-C and trypsin digestion and labeled with [32P]GTP indicated that a lysine residue between Met-189 and Lys-228 in the polymerase was cross-linked to NTP. Nucleotide binding was exploited for rapid purification of the enzyme by GTP-agarose affinity chromatography. In addition, a set of cloned, modified polymerase molecules with reduced or absent polymerization activity was analyzed for binding efficiency to a GTP-agarose column. Some mutations eliminated GTP binding, whereas others generated proteins with varying affinities for GTP. Incubation of the poliovirus polymerase with high concentrations of NTP, particularly GTP, resulted in a dramatic protection against heat denaturation and activity loss. These data suggest that nucleotide binding results in an alteration of the enzyme conformation or the stabilization of an ordered conformation.     

Reovirus progeny subviral particles synthesize uncapped mRNA.

Reovirus progeny subviral particles were isolated from L-cells at late times postinfection. It has been shown (D. Skup and S. Millward, J. Virol. 34: 490--496, 1980) that these progeny subviral particles have masked capping enzymes, indicating that mRNA synthesized by these particles should be uncapped. When progeny subviral particles were used for mRNA synthesis in vitro, they failed to incorporate the beta-phosphate of [beta-32P]GTP into the 5' terminal. Direct analysis of reovirus mRNA synthesized by progeny subviral particles in the presence of either [alpha-32P]GTP or [alpha-32P]CTP indicated that the 5' terminal was uncapped, having the structure pGpC... The implications of this finding to the reovirus replicative cycle are discussed.     

Guanidine-resistant defective interfering particles of poliovirus.

A mixture containing standard poliovirus and D3 particles (mutants with deletions in the capsid locus) was serially passaged in the presence of guanidine. Within five growth cycles, the standard virus was guanidine resistant, but the D3 particles were guanidine sensitive, even after 21 passages with the inhibitor. By passage 40 with guanidine, D3 particles were eliminated, and a new deletion mutant (DX) appeared in the virus population. D3 particles contained a 15% deletion, and DX particles contained a 6% deletion in the capsid locus. Although neither mutant induced the synthesis of NCVP1a or a complete complement of capsid proteins after infection, cells infected with DX particles produced two novel proteins, which had molecular weights of approximately 68,000 and 25,000.     

Antigenic and immunogenic properties of recombinant hepatitis A virus 14S and 70S subviral particles.

Hepatitis A virus (HAV) has an immunodominant neutralization antigenic site. By using a panel of monoclonal antibodies targeted against the HAV neutralization antigenic site, it was shown that three epitopes within this site are present on 14S subunits (pentamers of the structural unit). In contrast, two other epitopes within this site are formed upon assembly of 14S subunits into capsids. Thus, the epitopes recognized by these two monoclonal antibodies are formed either by a conformational change in the antigenic site or by the juxtaposition of epitope fragments present on different 14S subunits during assembly of 14S into 70S particles. Both 14S and 70S particles elicited HAV-neutralizing antibodies in mice; thus, these particles may be useful for HAV vaccine development.     

mRNA capping enzymes are masked in reovirus progeny subviral particles.

We examined the enzyme activities associated with progeny subviral particles isolated from L-cells infected with reovirus at 12 h postinfection. Activities normally present in reovirus cores were also found to be present in the progeny subviral particles, with the exception of the capping enzymes. The methylase and guanyl transferase activities, which constitute the capping system, were present in a masked form that could be activated by chymotrypsin digestion. The appearance of these progeny subviral particles in infected cells coincided with the time when mRNA synthesis was maximal, suggesting that viral mRNA synthesized at later times is uncapped.     

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.     

Properties of subviral particles of hepatitis B virus

In the sera of patients infected with hepatitis B virus (HBV), in addition to infectious particles, there is an excess (typically 1,000- to 100,000-fold) of empty subviral particles (SVP) composed solely of HBV envelope proteins in the form of relatively smaller spheres and filaments of variable length. Hepatitis delta virus (HDV) assembly also uses the envelope proteins of HBV to produce an infectious particle. Rate-zonal sedimentation was used to study the particles released from liver cell lines that produced SVP only, HDV plus SVP, and HBV plus SVP. The SVP made in the absence of HBV or HDV were further examined by electron microscopy. They bound efficiently to heparin columns, consistent with an ability to bind cell surface glycosaminoglycans. However, unlike soluble forms of HBV envelope protein that were potent inhibitors, the SVP did not inhibit the ability of HBV and HDV to infect primary human hepatocytes.     

Internal binding of eucaryotic ribosomes on poliovirus RNA: translation in HeLa cell extracts.

Translation initiation on poliovirus mRNA in poliovirus-infected cells has been shown to occur by internal binding of ribosomes to the 5' noncoding region (J. Pelletier and N. Sonenberg, Nature [London] 334:320-325, 1988). Here we show that internal ribosome binding can occur in HeLa cell extracts in vitro. Internal binding to the 5' noncoding region of poliovirus mRNA in a bicistronic context was independent of the upstream open reading frame and did not require poliovirus proteins.     

Morphogenesis of hepatitis A virus: isolation and characterization of subviral particles.

The morphogenesis of hepatitis A virus (HAV) in BS-C-1 cells was examined by immunoblotting with antisera to capsid proteins and labeling of virus-specific proteins with L-[35S]methionine. Antiserum to VP2 detected two virus-specific proteins with apparent molecular masses of 30.6 and 30 kDa, representing VP0 and VP2, while antiserum to VP1 detected proteins with molecular masses of 33 and 40 kDa, representing VP1 and a virus-specific protein which we designated PX, respectively. Sedimentation of cell lysates revealed the presence of virions, procapsids, and pentamers, but particles analogous to the protomers of other picornaviruses were not detected. Although provirions and virions were not found as discrete species in our gradient system, it was evident that the rate of sedimentation was proportional to the relative amounts of VP0 and VP2 in particles, with slower-sedimenting particles (provirions) containing predominantly VP0 rather than VP2. Procapsids contained VP0 in addition to VP1 and VP3. Pentamers also contained VP0, but PX was present rather than VP1. These results suggest that PX is a precursor to VP1 and is most likely 1D2A. Primary cleavage of the viral polyprotein also occurs at the 2A-2B junction in cardioviruses and aphthoviruses, but assembly of pentamers containing 1D2A has not been reported for those viruses. The absence of detectable levels of protomers suggests a high efficiency of pentamer formation, which may be related to the high efficiency of viral RNA encapsidation for HAV (D.A. Anderson, B.C. Ross, and S.A. Locarnini, J. Virol. 62:4201-4206, 1988). The results of this study reveal further unusual aspects of the HAV replicative cycle which distinguish it from other picornaviruses and may contribute to its restricted replication in cell culture.     

Functional oligomerization of poliovirus RNA-dependent RNA polymerase.

Using a hairpin primer/template RNA derived from sequences present at the 3' end of the poliovirus genome, we investigated the RNA-binding and elongation activities of highly purified poliovirus 3D polymerase. We found that surprisingly high polymerase concentrations were required for efficient template utilization. Binding of template RNAs appeared to be the primary determinant of efficient utilization because binding and elongation activities correlated closely. Using a three-filter binding assay, polymerase binding to RNA was found to be highly cooperative with respect to polymerase concentration. At pH 5.5, where binding was most cooperative, a Hill coefficient of 5 was obtained, indicating that several polymerase molecules interact to retain the 110-nt RNA in a filter-bound complex. Chemical crosslinking with glutaraldehyde demonstrated physical polymerase-polymerase interactions, supporting the cooperative binding data. We propose a model in which poliovirus 3D polymerase functions both as a catalytic polymerase and as a cooperative single-stranded RNA-binding protein during RNA-dependent RNA synthesis.     

Reaction of poliovirus RNAs with antibodies to double-stranded RNA demonstrated by an immunochemical binding assay.

An immunochemical binding assay was used to investigate the reactivity of radioactively labeled viral RNAs from poliovirus-infected cells with antibodies to the synthetic double-stranded RNA, poly(I)-poly(C). A RNase-free antibody-containing serum fraction was employed. Poliovirus replicative form reacted with the antibodies to poly(I)-poly(C) as well as or better than poly(I)-poly(C). Poliovirus replicative intermediate reacted with the antibodies to a greater extent than poliovirus single-stranded RNA, but both were less reactive than replicative form. The use of the immunochemical binding assay with sucrose-gradient fractions demonstrated that for both poliovirus single-stranded RNA and replicative form the peak of reactivity with the antibodies was coincident with the peak of radioactive material precipitated by trichloroacetic acid. The proportion of replicative intermediate that reacted with the antibody increased in sucrose-gradient fractions containing the more slowly sedimenting RI RNA.     

Host cell proteins binding to domain IV of the 5' noncoding region of poliovirus RNA.

Translation of poliovirus RNA occurs by the binding of ribosomes to an internal segment of RNA sequence within the 5' untranslated region of the viral RNA. This region is predicted to consist of six domains (I to VI) that possess complex secondary and tertiary structures. Domain IV is a large region in which alterations in the sequence or structure markedly reduce translational efficiency. In this study, we employed RNA mobility shift assays to demonstrate that a protein(s) from uninfected HeLa cell extracts, as well as from neuroblastoma extracts, interacts with the domain IV structure. A mutation in domain IV caused reduced binding of HeLa cell proteins and reduced translation both in vitro and in vivo, suggesting that the binding of at least one of these proteins plays a role in the mechanism of viral translation. UV cross-linking indicated that a protein(s) with a size of approximately 40 kDa interacted directly with the RNA. Using streptavidin beads to capture biotinylated RNA bound to proteins, we were able to visualize a number of HeLa and neuroblastoma cell proteins that interact with domain IV. These proteins have molecular masses of approximately 39, approximately 40, and approximately 42 kDa.     

In vitro construction of poliovirus defective interfering particles.

To construct poliovirus defective interfering (DI) particles in vitro, we synthesized an RNA from a cloned poliovirus cDNA, pSM1(T7)1, which carried a deletion in the genome region corresponding to nucleotide positions 1663 to 2478 encoding viral capsid proteins, by using bacteriophage T7 RNA polymerase. The RNA was designed to retain the correct reading frame in nucleotide sequence downstream of the deletion. HeLa S3 monolayer cells were transfected with the deletion RNA and then superinfected with standard virus as a helper. The DI RNA was observed in the infected cells after three passages at high multiplicity of infection. The sequence analysis of RNA extracted from the purified DI particle clearly showed that this DI RNA had the same deletion in size and location as that in the RNA used for the transfection. Thus, we succeeded in construction of a poliovirus DI particle in vitro. To gain insight into the mechanism for DI generation, we constructed poliovirus cDNAs pSM1(T7)1a and pSM1(T7)1b that, in addition to the same deletion as that in pSM1(T7)1, had insertion sequences of 4 bases and 12 bases, respectively, at the corresponding nucleotide position, 2978. The RNA transcribed from pSM1(T7)1a was not a template for synthesis of poliovirus nonstructural proteins and therefore was inactive as an RNA replicon. On the other hand, the RNA from pSM1(T7)1b replicated properly in the transfected cells. Superinfection of the transfected cells with standard virus resulted in production of DI particles derived from pSM1(T7)1b and not from pSM1(T7)1a. These observations indicate that deletion RNAs that are inactive replicons have little or no possibility of being genomes of DI particles suggesting the existence of a nonstructural protein(s) that has an inclination to function as a cis-acting protein(s). The method described here will provide a useful technique to investigate genetic information essential for poliovirus replication.