Bruce A. Phillips

In vitro assembly of polioviruses: I. Kinetics of the assembly of empty capsids and the role of extracts from infected cells

Abstract The kinetics of the in vitro assembly of 14 S particles into 73 S empty capsids was studied in cytoplasmic extracts made from poliovirus-infected HeLa cells. The formation of 73 S particles is complete after 10–15 minutes at 37°. About 20% of the 14 S material appears to be defective. An attempt was made to identify the component(s) in infected cell extracts that are responsible for the assembly activity by testing its sensitivity to various agents. Extracts treated with sufficient ribonuclease to destroy the cell ribosomes are able to assemble 73 S particles from 14 S particles. Pretreatment of the extracts with metabolic poisons (NaCN, NaF) or inhibitors of protein synthesis has no affect on assembly activity. The assembly activity of extracts is inactivated by 50° for 20 minutes or pretreatment with trypsin or deoxycholate. Extracts in which the endogenous 14 S to 73 S assembly has been completed by preincubation retain their ability to assemble newly added 14 S particles.

In vitro assembly of poliovirus: II. Evidence for the self-assembly of 14 S particles into empty capsids

Abstract A small (14S) virus-specific particle, isolated from poliovirus-infected HeLa cells, is able to self-assemble into a 73S particle. This 73S particle, when negatively stained and examined in the electron microscope, resembles the empty capsids found in infected cells. The kinetics of the self-assembly reaction was determined and found to be similar, but not identical, to the extract-mediated assembly of 14S particles into 73S particles. The self-assembly reaction, unlike the extract-mediated reaction, exexhibits a marked dependence on the initial concentration of 14S particles. The polypeptide content of 14S particles has been determined.

In vitro assembly of polioviruses: III. Assembly of 14 S particles into empty capsids by poliovirus-lnfected HeLa cell membranes

Abstract Rough membranes obtained from poliovirus-infected HeLa cells have the capacity to assemble 14 S particles into 73 S empty capsids in vitro . A corresponding fraction from uninfected cells did not possess this activity. When labeled 14 S particles were incubated with smooth membranes obtained from infected cells, a significant amount of radioactivity sedimented as heterogeneous material in the 30–70 S region of the gradient. Sucrose gradient analysis of 14 C-labeled rough and smooth membrane fractions lysed with deoxycholate (DOC) demonstrated the presence of 14 S particles, 73 S empty capsids, and a 110 S structure in the rough membrane fraction. These structures were found only in trace amounts in the smooth membrane fraction. In the absence of DOC treatment, no 14 S particles were found in any of the membrane fractions. The 73 S empty capsids, on the other hand, were detected in the presence or absence of DOC treatment in the rough-membrane fraction. Therefore, it appears that 14 S particles are associated with the rough membranes where they are assembled into 73 S empty capsids and/or complete virions.

Absence of subviral particles and assembly activity in HeLa cells infected with defective-interfering (DI) particles of poliovirus

Abstract HeLa cells infected with defective-interfering (DI) particles of poliovirus were examined for their capacity to synthesize viral proteins, viral-related particles, and the viral factor(s) that promotes the assembly of 14 S particles into empty capsidsin vitro. As reported by C. N. Cole and D. Baltimore (1973,J. Mol. Biol.76, 325–343) cells infected with purified DI particles failed to synthesize the capsid precursor NCVP 1a or any of the capsid polypeptides VP 0, VP 1, VP 2, or VP 3. Consequently, no 14 S particles, empty capsids, or virions were formed at low to moderate multiplicities. Cytoplasmic extracts prepared from cells infected with purified DI particles do not promote the assembly of viral 14 S particles into empty capsids. We conclude that the presence of assembly activity is dependent on the formation of the capsid precursor protein (NCVP-1a) or its cleavage products.

In vitro assembly of poliovirus. V. Evidence that the self-assembly activity of 14 S particles is independent of extract assembly factor(s) and host proteins.

Abstract Experiments were performed to determine whether or not the self-assembly of 14 S precursor particles into empty capsids was caused by the contamination of certain 14 S isolates with assembly factor(s) present in poliovirus-infected cell extracts. The self-assembly capacity of 14 S particle preparations was found to be directly related to the amount of viral-specific protein present. Dilution of 14 S particles markedly inhibited their self-assembly activity, whereas using ultrafiltration methods to concentrate 14 S particles increased their self-assembly activity. No consistent relationship was found for the presence or absence of particular viral or host proteins and the ability of 14 S particle preparations to self-assemble. The self-assembly capacity of 14 S particles was more sensitive to uv-inactivation than their ability to assemble into empty capsids in the presence of infec cted cell extracts. On the basis of these data and a detailed reanalysis of the effect of relative initial 14 S particle concentration on the rate of formation of empty capsids in extracts, we propose that the assembly of 14 S particles into empty capsids occurs in two steps, an initiation event and subsequent polymerization, and that extracts act by promoting the initiation event.

Use of temperature-sensitive mutants to study the morphogenesis of poliovirus

Three temperature-sensitive (ts) mutants of poliovirus (type 1 Mahoney) were isolated after nitrous acid treatment and characterized as phenotypically RNA+. When cells were infected at 37 degrees with two of the three RNA+ ts mutants (ts109 and ts739), reduced levels of 14 S particles were synthesized. One RNA+ mutant (ts520) synthesized significant amounts of viral 14 S particle subunits. All of the mutants synthesized reduced amounts of procapsids and virions at 37 degrees. At 39.5 degrees, with all three ts mutants, the production of all virus-related particles in infected cells was markedly suppressed. Isoelectric focusing of the viral-related particles produced at 37 degrees by the ts mutants and electrophoretic analysis of their structural polypeptides revealed the following: (i) ts739 synthesized an altered VP0 polypeptide and produced 14 S particles with an altered isoelectric point; (ii) ts109 produced 14 S particles with a normal pI but containing what appeared to be an altered VP1; (iii) ts520 produced normal 14 S particles as demonstrated by their pI, the electrophoretic behavior of their constituent structural polypeptides in SDS-PAGE, their ability to self-assemble, and their ability to form procapsid-like structures when incubated in extracts from wild-type (wt) virus-infected cells. However, ts520-infected cells contained few, if any, procapsids and extracts made therefrom were unable to assemble ts520 or wt 14 S particles into detectable amounts of pI 6.8 empty capsids. These and other findings are consistent with ts739 (and probably ts109) possessing an altered structural protein and ts520 being mutant in its morphopoietic factor.

In vitro assembly of polioviruses. IV. Evidence for the existence of two assembly steps in the formation of empty capsids from 14 S particles.

Abstract Evidence obtained from kinetic studies indicated that the assaembly reaction mediated by cytoplasmic extract prepared from poliovirus-infected HeLa cells was conposed of at least two distinct activities. One activity (assembly activity) converted mature 14 S particles into 73 S empty capsids. The other activity (activation) seemed to act on “immature” 14 S particles, enabling them to be assembled. Unlike the assembly activity, the activation reaction was depleted rapidly during the reaction in vitro. The activation factor(s) was not associated with isolated 14 S particles themselves but, like the assembly activity, appeared to be associated with the rough membrane fraction obtained from infected cells.

Unusual attachment behavior exhibited by a urea-resistant mutant of poliovirus type 1.

Abstract A urea-resistant mutant of type 1, LSc poliovirus, called LSc-U r , always produced about 30% of the progeny and 50% of the viral-specific RNA in infected cells as compared to its parent virus. LSc-U r virus was found to attach normally at low multiplicities of infection (m.o.i.) but exhibited autointerference at higher m.o.i. For example, at an m.o.i. of 10, less than 25% of the added virions was cell associated after 90 min. Neither parental LSc nor Mahoney poliovirus exhibited unusual attachment kinetics under comparable conditions. In addition, LSc-U r (m.o.i. = 10) interfered markedly with the attachment of Mahoney poliovirus. These findings were interpreted as resulting from a high particle/PFU ratio for the LSc-U r virus. LSc-U r virus partially purified by differential and sucrose gradient centrifugations still exhibited attachment autointerference at high m.o.i. in contrast to virus purified by CsCl banding which showed normal attachment and a much reduced ability to interfere with the attachment of other polioviruses. These results provide circumstantial evidence that LSc-U r virus preparations contain virion-like particles ( s 20,w of 120–150 S) which compete with the attachment of virions to cell receptors and which are degraded by high concentrations of CsCl.