Virus protein changes and RNA termini alterations evolving during persistent infection

Cloned infectious vesicular stomatitis virus isolated following 5 years of persistent infection of BHK21 cells in vitro exhibits a number of peptide map changes in the G protein (spike glycoprotein), the M protein (membrane matrix protein) and the N protein (nucleocapsid structural protein). Only slight alterations have occurred in the peptide maps of the two VSV polymerase-associated proteins L and NS. Dideoxy sequencing of the 3′ ends of the cloned virus originally used to establish the persistent infection, and of the cloned virus recovered following 5 years of persistence, shows one base substitution in the three base junction between the 3′ leader sequence and the N protein-coding region. Repeated lytic passages of virus recovered from persistent infection led to no oligonucleotide map changes after 30 passages, but two map changes were present after 102 and remained after 133 lytic passages in BHK21 cells in vitro. Only one of these represented reversion to the original map position, and this “mutant” virus still exhibited a temperature-sensitive small plaque phenotype. Finally, the mutated virus recovered after more than $\text{5}\text{1}\text{2}$ years of persistent infection is now so slow-growing that it can establish persistent infection of BHK21 cells in the absence of DI particles (although DI particles are present constantly once the cells recover from the initial cytopathology).     

trans rescue of a mutant poliovirus RNA polymerase function.

A series of three-nucleotide insertions was engineered into the P2 and P3 coding regions of the T7 expression plasmid pT7(tau)-PV1, which encodes a full-length copy of poliovirus type 1 (Mahoney) cDNA. When RNA derived in vitro from these mutated templates was used to transfect HeLa cells, viable virus mutants were recovered. One mutant, Sel-3D-18, which contained a single amino acid insertion in the 3Dpol coding region, was temperature sensitive for growth at 39 degrees C and showed defects in both RNA synthesis and P1 protein processing at the nonpermissive temperature. The RNA replication defect in Se1-3D-18 was identified at the level of RNA chain elongation. A highly specific and sensitive method was developed for analyzing the ability of mutant RNA templates to replicate in the presence or absence of helper functions provided in trans. This approach was used to demonstrate that RNA synthesis in Se1-3D-18 can be rescued by helper functions provided in trans.     

In vitro translation of poliovirus RNA: utilization of internal initiation sites in reticulocyte lysate.

The translation of poliovirus RNA in rabbit reticulocyte lysate was examined. Translation of poliovirus RNA in this cell-free system resulted in an electrophoretic profile of poliovirus-specific proteins distinct from that observed in vivo or after translation in poliovirus-infected HeLa cell extract. A group of proteins derived from the P3 region of the polyprotein was identified by immunoprecipitation, time course, and N-formyl-[35S]methionine labeling studies to be the product of the initiation of protein synthesis at an internal site(s) located within the 3'-proximal RNA sequences. Utilization of this internal initiation site(s) on poliovirus RNA was abolished when reticulocyte lysate was supplemented with poliovirus-infected HeLa cell extract. Authentic P1-1a was also synthesized in reticulocyte lysate, indicating that correct 5'-proximal initiation of translation occurs in that system. We conclude that the deficiency of a component(s) of the reticulocyte lysate necessary for 5'-proximal initiation of poliovirus protein synthesis resulted in the ability of ribosomes to initiate translation on internal sequences. This aberrant initiation could be corrected by factors present in the HeLa cell extract. Apparently, under certain conditions, ribosomes are capable of recognizing internal sequences as authentic initiation sites.     

A mutant poliovirus containing a novel proteolytic cleavage site in VP3 is altered in viral maturation.

A six-amino-acid insertion containing a Q-G amino acid pair was introduced into the carboxy terminus of the capsid protein VP3 (between residues 236 and 237). Transfection of monkey cells with full-length poliovirus cDNA containing the insertion described above yields a mutant virus (Sel-1C-02) in which cleavage occurs almost entirely at the inserted Q-G amino acid pair instead of at the wild-type VP3-VP1 cleavage site. Mutant Sel-1C-02 is delayed in the kinetics of virus production at 39 degrees C and exhibits a defect in VP0 cleavage into VP2 and VP4 at 39 degrees C. Sucrose gradient analysis of HeLa cell extracts prepared from cells infected by Sel-1C-02 at 39 degrees C shows an accumulation of fast-sedimenting replication-packaging complexes and a significant amount of uncleaved VP0 present in fractions containing mature virions. Our data provide in vivo evidence for the importance of determinants other than the conserved amino acid pair (Q-G) for recognition and cleavage of the P1 precursor by proteinase 3CD and show that an alteration in the carboxy terminus of VP3 or the amino terminus of VP1 affects the process of viral maturation.     

Attenuation stem-loop lesions in the 5' noncoding region of poliovirus RNA: neuronal cell-specific translation defects.

The nucleotide at position 480 in the 5' noncoding region of the viral RNA genome plays an important role in directing the attenuation phenotype of the Sabin vaccine strain of poliovirus type 1. In vitro translation studies have shown that the attenuated viral genomes of the Sabin strains direct levels of viral protein synthesis lower than those of their neurovirulent counterparts. We previously described the isolation of pseudorevertant polioviruses derived from transfections of HeLa cells with genome-length RNA harboring an eight-nucleotide lesion in a stem-loop structure (stem-loop V) that contains the attenuation determinant at position 480 (A. A. Haller and B. L. Semler, J. Virol. 66:5075-5086, 1992). This stem-loop structure is a major component of the poliovirus internal ribosome entry site required for initiation of viral protein synthesis. The eight-nucleotide lesion (X472) was lethal for virus growth and gave rise only to viruses which had partially reverted nucleotides within the original substituted sequences. In this study, we analyzed two of the poliovirus revertants (X472RI and X472R2) for cell-type-specific growth properties. The X472RI and X472R2 RNA templates directed protein synthesis to wild-type levels in in vitro translation reaction mixtures supplemented with crude cytoplasmic HeLa cell extracts. In contrast, the same X472 revertant RNAs displayed a decreased translation initiation efficiency when translated in a cell-free system supplemented with extracts from neuronal cells. This translation initiation defect of the X472R templates correlated with reduced yields of infectious virus particles in neuronal cells compared with those obtained from HeLa cells infected with the X472 poliovirus revertants. Our results underscore the important of RNA secondary structures within the poliovirus internal ribosome entry site in directing translation initiation and suggest that such structures interact with neuronal cell factors in a specific manner.     

A cellular cofactor facilitates efficient 3CD cleavage of the poliovirus P1 precursor.

The production of poliovirus capsid proteins from a capsid protein precursor (P1) is mediated by virus-encoded proteinase 3CD and involves a complicated set of proteinase-substrate interactions. In addition to substrate and enzymatic determinants required for this interaction, we describe a cellular cofactor, which facilitates 3CD recognition of the P1 precursor. Cellular cofactor activity is 3CD dependent and salt dependent. Our analysis shows that proteolytic cleavage of the P1 precursor at the VP0/VP3 cleavage site exhibits a greater dependency on the cellular cofactor than cleavage at the VP3/VP1 site. Such a greater dependency on cellular cofactor activity can be relieved (in part) by the substitution of an Ala residue for the Pro residue at the -4 position of the VP0/VP3 cleavage site. However, mutant viruses containing Pro-to-Ala substitutions at the -4 position of the VP0/VP3 site exhibit defects in viral growth.     

Replication-competent picornaviruses with complete genomic RNA 3' noncoding region deletions.

The genomic RNA 3' noncoding region is believed to be a major cis-acting molecular genetic determinant for regulating picornavirus negative-strand RNA synthesis by promoting replication complex recognition. We report the replication of two picornavirus RNAs harboring complete deletions of the genomic RNA 3' noncoding regions. Our results suggest that while specific 3'-terminal RNA sequences and/or secondary structures may have evolved to promote or regulate negative-strand RNA synthesis, the basic mechanism of replication initiation is not strictly template specific and may rely primarily upon the proximity of newly translated viral replication proteins to the 3' terminus of template RNAs within tight membranous replication complexes.     

Expression of a cloned gene segment of poliovirus in E. coli: evidence for autocatalytic production of the viral proteinase

The poliovirus polyprotein is proteolytically processed predominantly by a virus-encoded proteinase (P3-7c) that cleaves glutamine-glycine amino acid pairs. The biosynthesis of the viral proteinase, itself a product of glutamine-glycine cleavages, was studied by constructing a bacterial expression plasmid that contained a cloned segment of the poliovirus genome slightly larger than the coding region for P3-7c. The induction of expression of this plasmid in E. coli produced several poliovirus-specific polypeptides. One polypeptide, an unstable protein called 3i, was the product of fortuitous in-phase initiation of translation within the coding region of P3-7c. Three other induced polypeptides were products of proteolytic cleavages, the smallest (polypeptide 3) having the properties (amino-terminal amino acids, carboxy-terminal amino acids, size, antigenicity) of P3-7c. Insertion of a DNA linker into the P3-7c coding region results in the loss of P3-7c-specific glutamine-glycine cleavage activity. We conclude that P3-7c was produced by autocatalytic cleavage.