Genetic stability determinants of temperature sensitive, live attenuated respiratory syncytial virus vaccine candidates.

An intranasally delivered, live attenuated, temperature sensitive (ts) respiratory syncytial virus vaccine candidate, rA2cp248/404/1,030DeltaSH, exhibits a low level of genetic instability in clinical studies, in contrast to the relatively high stability of two similar candidates, cpts248/404 and rA2cp248/404DeltaSH. The latter strains, containing two ts mutations (248ts and 404ts), are partially growth restricted at 37 degrees C, whereas, rA2cp248/404/1,030DeltaSH contains an additional ts mutation (1,030ts) that increases attenuation and partially restricts virus growth at 35 degrees C. Since the maximum human airway temperature is 35.5 degrees C, we investigated whether growth restriction at 35 degrees C contributes to genetic instability of rA2cp248/404/1,030DeltaSH in vitro. We conducted in vitro passage studies with the three strains at 32 degrees C (a fully permissive growth temperature) and 35 degrees C (restrictive for only rA2cp248/404/1,030DeltaSH). Instability of the ts phenotype was observed only in rA2cp248/404/1,030DeltaSH passaged at 35 degrees C, and corresponded with reversion at the 248ts or 1,030ts mutation sites, as observed in clinical studies. This study indicates that ts mutations that partially restrict replication at physiologic temperatures may contribute to genetic instability of viruses in vivo. In vitro passage studies performed at appropriate temperatures can be used to assess genetic stability and to prioritize ts vaccine candidates for clinical evaluation.

A cell-free system for the synthesis of Sindbis virus subgenomic RNA: importance of the concentration of the initiating NTP.

We describe here an in vitro system for template-dependent initiation and synthesis of a Sindbis virus (SV) subgenomic (SG) RNA transcript. The critical components of this system were (1) a minus-strand promoter-template corresponding to the region of the SV genome from nt 7441 to nt 7772 (-157 to +175 relative to the SG RNA transcription initiation site at nt 7598), and (2) a p15 fraction from cells infected with recombinant vaccinia viruses expressing the SV nonstructural proteins, P123 and nsP4 (the nsP2 coding region in P123 contained a mutation which results in more rapid than normal processing of P123). Our data indicate that the SG RNA transcript is of the expected size, of positive polarity, and is initiated at the expected site. Changing the +1 nt from A to G, U, or C resulted in decreased synthesis of the SG RNA transcript. However, in each case, increasing the concentration of the initiating NTP restored synthesis of the transcript to the wild-type level. This is the first demonstration of an in vitro synthesis of an alphavirus SG RNA transcript which is dependent on the addition of an exogenous promoter-template. As such, it will make possible new approaches for learning how the synthesis of SG RNA is regulated.

Restriction of a Sindbis virus mutant in BHK cells and relief of the restriction by the addition of adenosine.

SV(PZF) is a mutant of Sindbis virus (SV) which we selected on the basis of its ability to replicate in mosquito cells treated with pyrazofurin (PZF), a drug which inhibits pyrimidine nucleotide biosynthesis (Lin et al., 2000, Virology 272, 61-71). Three mutations, A6627U, A7543U, and C7593A, were identified in the nsP4 (the viral RNA polymerase) coding region, which were required for the PZF-resistant phenotype. We report here that SV(PZF) has a second phenotype. Its replication in BHK cells is severely restricted; yields of SV(PZF) from BHK cells are 100- to 1000-fold lower than the yields of standard SV (SV(STD)). However, addition of adenosine to the SV(PZF)-infected cultures completely relieves this restriction and results in yields comparable to those observed with SV(STD). Adenosine has no effect on the yield of SV(STD) from BHK cells. Synthesis of the viral structural proteins is markedly depressed in SV(PZF)-infected BHK cells, as is synthesis of the viral subgenomic (SG) RNA from which these proteins are translated. In contrast, normal amounts of genomic RNA are made. Experiments with mutagenized viruses indicated that the SV(PZF) mutation, C7593A, by itself, was sufficient to produce the restriction phenotype. However, this mutation not only changes Pro 609 of nsP4 to Thr, it also changes the nucleotide at the minus sign5 position of the SG promoter. To evaluate the relative contributions of the change in nsP4 and the change in the SG promoter to the restriction phenotype, we made use of double SG viruses, in which nsP4 and the promoter for the SG RNA which encodes the structural proteins can be changed independent of each other. Our results indicated that both the change in nsP4 and the change in the SG promoter were required to produce the full restriction phenotype. We suggest that the changes in nsP4 and the SG promoter destabilize the RNA initiation complex assembled at the SG promoter and that since ATP is the initiating nucleotide in the SG RNA transcript, the increased level of ATP resulting from the addition of adenosine is able to compensate for this destabilization and restore the synthesis of SG RNA to normal levels.

A mutant of Sindbis virus which is able to replicate in cells with reduced CTP makes a replicase/transcriptase with a decreased Km for CTP.

We reported earlier the isolation and characterization of a Sindbis virus mutant, SV(PZF), that can grow in mosquito cells treated with pyrazofurin (PZF), a compound that interferes with pyrimidine biosynthesis (Y. H. Lin, P. Yadav, R. Ravatn, and V. Stollar, Virology 272:61-71, 2000; Y. H. Lin, H. A. Simmonds, and V. Stollar, Virology 292:78-86, 2002). Three amino acid changes in nsP4, the viral RNA polymerase, were required to produce this phenotype. We now describe a mutant of Sindbis virus, SVCPC, that is resistant to cyclopentenylcytosine (CPC), a compound that interferes only with the synthesis of CTP. Thus, in contrast to SVPZF, which was selected for its ability to grow in mosquito cells with low levels of UTP and CTP, SVCPC was selected for its ability to grow in cells in which only the level of CTP was reduced. Although SV(PZF) was cross-resistant to CPC, SVCPC was not resistant to PZF. Only one amino acid change in nsP4, Leu 585 to Phe, was required for the CPC resistance phenotype. The viral replicase/transcriptase generated in SVCPC-infected mosquito cells had a lower Km for CTP (but not for UTP) than did the enzyme made in SVSTD-infected mosquito cells. SV(PZF) and SVCPC represent the first examples of viral mutants selected for the ability to grow in cells with low levels of ribonucleoside triphosphates (rNTPs). Further study of these mutants and determination of the structure of nsP4 should demonstrate how alterations in an RNA-dependent RNA polymerase permit it to function in cells with abnormally low levels of rNTPs.