TABLE 3.
Temperature sensitivity and sequence analysis of rHPIV1 mutants with the L protein Y942H mutation (single nucleotide substitution) or the Y942A mutation (three nucleotide substitutions) following passage at restrictive temperatures
| Virus | Passage seriesa | Passage level analyzedb (°C) | Mean titer (log10 TCID50/ml) at 32°C | Reduction in virus titer at the indicated tempc
|
Sequence of L gene and proteind
|
|||
|---|---|---|---|---|---|---|---|---|
| 38°C | 39°C | Codon 942 (amino acid assignment) | Extent of sequencinge | Second-site mutation | ||||
| rHPIV1 wild type | 8.0 | 1.2 | 3.4 | TAT (Tyr) | F | None | ||
| rHPIV1-Y942H | Unpassaged | 6.7 | 3.7f | >5.2g | CAC (His) | C | None | |
| rHPIV1-Y942H | 1 | p10-32 | 7.7 | 3.5 | 5.5 | CAC (His) | C | None |
| rHPIV1-Y942H | 2 | p4-35 | 7.5 | 3.3 | 5.3 | CAC (His) | C | None |
| rHPIV1-Y942H | 2 | p6-36 | 7.2 | 2.0 | 4.0 | TAC (Tyr) | C | None |
| rHPIV1-Y942H | 2 | p8-37 | 7.0 | 0.3 | 3.5 | TAC (Tyr) | C | None |
| rHPIV1-Y942H | 2 | p9-38 | 6.5 | 1.0 | 3.3 | TAC (Tyr) | F | None |
| rHPIV1-Y942H | 2 | p10-39 | 5.2 | 1.0 | 3.0 | TAC (Tyr) | F | None |
| rHPIV1-Y942A | Unpassaged | 6.5 | 4.8 | >5.0g | GCG (Ala) | P | None | |
| rHPIV1-Y942A | 3 | p10-32 | 7.5 | 4.0 | 4.5 | GCG (Ala) | P | None |
| rHPIV1-Y942A | 4 | p4-35 | 7.0 | 4.3 | >5.5g | GCG (Ala) | P | None |
| rHPIV1-Y942A | 4 | p6-36 | 7.0 | 4.0 | 4.8 | GCG (Ala) | P | V1016L |
| rHPIV1-Y942A | 4 | p8-37 | 6.7 | 3.5 | >5.2g | GCG (Ala) | P | V1016L/N1125Dh |
| rHPIV1-Y942A | 4 | p9-38 | 6.2 | 3.5 | >4.7g | GCG (Ala) | F | V1016L/N1125D |
| rHPIV1-Y942A | 5 | p10-39 | 5.2 | 3.0 | >3.7g | GCG (Ala) | F | S1328P |
Series 1 and 2 involve the Y942H mutant at the permissive and restrictive temperature regimens, respectively (Materials and Methods). Series 3 involves the Y942A mutant at the permissive regimen, while 4 and 5 are independent parallel series at the restrictive regimen.
Samples were analyzed from various passage levels within series 1 to 5: each passage level is identified by its number (p1 to p10) and its temperature (32 to 39, representing 32°C to 39°C).
Data shown are the results of a single experiment. Because of the HPIV1 requirement for the LLC-MK2 cells to be incubated in the presence of trypsin, values at higher temperatures can vary from experiment to experiment.
Consensus sequence determined from uncloned RT-PCR products.
The amount of the L gene sequenced was F (full), the entire L gene ORF; P, partial, nucleotides 10,000 to 13,300; C, codon-proximal, the region immediately surrounding codon 942.
Titer reduction is compared to titer at 32°C. A mutant is considered to be temperature sensitive if its reduction in titer at a nonpermissive temperature is 100-fold greater than the reduction of titer of the wild-type HPIV1 tested at that temperature (indicated by values in bold).
No detectable virus.
Sequencing electropherogram indicated 50% of each nucleotide (A or G) at nucleotide 12,143 in codon 1,125.