A single codon in the nucleocapsid protein C terminus contributes to in vitro and in vivo fitness of Edmonston measles virus

Abstract

The major inducible 70-kDa heat shock protein (hsp72) increases measles virus (MV) transcription and genome replication. This stimulatory effect is attributed to hsp72 interaction with two highly conserved hydrophobic domains in the nucleocapsid protein (N) C terminus of Edmonston MV. These domains are known as Box-2 and Box-3. A single amino acid substitution in Box-3 of Edmonston MV (i.e., N522D) disrupts hsp72 binding. The prevalence of the N522D substitution in contemporary wild-type MV isolates suggests that this sequence has been positively selected. The present work determined if the N522D substitution enhances viral fitness and the degree to which any fitness advantage is influenced by hsp72 levels. Both parent Edmonston MV (Ed N) and an N522D substitution mutant (Ed N-522D) exhibited similar growth on Vero and murine neuroblastoma cells and in cotton rat lung, although Ed N-522D virus exhibited an attenuated in vitro response to hsp72 overexpression. In contrast, mixed infections showed a significantly reduced in vitro and in vivo fitness of Ed N-522D virus. Results support the involvement of additional selectional pressures that maintain the circulation of virus containing N-522D despite the cost to viral fitness.

FIG. 1.

Constitutive overexpression of hsp72 in stably transfected murine neuroblastoma cells (N2a). (A) Western blot analysis of total cell protein using an antibody recognizing the constitutively expressed and major inducible 70-kDa heat shock proteins (i.e., hsp73 and hsp72, respectively). hsp72 is expressed in construct-transfected cell lines N2a-HSP1₁ and N2a-HSP₂ but not vector-transfected control cell lines N2a-V₁ and N2a-V₂. (B) Thermotolerance of hsp72-expressing and control N2a cells as a measure of hsp72 function. The hsp72-expressing cell lines exhibited enhanced colony-forming ability relative to vector-transfected cell lines following exposure to 45°C for 1.5 to 4.5 h. (C) Northern blot analysis of Ed N viral transcripts in total RNA from infected hsp72-expressing and control N2a cells. Cells were infected at an MOI of 1.0, and the RNA was harvested at 24 h p.i. Phosphorimage analysis of signal intensities, corrected for variations in the loading control (GAPDH), showed that hsp72-expressing cells support stimulation of viral transcript production to a similar degree (i.e., approximately twofold); the mean increase was statistically significant (P < 0.05, t test).

FIG. 2.

Cell-free infectious viral progeny release by hsp72-expressing N2a-HSP₁ and control N2a-V₂ murine neuroblastoma cells infected with either Ed N or Ed N-522D virus. Cells were infected at an MOI of 0.05, and progeny were measured between 36 and 84 h p.i. Results are an average of three consecutive experimental trials and are represented by the mean titer ± the standard error of the mean. Progeny release was increased on hsp72-expressing cells, although the increase was statistically significant (*) only for cells infected with the Ed N virus (P < 0.05, t test).

FIG. 3.

(A) Northern blot analysis of viral genome and transcript levels in hsp72-expressing (N2a-HSP₁) and control (N2a-V₂) neuroblastoma cells infected with either Ed N or Ed N-522D virus at an MOI of 0.05. hsp72 expression enhanced genome and transcript levels for both Ed N and Ed N-522D virus, although the increase in transcript levels was attenuated for N-522D. The latter resulted in a greater transcript/genome ratio for Ed N relative to Ed N-522D virus on hsp72-expressing cells. (B) Ratios reflect phosphorimage signal intensities corrected for variations in GAPDH signal intensity, calculated independently for the N and H transcript, and represent the mean ± SD for samples harvested at 48, 72, and 84 h p.i. (i.e., time points providing readily quantifiable genome signals). Differences between Ed N and Ed N-522D were statistically significant (*, P < 0.05; t test). Results are representative of three experimental trials.

FIG. 4.

Linear relationship between the ratio of infectious viral variants Ed N and Ed N-522D and virus-specific RT-PCR amplicon yield. Individual pools of titrated Ed N and Ed N-522D virus were combined at an N-522D/N ratio of 1:1, 1:2, 1:5, or 1:10. Total RNA was extracted, and a 267-nt genomic sequence spanning the N-P junction was amplified by RT-PCR. (A) The proportion of Ed N and Ed N-522D represented in the amplicons was based upon BsmAI restriction fragment length polymorphism. The 267-bp amplicon derived from Ed N virus is not cleaved, whereas the corresponding amplicon from Ed N-522D is cleaved to yield 134- and 133-bp fragments; these fragments resolve as a single band following 2% agarose gel electrophoresis. Reactions lacking RT were used as a negative control (-) for the RT-PCR. (B) Linear regression analysis of the ratio of N-522D and N signal intensities (i.e., ethidium bromide staining intensities of virus-specific amplicons) expressed as a function of the ratio of N-522D and N virus used in the assay. Mean signal ± SD was based upon three separate analyses. The line describing the relationship between the ratio of infectious viral variants and the ratio of virus-specific RT-PCR amplicons (y = 0.55x + 0.42) exhibits an excellent fit to the experimental data (R² = 0.99).

FIG. 5.

Relative fitness of Ed N-522D compared to Ed N based upon coinfection of hsp72-expressing N2a-HSP₁ and control N2a-V₂ cells. The inocula contained a 1:1 ratio of each virus for a combined MOI of 0.1, 1.0, or 10.0. The proportion of Ed N and Ed N-522D in the viral progeny was based upon BsmAI restriction fragment length polymorphisms of amplicons derived from RT-PCR of cell-free viral genomic RNA. Yield of amplicon was based upon ethidium bromide staining intensity of BsmAI-digested products resolved by gel electrophoresis (A). The ratio of the 133-/134- and the 267-bp products was used as a correlate of the relative fitness of Ed N-522D compared to Ed N (B). Results reflect the means of three experimental trials ± SD. Differences in ratios calculated from infection of N2a-HSP₁ and N2a-V₂ cells were not statistically significant. Controls for the infection were individual inoculations with Ed N or Ed N-522D virus, and reactions lacking RT were used as a negative control (-) for the RT-PCR.

FIG. 6.

(A) Reduced fitness of Ed N-522D relative to Ed N was also illustrated by coinfecting hsp72-expressing N2a-HSP₁ and control N2a-V₂ cells with each variant at a total MOI of 1.0, using progressively increased proportions of Ed N-522D (i.e., from a 1:1 ratio with Ed N to a 100:1 ratio). The proportion of each viral variant in cell-free progeny was based upon BsmAI restriction fragment length polymorphisms of RT-PCR amplicons derived from genomic RNA, quantifying ethidium bromide staining intensities of products resolved on 2% agarose gels. Increasing the relative amount of Ed N-522D/N in the inoculum to 20:1 resulted in increased recovery of Ed N-522D in the progeny, although further increasing Ed N-522D in the inoculum was without effect. Results are representative of two experimental trials. Controls for the infection were individual inoculations with Ed N or Ed N-522D virus, and reactions lacking RT were used as a negative control (-) for the RT-PCR. (B) Recovery of Ed N-522D in the progeny of mixed infections was also enhanced by transfecting cells with plasmid supporting the expression of N-522D. Cells were infected with a 1:1 ratio of Ed N-522D and Ed N (combined MOI of 1.0) at 2 h posttransfection. Negative controls included nontransfected cells (-) and cells transfected with empty plasmid vector (V). Western blot analysis of uninfected cell lysates processed in parallel showed expression of the N-522D protein. Infections were performed in triplicate, and the Ed N-522D/N RT-PCR signal ratio is expressed as the mean ± SD.

FIG. 7.

(A) Infectious viral progeny release for parental Ed N compared to the Ed N-522D variant in control (C) and preconditioned (PC) Vero cells following infection at an MOI of 0.01. Results are an average of two separate experimental trials and expressed as the mean ± standard error of the mean (SEM). Preconditioning of Vero cells was achieved by exposing cells in the log phase of growth to 43°C for 1.5 h, a treatment elevating cytoplasmic hsp72 for 24 to 48 h posttreatment. Cells were infected at 16 h posttreatment. Peak infectious progeny release was statistically significantly elevated for Ed N but not Ed N-522D virus, representing increases of 5- and 1.5-fold, respectively. (B) Relative fitness of Ed N-522D compared to Ed N based upon coinfection of preconditioned and control Vero cells. The inocula contained a 1:1 ratio of each virus for a combined MOI of 0.01, or 1.0. The proportion of Ed N and Ed N-522D in the viral progeny was based upon BsmAI restriction fragment length polymorphisms of amplicons derived from RT-PCR of cell-free viral genomic RNA. Yield of amplicon was based upon ethidium bromide staining intensity of BsmAI-digested products resolved by gel electrophoresis.

FIG. 8.

Nested RT-PCR amplification of viral sequence from total lung RNA of cotton rats infected with 1 × 10⁵ TCID₅₀ of Ed N, Ed-N522D, or a 1:1 ratio of Ed N and N-522D. Samples were processed at 4 days p.i. Amplicons were digested with BsmAI to determine the proportion of the 267-bp (Ed N) versus 133/134-bp (Ed N-522D) product on 2% agarose gels stained with ethidium bromide. Negative controls included total lung RNA from uninfected rats (U) and infected rats in which the RT step was omitted (-). Ed-N is the predominant virus identified as a consequence of mixed infections.