Human parainfluenza virus evolution during lung infection of immunocompromised individuals promotes viral persistence

Abstract

The capacity of respiratory viruses to undergo evolution within the respiratory tract raises the possibility of evolution under the selective pressure of the host environment or drug treatment. Long-term infections in immunocompromised hosts are potential drivers of viral evolution and development of infectious variants. We showed that intrahost evolution in chronic human parainfluenza virus 3 (HPIV3) infection in immunocompromised individuals elicited mutations that favored viral entry and persistence, suggesting that similar processes may operate across enveloped respiratory viruses. We profiled longitudinal HPIV3 infections from 2 immunocompromised individuals that persisted for 278 and 98 days. Mutations accrued in the HPIV3 attachment protein hemagglutinin-neuraminidase (HN), including the first in vivo mutation in HN's receptor binding site responsible for activating the viral fusion process. Fixation of this mutation was associated with exposure to a drug that cleaves host-cell sialic acid moieties. Longitudinal adaptation of HN was associated with features that promote viral entry and persistence in cells, including greater avidity for sialic acid and more active fusion activity in vitro, but not with antibody escape. Long-term infection thus led to mutations promoting viral persistence, suggesting that host-directed therapeutics may support the evolution of viruses that alter their biophysical characteristics to persist in the face of these agents in vivo.

Keywords: Influenza; Virology.

Figure 1. Longitudinal sampling of long-term HPIV3 infections in vivo.

Sampling time series data with associated Ct values shown for patient 1 (A) and patient 2 (B). Samples collected by nasal swabs are represented by dots; black dots are HPIV3-positive samples with associated sequencing data, gray dots are HPIV3-positive samples that were not available for sequencing, and empty dots tested negative for HPIV3. HPIV3-positive bronchoalveolar lavage (BAL) samples are represented by asterisks. Light blue boxes indicate time periods in which patient 1 was treated with DAS181. Amino acid sequences of the HN protein for patient 1 and patient 2 are placed in the context of circulating strains that were downloaded from NCBI’s GenBank (C). All sequences are labeled with GenBank accession number followed by collection location and collection date. Consensus support values are shown next to branch points. The laboratory-adapted HPIV3 reference strain (NC_001796) is used as an outgroup. Amino acid alignment of the day 0 consensus sequences of the HN and F attachment proteins for each patient were aligned with the clinical isolate used in functional assays (D). Only amino acids that differ among these isolates are depicted.

Figure 2. Maximum allele frequency plots for nonsynonymous mutational changes across the whole genome during persistent HPIV3 infection.

Maximum allele frequency noted for nonsynonymous mutational changes across whole genome during persistent HPIV3 infection for patient 1 (A) and patient 2 (B). All changes are relative to the day 0 majority consensus for each patient. Mutational changes were determined using LAVA. Plots show nonsynonymous mutations present in at least 2 longitudinal time points that had minor allele frequency more than 5% and depth more than 10 reads and passed manual review for tagmentation artifacts. Nucleotide position is listed across the x axis and maximum allele frequency observed is plotted on the y axis. Alternating green and blue rectangles denote protein coding regions. Mutational changes observed in the HN protein and those above 50% maximum allele frequency are labeled with consensus amino acid, amino acid position relative to protein start, and amino acid change.

Figure 3. Nonsynonymous mutational changes and allele frequencies in HN protein across long-term persistent HPIV3 infection.

Patient 1 (A); patient 2 (B). The mutational change is shown above each subplot, with all changes relative to the day 0 majority consensus for that patient. Variants above an allele frequency of 5% and a depth of 10 reads in at least 2 samples for patient 1 and 1 sample for patient 2 are depicted, given the increased number of samples. Light blue boxes indicate time periods in which patient 1 was treated with DAS181. Sample collection dates are given relative to the first sample and plotted on the x axis for each plot. Samples collected by nasal swab are represented by dots, and BAL samples are marked with an asterisk.

Figure 4. Location of mutated residues in HN that arose during long-term infection.

(A) Overview of HN (PDBID:4MZA) and F (PDBID:6MJZ) on the viral surface. Residues that showed variant changes of greater than 25% in the HN protein during long-term infection for (B) patient 1 and (C) patient 2 are labeled. The backbone is colored in green, and the side chains are labeled by standard atom coloring. Residues are labeled with the original reference amino acid and not the changes observed. Note the localization of minor variants to 2 of the known active sites of HPIV3 HN protein.

Figure 5. Patient-derived HPIV3 HN proteins have altered receptor-binding and receptor-cleaving properties.

(A) Neuraminidase activity of patient-derived HPIV3 HNs. HEK293T cells were transfected with plasmids containing corresponding sequences of clinically isolated HNs. Neuraminidase activity was quantified by measuring amount of cleavage of 4-MUNANA. Results depict representative experiments from 3 biological replicates. Data indicate the mean ± SD. A 1-way ANOVA was performed to determine significance; P values are specified as follows: *P ≤ 0.05; ****P ≤ 0.0001. FC, fold change. (B) Release kinetics of patient-derived HPIV3 HNs binding sialic acid–containing RBCs at 37°C. HEK293T cells transiently expressing HNs were incubated with RBCs at 4°C for 30 minutes, washed, and transferred to 37°C. Supernatant was collected at 0, 5, 10, 15, 45, and 60 minutes and percentage of RBCs released at each time point was determined by quantification of relative absorbance at 410 nm. Results depict representative experiments from 3 biological replicates.

Figure 6. Fusion activity of clinically isolated HPIV3 HN and F proteins.

Fusion activity was assessed using a β-galactosidase complementation assay. HEK293T cells were cotransfected with either (A) HN sequence of the indicated patients, lab-adapted HPIV3 F, and the α subunit of β-galactosidase, or (B) F sequence of the indicated patients, lab-adapted HPIV3 HN, and the α subunit of β-galactosidase. Cells were then incubated for 6 hours with HEK293T cells expressing the Ω subunit of β-galactosidase. Fusion is depicted by luminescence relative to WT patient HN (A) or relative to mock-transfected cells (B) containing only lab-adapted HPIV3 HN and the Ω subunit of β-galactosidase. Results depict representative experiments from 3 biological replicates. Data indicate the mean ± SD. A 2-way ANOVA was performed to determine significance; P values are specified as follows: *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, and ****P ≤ 0.0001.

Figure 7. Growth of recombinant CI-1 HPIV3 EGFP containing SC9779 HN/F in HAE cells.

(A) HAE cells were infected with 200 PFU of CI-1 HPIV3 EGFP containing SC9779 HN/F, SC9779 HN T193A-H552Q/F, or HN H552Q/F at the apical surface. Viruses were collected from the apical surface of the HAE cells on days 1, 2, 3, 5, and 7 after initial infection. (B) Titration of HPIV-3 CI-1 EGFP viruses (WT CI-1 or HN mutants) of virus collected from HAE cells 1, 2, 3, 5, and 7 days after initial infection (PFU/mL). Results depict a representative experiment from 3 biological replicates; data shown as mean ± SEM.

Figure 8. In vivo infectivity of patient-derived viruses.

Cotton rats were infected intranasally with CI-1, CI-1 HN H552Q, CI-1 HN/F SC9779, and CI-1 HN/F SC9779 H552Q-T193A, and the left lungs were collected after 3 and 5 days. A 1-way ANOVA was performed to determine significance; P values are specified as follows: *P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.001. TCID₅₀, 50% tissue culture infectious dose.