gD-Independent Superinfection Exclusion of Alphaherpesviruses

Abstract

Many viruses have the capacity to prevent a cell from being infected by a second virus, often termed superinfection exclusion. Alphaherpesviruses, including the human pathogen herpes simplex virus 1 (HSV-1) and the animal herpesvirus pseudorabies virus (PRV), encode a membrane-bound glycoprotein, gD, that can interfere with subsequent virion entry. We sought to characterize the timing and mechanism of superinfection exclusion during HSV-1 and PRV infection. To this end, we utilized recombinant viruses expressing fluorescent protein (FP) markers of infection that allowed the visualization of viral infections by microscopy and flow cytometry as well as the differentiation of viral progeny. Our results demonstrated the majority of HSV-1- and PRV-infected cells establish superinfection exclusion by 2 h postinfection. The modification of viral infections by virion inactivation and phosphonoacetic acid, cycloheximide, and actinomycin D treatments indicated new protein synthesis is needed to establish superinfection exclusion. Primary infection with gene deletion PRV recombinants identified that new gD expression is not required to establish superinfection exclusion of a secondary viral inoculum. We also identified the timing of coinfection events during axon-to-cell spread, with most occurring within a 2-h window, suggesting a role for cellular superinfection exclusion during neuroinvasive spread of infection. In summary, we have characterized a gD-independent mechanism of superinfection exclusion established by two members of the alphaherpesvirus family and identified a potential role of exclusion during the pathogenic spread of infection.

Importance: Superinfection exclusion is a widely observed phenomenon initiated by a primary viral infection to prevent further viruses from infecting the same cell. The capacity for alphaherpesviruses to infect the same cell impacts rates of interviral recombination and disease. Interviral recombination allows genome diversification, facilitating the development of resistance to antiviral therapeutics and evasion of vaccine-mediated immune responses. Our results demonstrate superinfection exclusion occurs early, through a gD-independent process, and is important in the directed spread of infection. Identifying when and where in an infected host viral genomes are more likely to coinfect the same cell and generate viral recombinants will enhance the development of effective antiviral therapies and interventions.

FIG 1

HSV superinfection exclusion. (A) A diagram of the timeline of experimentation. CFP-expressing virus (termed primary virus) is added at T−1. YFP-expressing virus (termed the second virus), is either added at the same time, or applied at later times postinfection (T1, T2, or T3). Cells then are analyzed for FP expression between 6 and 8 h postinfection (hpi). (B) Fluorescent micrographs of infected Vero cells. Cells were infected at the time points indicated in panel A at an MOI of 10 of either HSV OK12 or HSV MT01. At 8 hpi, FP expression was imaged with identical exposure settings under ×200 magnification (the scale bar is 50 μm). CFP and YFP channels are monochrome, while the two-channel merged image is in color (CFP in blue, YFP in green). Experiments were performed twice with triplicate samples for each condition. Representative images are presented. (C) Flow cytometry of YFP expression in populations of HSV-1-coinfected cells. Coinfected populations of Vero cells were tracked for YFP expression (green-line histogram) compared to in mock-infected cells (filled gray histogram). A total of 100,000 events were collected per sample. Representative data from duplicate experiments are depicted. (D) Coinfected cells were harvested at 10 hpi, and progeny virus was subjected to limiting dilution. Plaques were visualized and scored for FP expression. Replicates of three were performed per condition, with a minimum of 100 plaques counted per sample. The ratio of YFP and CFP to total plaques counted is displayed.

FIG 2

PRV superinfection exclusion. (A) PRV 287 and 289 were used to coinfect PK15 cells at an MOI of 10 for each virus under the conditions indicated. FP expression was imaged with identical exposure settings under ×200 magnification (the scale bar is 50 μm). CFP and YFP channels are monochrome, while the two-channel merged image is in color (CFP in blue, YFP in green). Experiments were performed twice with triplicate samples for each condition. (B) Flow cytometry of YFP expression in populations of PRV-coinfected cells. Coinfected populations of PK15 cells were tracked for YFP expression (green-line histogram) compared to mock-infected cells (filled gray histogram). A total of 100,000 events were collected per sample. (C) Coinfected cells were harvested at 10 hpi, and progeny virus was subjected to limiting dilution. Plaques were visualized and scored for FP expression. Replicates of three were performed per condition, with a minimum of 100 plaques counted per sample. The ratio of YFP and CFP to total plaques counted is displayed.

FIG 3

Chemical inhibition of PRV coinfection. Microscopy of T2 coinfections using PRV 289 (primary inoculum; MOI of 10) and PRV 287 (delayed inoculum; MOI of 10). Cells were imaged for CFP (monochrome channel, left) and YFP (monochrome channel, middle) at 6 to 8 hpi. Also depicted is a merged image of the two channels (CFP is blue, YFP is green). (A) Mock-treated cells. (B) Primary inoculum subjected to UV irradiation. (C and D) Cells treated with CHX at 100 μg/ml (C) or PAA at 400 μg/ml (D) for 2 h between the primary and secondary infections. (E) Following primary infection, cells were treated sequentially with CHX and ActD at 100 μg/ml for 2 h each before application of the PRV 287 at T4. (F) Western blot analysis of VP5 expression from PRV-infected cells during chemical treatment. Cells were either mock infected or infected with PRV 289 at an MOI of 10. Following infection, cells were mock treated or treated with CHX, ActD, or PAA as previously described. Cells were harvested at 6 hpi, and proteins were subjected to Western blot analysis with anti-VP5 or anti-beta-actin antibodies. (G) Quantitative real-time PCR analysis of viral gene transcription. Cells were infected with PRV 289 and subjected to CHX, PAA, and ActD treatment as previously described. Cells were harvested at 2 or 4 hpi, and RNA was differentially extracted and analyzed by qRT-PCR with primers to detect PRV EP0 transcripts. Relative threshold cycle (C_T) values for each sample were calculated and then normalized to 28S rRNA controls. Fold change values are relative to values for 2-hpi samples (relative quantitation [Rq] values) for replicate samples (gray dots) along with average relative Rq values (black bars).

FIG 4

Dependence of superinfection exclusion on gD expression. PK15 cells were infected with wild-type (Wt) PRV Becker (A) or PRV GS6127, a gD-null virus, and coinfected with YFP-expressing PRV 287 (B). Viruses were applied simultaneously under T−1 conditions, or PRV 287 was applied at 2 hpi of the initial inoculum under T2 conditions. At 6 hpi, cells were imaged by phase contrast and YFP fluorescence illumination. (Left) Phase and YFP merged images. (Right) YFP alone. (B) PK15 cells were either mock infected or were infected with PRV Becker, PRV GS6127, or PRV GS442. The expression of gD or the major capsid protein VP5 was detected from infected cell extracts by Western blotting.

FIG 5

Coinfection during axon-to-cell spread. (A) Diagram of the compartmentalized neuronal cultures. The red box indicates the relative area where images were acquired. (B) Two micrographs of PK15 cells with mRFP-labeled capsids accumulating on the nuclear envelope. The top image is a phase-contrast and RFP merge. The bottom image is the RFP channel alone, with the outline of the cell border and nucleus (dashed) outlined in white. (C) Out of 153 infection events imaged, 57 cells were infected by more than 1 virion. The times between detection of the first and last capsid were calculated and cells were grouped in 30-min intervals.