Superinfection Exclusion between Two High-Risk Human Papillomavirus Types during a Coinfection

Abstract

Superinfection exclusion is a common phenomenon whereby a single cell is unable to be infected by two types of the same pathogen. Superinfection exclusion has been described for various viruses, including vaccinia virus, measles virus, hepatitis C virus, influenza A virus, and human immunodeficiency virus. Additionally, the mechanism of exclusion has been observed at various steps of the viral life cycle, including attachment, entry, viral genomic replication, transcription, and exocytosis. Human papillomavirus (HPV) is the causative agent of cervical cancer. Recent epidemiological studies indicate that up to 50% women who are HPV positive (HPV⁺) are infected with more than one HPV type. However, no mechanism of superinfection exclusion has ever been identified for HPV. Here, we show that superinfection exclusion exists during a HPV coinfection and that it occurs on the cell surface during the attachment/entry phase of the viral life cycle. Additionally, we are able to show that the minor capsid protein L2 plays a role in this exclusion. This study shows, for the first time, that superinfection exclusion occurs during HPV coinfections and describes a potential molecular mechanism through which it occurs.IMPORTANCE Superinfection exclusion is a phenomenon whereby one cell is unable to be infected by multiple related pathogens. This phenomenon has been described for many viruses and has been shown to occur at various points in the viral life cycle. HPV is the causative agent of cervical cancer and is involved in other anogenital and oropharyngeal cancers. Recent epidemiological research has shown that up to 50% of HPV-positive individuals harbor more than one type of HPV. We investigated the interaction between two high-risk HPV types, HPV16 and HPV18, during a coinfection. We present data showing that HPV16 is able to block or exclude HPV18 on the cell surface during a coinfection. This exclusion is due in part to differences in the HPV minor capsid protein L2. This report provides, for the first time, evidence of superinfection exclusion for HPV and leads to a better understanding of the complex interactions between multiple HPV types during coinfections.

Keywords: HPV16; HPV18; coinfection; human papillomavirus; superinfection exclusion; virus.

FIG 1

A single cell can be infected with multiple HPV types. (A) HaCaT cells were infected with HPV16 only, HPV18 only, or HPV16 and HPV18 together, and E1∧E4 (left) and E1-E2 (right) mRNAs were detected via RNA-FISH. HPV16 mRNA is labeled with fluorescein isothiocyanate (FITC) and depicted in green, and HPV18 mRNA is labeled with Cy3 and depicted in red in the merged image. Nuclei are stained with Hoechst dye and depicted in blue in the merged image. Individual channels are shown in grayscale. The inset in the merged image is representative of a magnified portion of the merged image (indicated by a small white box within the image). (B) Quantitation of infected cells via RNA-FISH staining. All experiments were done two times with two different virus preparations. These results are representative of data from at least 40 images taken per experiment.

FIG 2

HPV18 infectivity is decreased in the presence of HPV16 during a coinfection. (A and B) HaCaT cells (A) or primary foreskin and cervical keratinocyte cells (B) were infected with HPV16, HPV18, or both HPV16 and HPV18. At 48 hpi, mRNA was harvested, and levels of the E1∧E4 splice transcript were determined by qRT-PCR. The levels of infection are relative, with values for single infections being set equal to 1. (C) HaCaT cells infected with OptiPrep-purified HPV16, HPV18, or both HPV16 and HPV18. At 48 hpi, mRNA was harvested, and levels of the E1∧E4 splice transcript were determined by qRT-PCR. The levels of infection are relative, with values for single infections being set equal to 1. (D) HaCaT cells coinfected with either HPV16 or HPV18 and the primary HFK lysate. At 48 hpi, mRNA was harvested, and the levels of the E1∧E4 splice transcript were determined by qRT-PCR. The levels of infection are relative, with values for single infections being set equal to 1. Panels A to D are representative of results from at least three individual experiments utilizing at least two different HPV16 and HPV18 preparations. (E) HaCaT cells were infected with OptiPrep-purified HPV16 and/or HPV18 QV at a MOI of 10. At 48 hpi, mRNA was harvested, and levels of the E1∧E4 splice transcript were determined by qRT-PCR. The levels of infection are relative, with values for single infections being set equal to 1. (F) HaCaT cells were coinfected with either HPV16 NV (MOI = 10) and HPV18 VLPs (equivalent to MOIs of 10, 25, 50, and 100) (left) or HPV18 NV (MOI = 10) and HPV16 VLPs (equivalent to MOIs of 10, 25, 50, and 100) (right). Both NVs and VLPs were OptiPrep purified. At 48 hpi, mRNA was harvested, and the levels of the E1∧E4 splice transcript were determined by qRT-PCR. The levels of infection are relative, with values for single infections being set equal to 1. Panels E and F are representative of data from at least 2 different experiments with at least 2 batches of NV, QV, and VLPs. Bars represent standard deviations. An asterisk denotes significance by Student's t test. Statistical significance was defined as a P value of ≤0.05.

FIG 3

HPV18 infectivity is recovered by infection with additional virions but not with prebinding. (A) HaCaT cells were infected with HPV16 only (MOI = 10), HPV18 only (MOI = 10 to 1,000), or HPV16 (MOI = 10) and HPV18 (MOI = 10 to 1,000) together, followed by harvesting of mRNA and measurement of infectivity via qRT-PCR amplifying the E1∧E4 splice transcript. Analysis was performed on both HPV18 samples (left) and HPV16 samples (right). Values for individual HPV18 infections at each MOI were set equal to 1, and the relative levels of the HPV18 transcript detected in coinfections are shown. The levels of HPV16 during coinfections are all measured relative to the level of E1∧E4 detected during a single HPV16 infection. (B) HPV18 (MOI = 10) was bound to HaCaT cells at 4°C, followed 1.5 h later by the addition of HPV16 (MOI = 10) at 4°C. RNA was harvested, and infectivity was measured via qRT-PCR amplifying the E1∧E4 splice transcript. (C) HaCaT cells were seeded as described above. The cells were then infected at time zero with either HPV16 or HPV18. At 24 h, uninfected cells were infected with either HPV16 or HPV18 and a subset of the infected cells was coinfected with the other HPV type. RNA was harvested, and the level of infection was measured via qRT-PCR as described above. (D and E) Cells that stably maintain the HPV16 (HPV16 WT:3)or HPV18 (HPV18c) genome were seeded and then infected with increasing MOIs of HPV18 and HPV16, respectively. RNA was then harvested, and the levels of E1∧E4 present for both HPV types were assessed via qRT-PCR as described above. (F and G) Primary cells and stable cell lines harboring either HPV18 or HPV16 were infected with HPV16 or HPV18, respectively, at a MOI of 10. RNA was harvested, and infections were analyzed by determining E1∧E4 splice transcript levels via qRT-PCR. The level of infection in primary cells was set equal to 1, and the level of infection in stable cell lines persistently maintaining a HPV genome was measured relative to primary cell infections. All data are representative of results from at least three individual experiments utilizing at least two different HPV16 and HPV18 preparations. Bars represent standard deviations. An asterisk denotes significance by Student's t test. Statistical significance was defined as a P value of ≤0.05.

FIG 3

HPV18 infectivity is recovered by infection with additional virions but not with prebinding. (A) HaCaT cells were infected with HPV16 only (MOI = 10), HPV18 only (MOI = 10 to 1,000), or HPV16 (MOI = 10) and HPV18 (MOI = 10 to 1,000) together, followed by harvesting of mRNA and measurement of infectivity via qRT-PCR amplifying the E1∧E4 splice transcript. Analysis was performed on both HPV18 samples (left) and HPV16 samples (right). Values for individual HPV18 infections at each MOI were set equal to 1, and the relative levels of the HPV18 transcript detected in coinfections are shown. The levels of HPV16 during coinfections are all measured relative to the level of E1∧E4 detected during a single HPV16 infection. (B) HPV18 (MOI = 10) was bound to HaCaT cells at 4°C, followed 1.5 h later by the addition of HPV16 (MOI = 10) at 4°C. RNA was harvested, and infectivity was measured via qRT-PCR amplifying the E1∧E4 splice transcript. (C) HaCaT cells were seeded as described above. The cells were then infected at time zero with either HPV16 or HPV18. At 24 h, uninfected cells were infected with either HPV16 or HPV18 and a subset of the infected cells was coinfected with the other HPV type. RNA was harvested, and the level of infection was measured via qRT-PCR as described above. (D and E) Cells that stably maintain the HPV16 (HPV16 WT:3)or HPV18 (HPV18c) genome were seeded and then infected with increasing MOIs of HPV18 and HPV16, respectively. RNA was then harvested, and the levels of E1∧E4 present for both HPV types were assessed via qRT-PCR as described above. (F and G) Primary cells and stable cell lines harboring either HPV18 or HPV16 were infected with HPV16 or HPV18, respectively, at a MOI of 10. RNA was harvested, and infections were analyzed by determining E1∧E4 splice transcript levels via qRT-PCR. The level of infection in primary cells was set equal to 1, and the level of infection in stable cell lines persistently maintaining a HPV genome was measured relative to primary cell infections. All data are representative of results from at least three individual experiments utilizing at least two different HPV16 and HPV18 preparations. Bars represent standard deviations. An asterisk denotes significance by Student's t test. Statistical significance was defined as a P value of ≤0.05.

FIG 4

HPV16 does not block HPV18 during transcription. HaCaT cells were transfected with HPV16 only, HPV18 only, or both HPV16 and HPV18 genomic DNAs. At the indicated time points posttransfection, mRNA was harvested, and qRT-PCR was utilized to detect the amount of the E1∧E4 splice transcript as a measure of active transcription. The level of transcription measured during cotransfections at each time point is shown relative to the level of transcription during transfection with a single HPV type. Transfections were all done in triplicate. Bars represent standard deviations. An asterisk denotes significance by Student's t test. Statistical significance was defined as a P value of ≤0.05.

FIG 5

HPV16 blocks attachment of HPV18. (A and B) HaCaT cells (A) or primary keratinocytes (B) were incubated with HPV16, HPV18, or both types for 2 h at 4°C. Cells were then washed to remove any unbound particles, and total DNA was harvested. The number of virus particles bound to cells was quantified via qPCR against a standard curve of known concentrations of HPV genomes. (C) HaCaT cells were incubated with HPV16, HPV18, or both types for 2 h at 4°C. Cells were then washed, fixed, and stained for HPV16 and/or HPV18 L1 to detect virions bound to the cell surface. The inset in the merged image is representative of a magnified portion of the merged image (indicated by a small white box within the image). (D) Quantitation of HPV16 and HPV18 attachment to cells. Panels A and B are representative of data from at least three individual experiments, and panels C and D are representative of data from two individual experiments, all utilizing at least two different HPV16 and HPV18 preparations. Bars represent standard deviations. An asterisk denotes significance by Student's t test. Statistical significance was defined as a P value of ≤0.05.

FIG 6

HPV16 and HPV18 have differential binding abilities and entry times. (A) Attachment to the cell surface was measured by the addition of 1 × 10⁵ HaCaT cells to a suspension, followed by the addition of either HPV16 or HPV18 (MOI = 25). Attachment to the ECM was measured by removing cells from slides with EDTA, leaving only the ECM. Either HPV16 or HPV18 (MOI = 25) was then added and allowed to bind for 2 h, followed by washes to remove any unbound particles. After 2 h, bound virus was quantified by qPCR against a standard curve of known concentrations of HPV genomes. (B) HaCaT cells were seeded and allowed to grow for ∼2 days. Cells were then left untreated or treated with 10 mM EDTA to remove the cells. Cells were then stained with an antibody against laminin 5 (red) to identify the ECM and with Hoechst dye (blue) to identify the nucleus. (C) Either HPV16 or HPV18 was added to HaCaT cells for 2 h to allow binding, followed by washing of unbound particles. Cells were then stained with anti-L1 antibodies specific for HPV16 L1 (H16.V5) or HPV18 L1 (H18.J4) (green) and either CD147 or LN-5 (red). Nuclei were identified via Hoechst staining (blue). The inset in the merged image is representative of a magnified portion of the merged image (indicated by a small white box within the image). (D) The localization of attachment was quantified by using NIS-Elements Analysis software to analyze the signal overlap between stained virions and a localization marker (CD147 or LN-5). (E) Either HPV16 or HPV18 (MOI = 10) was added to cells for 2 h at 4°C. Unbound virus was then washed, and neutralizing antibody was added at the indicated time points postinfection. Samples were harvested after 48 h, and qRT-PCR to analyze the E1∧E4 splice transcript was done to measure infectivity. Panels A and E are representative of data from three individual experiments utilizing at least two different HPV16 and HPV18 preparations. Bars represent the standard deviations. An asterisk denotes significance by Student's t test. ns, nonsignificant. Statistical significance was defined as a P value of ≤0.05. Panel B is representative of data from two individual experiments. Panels C and D are representative of data from two individual experiments with at least two different HPV16 and HPV18 preparations. Images and quantifications are representative of results for at least 15 fields of view for each individual experiment, and standard deviations are shown for quantifications.

FIG 7

HPV16 L2 is involved in superinfection exclusion of HPV18 during a coinfection. HaCaT cells were infected with the chimeric virus HPV18-L2(16)L1(Δ18) only (A), the chimeric virus HPV18-L2(18/16)L1(Δ18) only (B), or both chimeric HPV18 and chimeric HPV16. At 48 hpi, mRNA was harvested, and infectivity was determined via qRT-PCR amplification of the E1∧E4 splice transcript. The levels of infection are relative, with values for single infections being set equal to 1. Data are representative of results from at least three individual experiments utilizing at least two different HPV16 and HPV18 preparations. Bars represent standard deviations. Statistical significance was defined as a P value of ≤0.05.