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, 87 (23), 12583-91

Seminal Plasma and Semen Amyloids Enhance Cytomegalovirus Infection in Cell Culture

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Seminal Plasma and Semen Amyloids Enhance Cytomegalovirus Infection in Cell Culture

Qiyi Tang et al. J Virol.

Abstract

Among the modes of transmission available to the cytomegalovirus (CMV) is sexual transmission, primarily via semen. Both male-to-female (M-F) and male-to-male (M-M) sexual transmission significantly contribute toward the spread of CMV infections in the global population. Semen plays an important role in carrying the viral particle that invades the vaginal or rectal mucosa, thereby initiating viral replication. Both semen and seminal plasma (SP) can enhance HIV-1 infection in cell culture, and two amyloid fibrils, semen-derived enhancer of viral infection (SEVI) and amyloids derived from the semenogelins (SEM amyloids), have been identified as seminal factors sufficient to enhance HIV-1 infection (J. Munch et al., Cell 131:1059-1071, 2007; N. R. Roan et al., Cell Host Microbe 10:541-550, 2011; F. Arnold et al., J. Virol. 86:1244-1249, 2012). Whether SP, SEVI, or SEM amyloids can enhance other viral infections has not been extensively examined. In this study, we found that SP, SEVI, and SEM amyloids strongly enhance both human CMV (HCMV) and murine CMV infection in cell culture. SEVI and SEM amyloids increased infection rates by >10-fold, as determined by both flow cytometry and fluorescence microscopy. Viral replication was increased by 50- to 100-fold. Moreover, viral growth curve assays showed that SP, SEVI, and SEM amyloids sped up the kinetics of CMV replication such that the virus reached its replicative peak more quickly. Finally, we discovered that SEM amyloids and SEVI counteracted the effect of anti-gH in protecting against CMV infection. Collectively, the data suggest that semen enhances CMV infection through interactions between semen amyloid fibrils and viral particles, and these interactions may prevent HCMV from being neutralized by anti-gH antibody.

Figures

Fig 1
Fig 1
Flow cytometry to detect viral infection rate. (A) MCMV infection in NIH 3T3 cells. NIH 3T3 cells were cultured in a six-well plate. When the cells were 80% confluent, they were mock infected or infected with MCMVE5gfp (12) at an MOI of 0.1. Virus was preincubated with 0, 2, 5, or 10 μg of SEM amyloids or SEVI/ml for 1 h at 37°C before infection. The cells were fixed at 16 h postinfection and measured by flow cytometry to detect the percentage of GFP-positive (infected) cells. The percentage of infected cells is shown in the lower right-hand corner of each plot. (B) HCMV infection in MRC-5 cells. MRC-5 cells were mock infected or infected with HCMVgfpSVH at an MOI of 0.1 for 16 h. Prior to infection, the virus was incubated with 0, 2, 5, or 10 μg of SEM amyloids or SEVI/ml at 37°C for 1 h. Infection rates were detected by flow cytometry, as described in panel A, and the percentage of infected cells is shown in the lower right-hand corner of each plot. Mock-infected cells in the absence or presence of 10 μg of SEM/ml or 10 μg of SEVI/ml were used to control for autofluorescence. These experiments were performed in duplicate, with one set of the results reported in the figure. The results are representative of one of three independent experiments.
Fig 2
Fig 2
Visualization of HCMV infection in U-251 MG cells. (A to C) The epithelial cell-like cell line U-251 MG was infected with HCMVgfpSVH at an MOI of 0.5 for 16 h in the absence of amyloids (A1 to A3) or after pretreatment with either 10 μg of SEM amyloids/ml (B1 to B3) or 10 μg of SEVI/ml (C1 to C3). The cells were fixed with 1% paraformaldehyde and stained with DAPI. The slides were observed under a fluorescence microscope (×10 amplification lens), and pictures were taken to show infected cells (GFP, A1 to C1) and total cells (DAPI, A2 to C2). The merged pictures are shown in A3 to C3. The percentages of GFP-positive (infected) cells are shown in the upper right-hand corners of A1 to C1. Scale bars, 20 μm. (D) Imaging experiments were performed three independent times. We counted a total of 1,000 cells for each experiment and averaged the percentages of GFP-positive cells. The averages and standard errors are shown in this panel. Statistically significant differences are indicated at the top (P < 0.005).
Fig 3
Fig 3
Western blot assay to detect viral protein production. (Left) NIH 3T3 cells were mock infected or infected with either MCMVE5gfp alone (MOI = 0.1) or MCMVE5gfp treated with SP, SEM amyloids, or SEVI for 24 h. Whole-cell lysates were prepared for Western blot with antibodies against GFP (IE3-GFP), IE1, M112-113, and tubulin (as loading control). (Right) MRC-5 cells were mock infected or infected either with HCMV alone or HCMV treated with SP, SEM amyloids, or SEVI; all infections were carried out for 24 h at an MOI of 0.1. Whole-cell lysates were prepared for Western blot with antibodies against IE1, IE2, UL112-113, and tubulin. By comparing the density of the IE3 bands of MCMV or the IE1 bands of HCMV with that of the virus alone (with all intensities normalized to the tubulin control), the fold increase of IE3 levels for MCMV (left) and that of IE1 for HCMV (right) were calculated (Quantity One 4.5.0 software; Bio-Rad Laboratories, Richmond, CA). Normalized IE3 (MCMV, left) or IE1 (HCMV, right) levels are shown below the corresponding Western blots.
Fig 4
Fig 4
Viral entry assay. (A) MCMV and HCMV infection rate. HCMV or MCMV was incubated with 10 μg of SEVI or SEM amyloids/ml or MEM (as a buffer control) at 37°C for 1 h. Pretreated virions were used to infect MRC-5 or NIH 3T3 cells (MOI = 0.1). Six hours later, the cells were fixed with 1% paraformaldehyde and immunostained for pp65 (HCMV tegument protein, right) or m25 (tegument protein of MCMV, left). The total cells and pp65-positive (or m25-positive) cells were counted by flow cytometry. Mock-infected cells treated with SEVI and SEM amyloids were used to control for autofluorescence. Based on statistical analysis, the average values (± standard deviations) of duplicate measurements from three independent experiments that yielded similar results were determined. (B) MRC-5 cells or NIH 3T3 cells were infected with HCMV or MCMV for 2 h and washed three times with MEM. The cells were then treated with MEM, with SEVI, or with SEM amyloids. After 24 h, whole-cell lysates were collected for Western blot analysis to assess for IE1 and IE2 (for HCMV, right) or IE1 and IE3 (for MCMV, left) expression.
Fig 5
Fig 5
Viral growth curve assay. (Upper panel) HCMV infection of MRC-5 cells. MRC-5 cells were infected with virus alone or with virus treated with SP (1:1,000 dilution), SEM amyloids (5 μg/ml), SEVI (5 μg/ml), or SEM amyloids (5 μg/ml) plus SEVI (5 μg/ml) at an MOI of 0.1 for 24 h (as indicated). Each day for a period of 9 days, one well of each culture was collected (along with medium) and stored at −80°C. The collected cells (with medium) were freeze-thawed for three cycles and then centrifuged at 8,000 × g for 20 min to remove the cellular debris. To perform a PFU assay, a 25-μl supernatant of each sample was used to infect the MRC-5 cells in triplicates. (Lower panel) MCMV infection in NIH 3T3 cells. The procedure is the same as that used for HCMV infection of MRC-5 cells, except that the infection period was 6 days. A Student t test was used to statistically analyze the difference between the groups versus virus alone (*, P < 0.001) and that of the groups of SEM+SEVI versus SEM or SEVI (**, P < 0.005).
Fig 6
Fig 6
Semen amyloids bind CMV. (Left panel) A total of 1 ml of HCMV (107/ml) was incubated with SEVI or SEM amyloid fibrils at 50 μg/ml for 1 h at 37°C. Controls include virus alone and virus incubated with 50 μg of Aβ(1-42) amyloids/ml. The mixtures were centrifuged, and the pellet was washed twice with MEM. The pellet was lysed in 2× Laemmli buffer and blotted with anti-IE1 (nonstructural protein), anti-gH (envelope protein), anti-gB (envelope protein), and anti-pp71 (tegument protein) antibodies. Whole-cell lysates (WCL) of HCMV-infected NIH 3T3 cells were used as input controls for the Western blot. (Right panel) Similar conditions, but using MCMV instead of HCMV. The tegument protein detected for MCMV was m25.
Fig 7
Fig 7
Semen amyloids overcome the antiviral effects of anti-CMV neutralizing antibodies. Five groups were set up as follows: MCMV or HCMV was (i) mock treated, (ii) treated with anti-gH antibody, (iii) treated with anti-gH antibody and then SEVI or SEM amyloids, (iv) treated with SEVI or SEM amyloids and then anti-gH antibody, or (v) treated with SEVI or SEM amyloids. Based on the statistical analysis, the average values (± standard deviations) of duplicate measurements from three independent experiments that yielded similar results were determined. Values were averaged from three independent experiments performed at different times. A Student t test was used to statistically analyze the difference between the groups. *, P < 0.005; **, P = 0.024; ***, P = 0.016.

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