Sexually transmitted diseases (STDs) are caused by microorganisms including bacteria, viruses, and protozoa, that colonize the female and male genital tracts, often causing only mild symptoms [1
]. STDs affect health and cause social and economic problems worldwide [2
]. Despite the development of antibiotics and vaccines and the existence of disease prevention and control programs, these pathogens remain important causes of acute and chronic diseases [1
]. Most STD pathogens have been detected in semen from asymptomatic and symptomatic men [4
]. Viral STD pathogens include human papilloma virus (HPV), human immunodeficiency virus (HIV), herpes simples virus (HSV), hepatitis virus B (HBV), hepatitis virus C (HCV), and cytomegalovirus (CMV), all of which have been detected in semen [4
HSV infects a large population (over 500 million worldwide) and is associated with a variety of diseases, including genital herpes, most of which cases are caused mostly by HSV-2 and partly by HSV-1 [5
]; ocular herpes, induced by HSV-1 and a leading cause of blindness worldwide [5
]; and neonatal herpes, which last is usually an infectious consequence of either HSV-1 or HSV-2 via vertical transmission [5
]. One of the characteristics of HSV infection is its ability to latently infect neurons so that they can be reactivated, thereby causing recurrent infections [8
]. People who are infected with HSV can shed the virus in their body fluids; for men, those fluids include semen [3
]. Viral load in semen is directly related to male-to-female (M-F) transmission of HSV, as well as to the virus’s transmission among men who have sex with men (MSM) [9
]. HSV-1 and -2 infections can both be transmitted sexually, with semen being the major carrier of viral particles. As stated above, most cases of sexually transmitted herpes are caused by HSV-2, but in recent years, the number of cases caused by HSV-1 has risen [10
]. Although the clinical symptoms of HSV-caused diseases can be controlled with antiviral drugs (acyclovir and valacyclovir), these drugs are not strong enough to stop subclinical transmission [6
]. No prophylactic or therapeutic vaccine against HSV is available. Topical microbicides that prevent the sexual transmission of viruses could significantly reduce sexually-transmitted diseases. By better understanding the mechanisms employed by HSV in sexual transmission, researchers will be able to design more effective preventive and/or therapeutic drugs against the infection.
To initiate viral infection, the virus and the to-be-infected cell must come into direct contact. The attachment of the virus to the cell can be either specific or non-specific; the type of attachment determines the pathway of viral entry. Viral entry is a critical step of viral transmission. HSV enters the cell via either of two different pathways: the fusion of the viral envelope with the cellular membrane or endocytosis [12
]. HSV entry via the fusion of the viral envelope with the plasma membrane requires the specific interaction of the virus with cellular surface receptors, while endocytosis is unspecific [12
]. Both pathways require that the virus become attached to the cell surface. Therefore, any enhancement of the attachment of viral particles to cell surfaces will strengthen viral infection, as has been demonstrated in the case of semen, which can enhance HIV and CMV infection [13
Semen contains proteolytic cleavage products of prostatic acid phosphatase (PAP) and semenogelins (SEM), each of which can form amyloid fibrils in semen. The first amyloid fibril found to strongly enhance HIV infection is made up of peptide fragments from PAP and was named semen-derived enhancer of viral infection
]. Additional peptides—a second set—that form HIV-enhancing amyloid fibrils are derived from SEM and are referred to as SEM amyloids
]. Both SEVI and SEM have been demonstrated to enhance CMV infection in different cells [15
]. The mechanism that SEVI and SEM use to enhance viral infection might be that the intrinsic positive charges of SEVI and SEM facilitate virion attachment to and fusion with target cells because the anionic polymers decrease the enhancement of HIV infection mediated by semen, SEVI, and SEM [14
]. If the positive charge of the amyloids is the only thing responsible for the enhancement of viral infection, then SEM, SEVI, and SP (semen plasma) should enhance the infection of all viruses with envelopes. To test the presumption, we expanded our investigation to human herpes simplex viruses.
In the present study, we show that SEM and SEVI amyloids as well as SP can enhance both the HSV-1 and the HSV-2 infection of permissive cells and that treating HSV with these substances not only enhances viral particle formation but also increases viral protein production. The enhancement is accomplished by increasing the entry of viruses into the infected cells. These experimental results imply a novel target for preventing HSV infection and, when that has not been or cannot be accomplished, its subsequent spread.
All of the currently defined viral STD pathogens have been detected in semen [4
]. Therefore, semen may be an important carrier in terms of viral sexual transmission. Of the eight human herpesviruses, four have been defined as STD pathogens, and they are EBV, CMV, HSV-1, and HSV-2 [18
]. HSV is one of the most common viruses, occurring in all human populations [4
], and genital HSV includes two distinct but closely related viruses: HSV-1 and HSV-2. HSV-1 causes oral and, occasionally, genital sores, while HSV-2 is the most common cause of genital herpes, which can be asymptomatic at the time of primary, initial, or recurrent infection [19
Human semen contains cationic amyloid fibrils that are aggregated from a wide variety of soluble proteins and peptides [20
]. These amyloid fibrils share certain properties, including self-assembly by a nucleation-dependent process and a non-branching fibrillar cross-β structure with β strands perpendicular to the fibril axis [21
]. Pioneering studies have revealed that two amyloid fibrils, SEVI and SEM, are linked to viral infection. SEVI is derived from prostatic acidic phosphatase (PAP) and SEM from semenogelin. Both SEVI and SEM were found to strongly enhance CMV and HIV-1 infections [13
]. In this report, we expand our investigations to determine whether semen plasma and semen amyloids can enhance the infection and replication of HSVs.
The fluid on the surface of the mucosa of the genital track contains different kinds of materials that act against viral infection, especially against viral entry [23
]. A high viral load and/or viral absorption on the genital mucosa are important for the success of viral infection. As we expect, SP and the semen amyloids (SEVI and SEM) were found to be able to enhance HSV replication through enhancing viral entry. The semen amyloids can directly interact with viral particles, as can be observed using the virus-amyloid binding assay (Figure 1
), which might result in multiple viral particles aggregating on an amyloid to more easily colonize on the surface of genital mucosa. This speculation is confirmed by our experimental studies (shown in Figure 3
and Figure 4
), which showed that semen plasma and both amyloids enhanced the HSV entry. The enhancement of viral entry accordingly strengthened viral gene expression (Figure 2
) and viral replication (Figure 5
). The results we obtained from cell culture experiments support the theory that semen acts as a vector for HSV’s sexual transmission, not only carrying infectious viral particles but also providing an environment that is beneficial for HSV infection. However, our current studies are only at the initial stage. In the future, it is necessary to test the enhancing effects of semen on clinical strains of HSV in different cells (including vaginal epithelial cells).
Developing novel strategies against HSV infection is a global public health priority. Three ways exist to fight HSV infection: inhibiting viral replication by anti-HSV drugs, killing or inactivating infectious viral particles using microbicides, and preventing viral infection with vaccines. Prevention is better than a cure, as antiviral drugs do not destroy their target pathogen; instead they inhibit their development. A novel strategy for HSV prevention is the development of mucosally delivered microbicides. In the case of semen-mediated viral transmission, developing compounds to interfere with the amyloids’ abilities to enhance viral entry might be significant in preventing HSV infection in the general population. Several chemicals have been used in preclinical trials to antagonize viral transmission via semen. The anionic polymer PRO-2000 showed robust activity in preclinical studies [1
] but failed to show efficacy in an MDP 301 trial [24
], possibly because its antiviral activity is inhibited by seminal plasma. New drugs are needed. Our findings that semen components strongly enhance viral infection and that SEM and SEVI protect CMV from being neutralized by anti-gB led us to investigate the extent to which antibodies against SEM, SEVI, and viral surface antigens can inhibit the semen-mediated enhancement of CMV infection in cell culture and in organ culture. These novel microbicide agents (antibodies) are expected to be able to avoid the side effects and toxicities because the antibodies will specifically interact with molecules in seminal fluid. The concept is also applicable to other sexually transmissive viruses, such as HSV-1 and -2. After this phase of the mechanistic studies of the effects of semen, SEVI, and SEM on herpes viral infection, we expect to continue our research with pre-clinical and/or clinical studies exploring how to prevent the sexual transmission (specifically, intercourse) of HCMV and HSV.
4. Materials and Methods
4.1. Tissue Culture and Viruses
Vero (ATCC, CCL-81, USA) and HEK293T (ATCC, USA) cells were used for the infection of HSV-1 and HSV-2. Vero cells were used because they adhere well to coverslips for immunofluorescent assays. HEK 293T cells were used because they are easily detached from cell culture dishes and facilitate flow cytometry assays. All cells were maintained in Dulbecco's Modified Eagle’s medium (DMEM) supplemented with 10% fetal calf serum (FCS) and 1% penicillin-streptomycin (PS). Wild-type HSV-1 17 was obtained from R.D. Everett and used previously [25
]. HSV-2 strain G was purchased from ATCC (VR-734). For immunohistochemical staining, cells were grown on round coverslips (Corning Incorporated, Corning, NY, USA) in 24-well plates (Falcon; Becton Dickinson Labware, Lincoln Park, NJ, USA).
SEVI was synthesized by the genomic and the proteomics core laboratories at the University of Pittsburgh (PA, USA). SEM amyloids (amino acid 49–107) [14
] were synthesized by Celtek Peptides and provided by Dr. Roan (University of California at San Francisco, USA). Synthetic peptides were dissolved in PBS or serum-free MEM and agitated overnight at 1400 rpm at 37 °C (using an Eppendorf Thermomixer), as described previously [13
]. A-beta (1–42) amyloids (Sigma, cat. #A9810) were purchased from Sigma–Aldrich (St. Louis, MO, USA). Semen samples were obtained from 10 different individuals and mixed. The pooled semen samples were centrifuged at 700 g for 5 min, and the supernatant (seminal plasma) was stored at −80 °C and for later use in the experiments. The obtaining and use of semen samples and the informed consent forms were fully reviewed and approved by the committee on human research at Ponce Health Sciences University before the initiation of the study (IRB number 081106-YY). Viral polymerase inhibitor phosphonoacetic acid (PAA) was purchased from Sigma–Aldrich (St. Louis, MO, USA).
The antibodies used for Western blot (WB) and immunofluorescence are listed below. A monoclonal antibody against tubulin (T-9026) was purchased from Sigma–Aldrich (St. Louis, MO, USA; 1:1000 for WB); polyclonal antibody against ICP8, and monoclonal antibodies against HSV ICP4, ICP27, and gD were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA).
4.4. Purification of Viruses
Herpesviruses were amplified in Vero cells. The viral supernatant was centrifuged at 8000 g for 20 min to remove cell debris. The clarified medium was transferred into SW27/28 ultra-clear centrifuge tubes that were underlain with 7 mL of 20% sorbitol buffer (20% D-sorbitol, 50 mM Tris-HCl, pH 7.2, and 1 mM MgCl) and centrifuged at 55,000 g for 1 hour. The purified viral pellet was resuspended in PBS.
4.5. Amyloid Fibril-Virus Binding Assay
Amyloid fibrils are large structures and can be pelleted by low-speed centrifugation (e.g., 1000 rpm on a table centrifuge) [16
]. Purified viral particles (HSV-1 and -2) were incubated with 50 μg of SEVI- or SEM-derived amyloid fibrils at 37 °C for 1 h. Controls for the experiment included virus in the absence of amyloids and virus incubated with 50 μg/mL of A-beta (1–42) amyloids (Sigma, cat. #A9810). Samples were then centrifuged at 1000 rpm for 5 min on a table-top centrifuge (Eppendorf 5424). The pellets were washed twice with serum-free MEM and resuspended in phosphate-buffered saline (PBS). The resuspended pellets were mixed with the same volume of 2× Laemmli buffer. After a 5 min heat treatment at 95 °C, the proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and analyzed by Western blot, as described below.
4.6. Immunoblot Analysis
Proteins were separated by SDS-PAGE (10 to 20 µg loaded in each lane), transferred to nitrocellulose membranes (Amersham Inc., Piscataway, NJ, USA), and blocked with 5% nonfat milk for 60 min at room temperature. Membranes were incubated overnight at 4 °C with primary antibody, followed by incubation with a horseradish peroxidase-coupled secondary antibody and detection with enhanced chemiluminescence (Pierce, Rockford, IL, USA), according to standard methods. Membranes were stripped with stripping buffer (100 mM 2-mercaptoethanol, 2% SDS, 62.5 mM Tris-HCl, pH 6.8), washed with PBS-0.1% Tween-20, and used to detect additional proteins.
4.7. Cell visualization by Confocal Microscopy
To visualize HSV-infected cells, the cells were seeded on coverslips and, 12 hours after infection, washed twice with phosphate-buffered saline (PBS), fixed in 1% paraformaldehyde for 10 min at room temperature, permeabilized with 0.2% triton-X 100 in ice for 20 min, and washed 2 more times with PBS. Then the cells were incubated with anti-ICP4 antibody and Texas Red (TR)-conjugated secondary antibody and stained with DAPI. Cells were examined at 10× magnification with a Leica TCS SPII confocal laser scanning system equipped with a water-cooled argon-krypton laser. Two channels (DAPI and TR) were recorded sequentially. DAPI staining was used to show the total number of cells recorded.
4.8. Flow Cytometry
For quantitation of infection efficiency, cells were infected for 12 h with viruses, trypsinized, and fixed in 1% paraformaldehyde. The cells were then stained using anti-ICP4 antibody (described as above) and analyzed by a FACSCalibur system with 2 lasers and 4 channels (BD Biosciences, MA, USA) to detect total cell numbers and cells with TR fluorescence. Mock-infected cells with or without treatment with amyloids served as autofluorescence controls.
4.9. Plaque Formation Unit (pfu) Assay
Viral titers were determined by the plaque assay, essentially as described [27
], but with some slight modifications. Supernatants containing serially diluted viral particles (total volume was 1 mL for each well) were added to confluent Vero cell monolayers in 6-well plates. After adsorption for 2 h, the medium was removed and the cells were washed twice with serum-free DMEM and overlaid with phenol-free DMEM containing 5% FCS, 0.5% low-melting point agarose (GIBCO), and 1% penicillin-streptomycin. Then, the number of plaques that were stained red was counted and reported as pfu per mL. Mean pfu was determined after averaging the number of pfu from different dilutions. Student’s t
-test was used to statistically analyze differences between the groups; a p
-value lower than 0.005 was used as the threshold for a significant difference.