Our studies confirmed previous reports that SEVI alone was capable of enhancing HIV-1 infection 
, and additionally revealed that the native PAP286
peptides and truncated forms (PAP266
) were also proviral. This may suggest additional situations in vivo
in which PAP derived peptides could exert activity in the absence of fully elongated amyloid fibrils. However, we also revealed that proteolytic mechanisms within SP could reduce the proviral effects of SEVI and PAP peptides under certain conditions. Moreover, differences in treatment of SP and semen might also affect concentrations of the antiviral cationic peptide components that we have reported 
. In vivo
, both pro- and antiviral situations could easily be explained by heretofore unknown donor-to-donor differences in PAP peptide concentration, protease concentration, and other factors that might affect the pro- and antiviral activity of SP directly or indirectly.
It is interesting to note that the ability of PAP-derived peptides to form amyloid fibrils is a common characteristic for many peptides and proteins given the correct conditions and time 
. Fibril formation follows a model nucleation-dependent elongation mechanism, initiated by a lag phase for nucleus seeding 
. When tested at a concentration 57-fold higher than the 35 µg/ml physiological concentration, SEVI exhibited a lag phase of ~10 h 
. Since concentration of the purified peptide plays a significant role in fibril formation, the spontaneous formation of SEVI from purified PAP286
observed in previous studies may be a prime example of this, due to supraphysiological stock concentrations (i.e. 10 mg/ml) used for fibril formation 
. Without agitation, it was found that fibril formation at lower concentrations of PAP286
may not occur or would require an exponentially longer lag phase time 
. Considering the lack of intense agitation post-ejaculation in vivo
and the significantly lower physiological concentration of PAP286
, the lag phase of SEVI formation might afford ample time for intrinsic inhibitors of SEVI to act.
As we observed, native proteases were responsible for the degradation of whole PAP as well as PAP peptides in the presence of SP. It is important to note that several protease incubation studies we conducted contained a significant excess of PAP or PAP peptides compared to SP or the protease of interest, suggesting that catalytic amounts of proteases in SP are responsible for PAP degradation in vitro. In addition, our SP samples contained greater than a 100-fold excess of PSA than would be necessary to degrade PAP286. One conclusion might be that the physiological concentration of SP would be sufficient to degrade PAP and PAP peptides in vivo. Conversely, it is plausible that an unknown promoter or stabilizer of SEVI formation might exist, which induces the formation of fibrils more rapidly in vivo. Notably, mechanisms behind the in vivo formation of SEVI warrant additional investigation, given that in vivo-formed SEVI fibrils themselves have not yet been reported.
It must be noted that while PSA is the primary candidate for the majority of PAP degradation, we also revealed other SP proteases that could proteolyze PAP286
. Likewise, proteases in human vaginal fluid, or mucosal proteases activated by the low pH in vaginal fluid might also degrade PAP286
or SEVI in the post-coital environment 
. While it is highly suggestive from our studies that PAP and PAP peptide degradation can occur, the level to which this occurs in vivo
may vary widely and be one of several reasons why the proviral effect has been reported to vary between individuals 
In our study, we have assessed the pro- and antiviral activity of PAP peptides and SEVI, under multiple conditions, many of which reproduced methods and techniques utilized by other groups 
. In short, the various testing conditions all had minor effects on the pro- and antiviral activities, yet the major finding that SP can abrogate part or all of the in vitro
proviral activity of SEVI was still substantiated. Syringe-filtered SP could confer HIV-1 enhancing activity under the right conditions, perhaps due to the loss of cationic peptides and proteins as a result of the filtration process. Both the concentration and duration of treatment influenced the overall activity of SP. Variable cell density led to differences in pro- and antiviral activity of SP and semen, as well as differences in cytotoxicity. While methodological nuances may help explain the disparity in data given between research groups, it is unclear which more closely represents the in vivo
environment of HIV-1 infection.
Previous studies have demonstrated the broad spectrum antimicrobial and antiviral activity of human seminal plasma 
. The heterosexual transmission of HIV is not efficient, occurring as infrequently as 1 in every 1000 coital acts 
, which might be rationalized in part by the observed antiviral activity of human SP and the cationic antimicrobial and antiviral peptides therein. Still, there are factors in SP that exhibit proviral activity in vitro
. PAP peptides have been confirmed in semen, and the extent of their HIV-1 enhancing activity varies on an individual donor basis 
. Our current studies suggest that the formation and activity of SEVI in vivo
might be challenging in the presence of SP, due to the natural degradation of PAP peptides by several intrinsic proteases within SP. While to date there is a lack of evidence confirming the ability of SEVI amyloid fibrils to form naturally in non-manipulated human SP under physiological conditions in vivo
, it still remains possible that PAP peptides can generate fibrils in vivo
and exhibit HIV-1 enhancing activity under the right circumstances. Together, we anticipate that our findings will not only spark intense discussion, but will unlock avenues for continued research on the proviral and antiviral aspects of human SP.