A Euro-American team of researchers has used mass spectrometry and HPLC analysis to find a protein in human semen that they say dramatically increases the chances of HIV infection.
While their findings, published in the Dec. 14 edition of Cell, don’t promise an immediate cure or treatment for the virus, or a vaccine against it, they offer new insight into the mechanisms of HIV, in particular, the role of sperm on infection.
The researchers from Germany, Spain, and the US had originally set out to find proteins in semen that inhibit HIV infection. But instead they discovered that an enzyme found in the cells called prostatic acidic phosphatase breaks off into fragments called fibrils, which then combine into tiny clumps.
These clumps, which the researchers dubbed Semen-derived Enhancer of Virus Infection, capture HIV particles, and deliver them to target cells. The results “enhance the infectious virus titer by several orders of magnitude,” the researchers write in their study.
They cannot say why the protein affects HIV infection so dramatically, but first author Jan Münch, a junior professor at the Institute of Virology at the University of Ulm, Germany, told ProteoMonitor this week that one possibility is that the virus binds “very efficiently” to the fibrils and the fibrils bind equally efficiently to target cells.
“Amyloidogenic PAP fragments are abundant in seminal fluid and boost semen-mediated enhancement of HIV infection,” Münch and his colleagues say in the study. “Thus, they may play an important role in sexual transmission of HIV and could represent new targets for its prevention.”
In addition, their work may shed light on a conundrum of HIV research: why the virus is highly contagious through sexual contact but appears to be less so in a laboratory setting. The researchers said that this could be the case because semen, as a result of the presence of SEVI, potently enhances HIV infection.
Carl Dieffenbach, acting director of the Division of AIDS at the National Institute of Allergy and Infectious Diseases at the National Institutes of Health, said the study serves as a good building block for additional work, but cautioned proper perspective for the moment.
There are modes of transmission, such as maternal-to-infant breast feeding and blood-borne transmissions through needles, where the presence of SEVI wouldn’t be a factor, so the paper provides no clues to the mechanisms of infection in those instances, he said.
In the US, the number of AIDS cases due to heterosexual and homosexual contact totaled 681,601 from the onset of the epidemic in the mid-1980s to 2005, according to the Centers of Disease Control and Prevention. The figure also includes transmission from both sexual contact and injection-drug use.
The number of cases of transmission solely from injection-drug use totaled 241, 364 while cases resulting from other modes such as perinatal and blood transfusions totaled 20,560.
And even for those modes in which SEVI may play a role, “ultimately HIV still has to bind to and enter a cell through its normal receptors, and all this does is get it closer to the cell,” Dieffenbach said. “So stopping this process probably wouldn’t eliminate transmission, [though] it may reduce it.”
Still, he said the study “gives us a new perspective in terms of thinking about … this notion of other facilitators or accelerators in the context of other modes of transmission. … It will stimulate research, that’s for sure.”
Most of the research into the virus has been directed toward factors that may potentially block transmission. Pawel Ciborowski, director of the mass spectrometry and proteomics core facility and assistant professor of biochemistry and molecular biology at the University of Nebraska, said that investigating what can aggravate transmission is an equally important approach.
“Knowing what enhances infection can lead us to understanding [the] mechanism of disease and in consequence further studies of prevention and treatment,” he said. In his own studies, he is taking a proteomic approach to search for biomarkers for HIV-1 infection of the brain.
Stumbling Toward SEVI
When the researchers set out on their work, their intent was to look for an HIV-inhibiting molecule. Building on a study published by Münch and colleagues earlier this year, they analyzed a complex peptide/proteins library derived from pooled human serum, encompassing 294 fractions, that the researchers say should be enough to represent all peptides and proteins in seminal fluid.
But rather than finding inhibiting molecules, they stumbled across one fraction that they found to significantly enhance HIV-1 infection.
“Nobody has screened a peptide library direct from semen so far as we know, so we were the first [to do] this, and [the result] was unexpected,” Münch said.
The fraction contained only small amounts of proteins and peptides, so he and his colleagues did an additional round of purification and identified several peptides in the active fractions by mass spectrometry.
“So stopping this process probably wouldn’t eliminate transmission, [though] it may reduce it.”
Peptide sequencing identified them as fragments of PAP. They then synthesized PAP peptides to verify that they enhance HIV-1 infection. In subsequent work to determine whether SEVI interacts directly with HIV-1 particles and how SEVI promotes viral infection, the researchers concluded that “SEVI enhances HIV-1 infection by capturing virions and promoting their physical interaction and fusion with target cells but does not bypass the requirement for the appropriate coreceptor.”
They also discovered that SEVI enhancement was “most pronounced” when the levels of infectious virus were low, resembling the conditions of sexually transmitted HIV-1.
Among the enhancing power of SEVI observed by the researchers was a 24-fold increase in HIV-1 particles of T cells when the HIV-1 particles were bound to dendritic cells.
Prior work had shown that seminal fluid enhances the binding of virions to epithelial cells in ex vivo cervicovaginal tissue. Based on that, the researchers did further work and found that while the human epithelial carcinoma cell line was “nonpermissive” for HIV-1 infection in the presence of SEVI, SEVI increased the ability of HeLa cells to transmit HIV-1 to T-cells via R5 and X4 proteins by 30- to 70-fold.
“Thus, SEVI may promote virus attachment to genital surfaces, penetration of the mucosal barrier, and subsequent dissemination to lymphoid organs by increasing HIV-1 virion binding to epithelial cells and to migrating [dendritic cells],” the researchers say.
Most of the work was done in vitro, but in one in vivo experiment using transgenic rats with human receptors, they found that SEVI “significantly enhanced the infectivity of R5 HIV-1.”
In conclusion, while HIV-1 has only limited ability to cross the mucosal barrier and infect a sufficient number of cells in the genital tract for sustained infection, the Cell study found that SEVI promotes virion-cell attachment and fusion, which dramatically enhances the infectiousness of the virus.
Their findings, the authors of the study say, “suggest that amyloidogenic peptides in [semen] may help HIV to pass the early ‘bottleneck’ in infection by assisting the virus to attach to genital surfaces, to establish a self-propagating infection at the point of entry, and to cross the mucosal barrier with migrating dendritic cells.”
In ongoing research, the authors have found at least one additional HIV-1 enhancing protein, Münch said, though he declined to provide greater details. They are also performing in vivo studies on the SEVI mechanism in mice and rats “to measure transmission of HIV-1 via vaginal administration of SEVI-primed virus” and plan to eventually go into monkey models.