Despite the advent of effective anti-Human Papillomavirus (HPV) vaccines, there are no biological agents to reliably prevent ~80 million Americans from transmitting their infectious HPV viral particles to sexual partners. Earlier we determined that the post-translational homocysteinylation of an mRNA-binding protein (heterogenous nuclear ribonucleoprotein-E1, hnRNP-E1) can transform hnRNP-E1 into a moiety with high affinity for a HPV16 57-nucleotide (nt) RNA cis-element under conditions of folate deficiency; this interaction led to a profound inhibition of both HPV16 L1 and L2 viral capsid proteins that are essential for HPV16- encapsidation (and infectivity). We have patented a powerful mutant of hnRNP-E1 [DomPos-E1(C293S)] that functions like homocysteinylated-hnRNP-E1 under folate-replete conditions. Because DomPos-E1(C293S)] has such a strong likelihood to eliminate HPV16 viral capsid proteins and thereby function as an anti-HPV agent, we wish to test its therapeutic potential both in vitro and in our novel HPV16-xenograft model in Beige Nude mice.
In Specific Aim 1 we will compare effects of the interaction of DomPos-E1(C293S)-protein [relative to control wild-type(wt)-like-E1(G292A)-proteins] with HPV16 57-nt cis-element in eliminating HPV16 L1 and L2 viral capsid protein expression. We will also extend these studies to assess the interaction of DomPos-E1(C293S) with similar cis-elements from low risk HPV6 and 11 and high-risk types (HPV18, 31, 33, 45, 52, 58). Next, we will confirm the greater impact of DomPos-E1(C293S)- over control wt-like-E1(G292A)- expression in reducing HPV16 L1 and L2 after stable transduction into HPV16-harboring keratinocytes that are also transformed into HPV16-organotypic rafts; then we can evaluate the extent in reduction of infectious HPV16 viral particles in 18- day old rafts and whether there is any increase in genomic integration by amplified capsid-less HPV16 episomes.
In Specific Aim 2, we will subcutaneously implant these DomPos-E1(C293S)- or control wt-like- E1(G292A))- expressing rafts in Beige Nude mice using our recently published model. This will allow us to assess the relative effects of DomPos-E1(C293S)- over control wt-like-E1(G292A) in reducing both HPV16 viral capsid proteins and infectious viral particles of HPV16 over the ensuing 8 weeks in vivo; evaluating if this reduces the capacity of the implanted HPV16-raft to auto-infect itself; determining changes in genomic integration by amplified capsid-less HPV16 episomes; and in prolonging the expected time of rafts to develop HPV16-cancers.
In Specific Aim 3, we will determine if DomPos-E1(C293S) is significantly more effective than control wt- like-E1(G292A) in preventing transmission of HPV16 to adjacent tissue. We will adapt our in vivo model to assess HPV16-infectivity wherein the effectiveness of transmission of infectious HPV16 from one donor tissue to an uninfected recipient tissue is assessed over 8 weeks in Beige Nude mice. These studies will help determine if DomPos-E1(C293S) or its mutant derivatives can be moved forward as first-in-class agents to help reduce transmission of infectious HPV16 viral particles from an infected host.
Young US Veterans are at risk for developing genitourinary and oropharyngeal warts and cancers which develop after sexual transmission of infectious HPV virus particles. Once such infections are established there are no effective agents to prevent transmission of the virus from such HPV-infected subjects to other uninfected individuals. We have recently patented a protein that has a high potential to prevent the transmission of HPV from one individual to another. This protein completely prevents HPV from developing into an intact infectious virus and has potential to be a first-in-class anti-HPV agent for use shortly after an HPV infection is established. So, we wish to test its therapeutic potential both in vitro and in a novel HPV-cancer model we recently developed in Beige Nude mice; the novelty of our mouse model is that we can compress the time to development of cancer from 10-20 years to just 8-12 weeks. The experiments proposed will therefore test the likelihood that this VA- sponsored patent could be advanced as a new agent to control transmission of HPV among our Veterans.
