A Polyamide Inhibits Replication of Vesicular Stomatitis Virus by Targeting RNA in the Nucleocapsid

Abstract

Polyamides have been shown to bind double-stranded DNA by complementing the curvature of the minor groove and forming various hydrogen bonds with DNA. Several polyamide molecules have been found to have potent antiviral activities against papillomavirus, a double-stranded DNA virus. By analogy, we reason that polyamides may also interact with the structured RNA bound in the nucleocapsid of a negative-strand RNA virus. Vesicular stomatitis virus (VSV) was selected as a prototype virus to test this possibility since its genomic RNA encapsidated in the nucleocapsid forms a structure resembling one strand of an A-form RNA duplex. One polyamide molecule, UMSL1011, was found to inhibit infection of VSV. To confirm that the polyamide targeted the nucleocapsid, a nucleocapsid-like particle (NLP) was incubated with UMSL1011. The encapsidated RNA in the polyamide-treated NLP was protected from thermo-release and digestion by RNase A. UMSL1011 also inhibits viral RNA synthesis in the intracellular activity assay for the viral RNA-dependent RNA polymerase. The crystal structure revealed that UMSL1011 binds the structured RNA in the nucleocapsid. The conclusion of our studies is that the RNA in the nucleocapsid is a viable antiviral target of polyamides. Since the RNA structure in the nucleocapsid is similar in all negative-strand RNA viruses, polyamides may be optimized to target the specific RNA genome of a negative-strand RNA virus, such as respiratory syncytial virus and Ebola virus. Negative-strand RNA viruses (NSVs) include several life-threatening pathogens, such as rabies virus, respiratory syncytial virus, and Ebola virus. There are no effective antiviral drugs against these viruses. Polyamides offer an exceptional opportunity because they may be optimized to target each NSV. Our studies on vesicular stomatitis virus, an NSV, demonstrated that a polyamide molecule could specifically target the viral RNA in the nucleocapsid and inhibit viral growth. The target specificity of the polyamide molecule was proved by its inhibition of thermo-release and RNA nuclease digestion of the RNA bound in a model nucleocapsid, and a crystal structure of the polyamide inside the nucleocapsid. This encouraging observation provided the proof-of-concept rationale for designing polyamides as antiviral drugs against NSVs.

Constraints of Viral RNA Synthesis on Codon Usage of Negative-Strand RNA Virus

“…Both of these experiments depict a large stabilization of the NLP by poly(rA). This is consistent with data from a previous study in which a polyamide (UMSL1011) that stabilizes the RNA in NLPs delayed RNA thermo-release by 2.04°C (24). These experimental results confirmed that nucleocapsid stability is dependent on the purine content of sequestered RNA, which may change the accessibility of the genomic RNA in the nucleocapsid to vRdRp to modulate its transcription/replication activities.…”

“…The results showed that the incorporation of poly(rA) in the NLP increased the stability by 2.33°C compared to the recombinantly purified NLP with a random RNA sequence (20) and by 2.96°C compared to the NLP with poly(rU). Furthermore, incorporation of poly(rA) in the NLP delayed the thermorelease of RNA by 7.86°C compared to the recombinantly purified NLP with a random RNA sequence and by 8.81°C compared to the NLP with poly(rU) (24). Both of these experiments depict a large stabilization of the NLP by poly(rA).…”

“…To verify that the stability of the nucleocapsid is related to the purine/pyrimidine content, thermal shift assays (TSAs) were carried out with recombinantly purified NLPs (20,24,25). A selected piece of RNA could be reconstituted in the NLP by removal of the original RNA with RNase A and subsequent incubation with the selected RNA (20).…”

“…NLPs containing RNA from recombinant purification, poly(rA), or poly(rU) were then subjected to TSA as previously described (24). Data availability.…”

Nucleocapsid Structure of Negative Strand RNA Virus

“…By thermal shift assays of nucleocapsid stability, it was shown that a nucleocapsid-like particle (NLP) containing poly(rA) is more stable than that containing random RNA sequences [67]. This is consistent with the observation that a compound stabilizing the nucleocapsid also inhibits viral RNA synthesis by VSV polymerase [68]. On the other hand, an NLP containing poly(rU) is less stable than that containing random RNA sequences, suggesting polymerase stuttering may be related to instability of the nucleocapsid.…”