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关键词： AIDS-related lymphomas   clonogenic p17 variants   HIV-1 matrix protein p17   HIV-1 mutants   viral quasi-species   virus evolution  

摘要： Since the introduction of combined antiretroviral therapy, acquired immune deficiency syndrome (AIDS)-related lymphomas account for a growing proportion of deaths among people living with human immunodeficiency virus (PLWHIV). In addition to the immune deficiency caused by AIDS and other cofactors, it has been shown that circulating HIV-1 proteins play a critical role in lymphoma development. The HIV-1 matrix protein p17 (refp17) is released from infected cells and accumulates in lymph nodes of PLWHIV, even during effective pharmacological control of viral replication. Circulating refp17 deregulates the biological activity of different immune cells. Moreover, p17 variants (vp17s) characterized by peculiar amino acid insertions occurring in the C-terminal region of the protein, differently from the refp17, also induce B-cell growth and clonogenicity. Notably, vp17s were found at a significantly higher prevalence in PLWHIV with than without lymphoma. HIV-1 mutants expressing clonogenic vp17s are actively spreading, and their prevalence is globally increasing worldwide. RNA viruses exist as a population of quasi-species, transmitted from one host to another, which ultimately leads to viral evolution by generating new master sequences. Here, we developed a next-generation sequence approach to evaluate the frequency of vp17 quasi-species in PLWHIV upon time and demonstrated that the incidence of vp17s also increases at quasi-species levels. Additionally, we established a regression model capable of predicting the insertions with higher probability to be fixed, further highlighting the evolutionary relevance of the C-terminal region in the adaptation of p17 to the human host.

关键词： RNA   SARS-CoV-2   antivirals   frameshifting   pseudoknot   small molecules  

摘要： Remaining SARS-CoV-2 vulnerable populations and the threat of viral resistance to existing immunity and COVID-19 vaccines highlight the need to expand treatment options. While the number of SARS-CoV-2 viral proteins is limited for therapeutic targeting, the RNA genome offers a wide range of conserved functional structures. Previous studies suggest that modulating the RNA-regulated programmed −1 ribosomal frameshift (−1 PRF) step in the coronavirus life cycle can inhibit viral replication. In this work, we aimed to identify small molecules that target the highly conserved SARS-CoV-2 frameshift site pseudoknot. An RNA-biased synthetic small molecule library screen against the RNA pseudoknot structure identified several candidate molecules. Of these, seven showed evidence of dose-dependent activity in in vitro and in cellulo frameshift assays and inhibited SARS-CoV-2 replication in cell culture. While binding was required for activity, affinity did not correlate with frameshift inhibition. Similarly, small molecule impacts to pseudoknot stability, as measured with an RT-qPCR assay, did not predict impacts on the frameshift efficiency. Comparison of the hits, as well as previously published inhibitors, across several assays supports the need for multiple characterization methods in small molecule–RNA targeting and the possibility of multiple modes of action for −1 PRF modulation. This work underscores the complexity of targeting dynamic RNA structures and the benefits of screening early for function and later optimizing for mode of action. The use of orthogonal techniques led to the identification of several promising hits and a lead antiviral compound.