All four isomeric series of novel 4-substituted pyrido-fused 7-deazapurine ribonucleosides possessing the pyridine nitrogen atom at different positions were designed and synthesized. The total synthesis of each isomeric fused heterocycle through multistep heterocyclizationw as followed by glycosylation and derivatization at position 4b y cross-coupling reactions or nucleophilic substitutions. All compoundsw ere tested for cytostatic and antiviral activity. The most active werep yrido[4',3':4,5]pyrimidine nucleosides bearing MeO, NH 2 ,M eS, or CH 3 groups at position 4, which showed submicromolar cytotoxic effects and good selectivity for cancer cells. The mechanism involved activation by phosphorylation and incorporation to DNA where the presence of the modified ribonucleosides causes double-strand breaksa nd apoptosis.

The mechanism of action of various viruses has been the primary focus of many studies. Yet, the data on RNA modifications in any type of virus are scarce. Methods for the sensitive analysis of RNA modifications have been developed only recently and they have not been applied to viruses. In particular, the RNA composition of HIV-1 virions has never been determined with sufficiently exact methods. Here, we reveal that the RNA of HIV-1 virions contains surprisingly high amount of the 1-methyladenosine. We are the first to use a liquid chromatography-mass spectrometry analysis (LC/MS) of virion RNA, which we combined with m 1 A profiling and deep sequencing. We found that m 1 A was present in the tRNA, but not in the genomic HIV-1 RNA and the abundant 7SL RNA. We were able to calculate that an HIV-1 virion contains per 2 copies of genomic RNA and 14 copies of 7SL RNA also 770 copies of tRNA, which is approximately 10 times more than thus far expected. These new insights into the composition of the HIV-1 virion can help in future studies to identify the role of nonprimer tRNAs in retroviruses. Moreover, we present a promising new tool for studying the compositions of virions.

NAD is a key component of cellular metabolism and also serves as an alternative 5'cap on short noncoding RNAs. The function of NAD in RNA, however, remains poorly understood. We investigated NAD capping of RNAs in HIV-1 infected cells, as HIV-1 is responsible for the depletion of the NAD/NADH cellular pool and causes intracellular pellagra. We used NAD captureSeq on HIV-1 infected/noninfected cells and revealed that four snRNAs (U1, U4ATAC, U5E, and U7) and four snoRNAs (SNORD3G, SNORD102, SNORA50A, and SNORD3B) lost their NAD cap when infected with HIV-1. Here, we provide evidence that the loss of the NAD cap increases the stability of the U1-HIV-1 pre-mRNA duplex. We also show that decreasing the amount of NAD-capped U1 snRNA by overexpressing the NAD RNA decapping enzyme DXO leads to a marked increase in HIV-1 infectivity. Moreover, an experimental increase of NAD capped RNAs improves the efficiency of splicing of HIV-1 and cellular RNAs and lowers HIV-1 infectivity. Our experiments show a dual role of U1 snRNA in HIV-1 infection and present the first clear role of NAD-capped RNAs in eukaryotic antiviral responses with a potential overlap into cellular splicing.

NAD is a key component of cellular metabolism and also serves as an alternative 5’cap on short noncoding RNAs. The function of NAD in RNA, however, remains poorly understood. We investigated NAD capping of RNAs in HIV-1 infected cells, as HIV-1 is responsible for the depletion of the NAD/NADH cellular pool and causes intracellular pellagra. We used NAD captureSeq on HIV-1 infected/noninfected cells and revealed that four snRNAs (U1, U4ATAC, U5E, and U7) and four snoRNAs (SNORD3G, SNORD102, SNORA50A, and SNORD3B) lost their NAD cap when infected with HIV-1. Here, we provide evidence that the loss of the NAD cap increases the stability of the U1-HIV-1 pre-mRNA duplex. We also show that decreasing the amount of NAD-capped U1 snRNA by overexpressing the NAD RNA decapping enzyme DXO leads to a marked increase in HIV-1 infectivity. Moreover, an experimental increase of NAD capped RNAs improves the efficiency of splicing of HIV-1 and cellular RNAs and lowers HIV-1 infectivity. Our experiments show a potential dual role of U1 snRNA in HIV-1 infection and present the first possible function of NAD-capped RNAs in eukaryotic antiviral responses.