Antiviral RNAi response against the insect-specific Agua Salud alphavirus

Arboviruses transmitted by mosquitoes are responsible for the death of millions of people each year. In addition to arboviruses, many insect-specific viruses (ISVs) have been discovered in mosquitoes in the last decade. ISVs, in contrast to arboviruses transmitted by mosquitoes to vertebrates, cannot replicate in vertebrate cells even when they are evolutionarily closely related to arboviruses. The alphavirus genus includes many arboviruses, although only a few ISVs have been discovered from this genus so far. Here, we investigate the interactions of a recently isolated insect-specific alphavirus, Agua-Salud alphavirus (ASALV), with its mosquito host. RNAi is one of the essential antiviral responses against arboviruses, although there is little knowledge on the interactions of RNAi with ISVs. Through knock-down of transcripts of the different key RNAi pathway (siRNA, miRNA and piRNA) proteins, we show the antiviral role of Ago2 (siRNA), Ago1 (miRNA), and Piwi4 proteins against ASALV in Aedes aegypti derived cells. ASALV replication increased in Dicer2 and Ago2 knock-out cells, confirming the antiviral role of the siRNA pathway. In infected cells, mainly ASALV-specific siRNAs are produced while piRNAs, with the characteristic nucleotide bias resulting from ping-pong amplification, are only produced in Dicer2 knock-out cells. Taken together, ASALV interactions with the mosquito RNAi response differs from arthropod-borne alphaviruses in some aspects, although they also share some commonalities. Further research is needed to understand whether the identified differences can be generalised to other insect-specific alphaviruses.

The HIV 5′ Gag Region Displays a Specific Nucleotide Bias Regulating Viral Splicing and Infectivity

Alternative splicing and the expression of intron-containing mRNAs is one hallmark of HIV gene expression. To facilitate the otherwise hampered nuclear export of non-fully processed mRNAs, HIV encodes the Rev protein, which recognizes its intronic response element and fuels the HIV RNAs into the CRM-1-dependent nuclear protein export pathway. Both alternative splicing and Rev-dependency are regulated by the primary HIV RNA sequence. Here, we show that these processes are extremely sensitive to sequence alterations in the 5’coding region of the HIV genomic RNA. Increasing the GC content by insertion of either GFP or silent mutations activates a cryptic splice donor site in gag, entirely deregulates the viral splicing pattern, and lowers infectivity. Interestingly, an adaptation of the inserted GFP sequence toward an HIV-like nucleotide bias reversed these phenotypes completely. Of note, the adaptation yielded completely different primary sequences although encoding the same amino acids. Thus, the phenotypes solely depend on the nucleotide composition of the two GFP versions. This is a strong indication of an HIV-specific mRNP code in the 5′ gag region wherein the primary RNA sequence bias creates motifs for RNA-binding proteins and controls the fate of the HIV-RNA in terms of viral gene expression and infectivity.

DeepCPP: a deep neural network based on nucleotide bias information and minimum distribution similarity feature selection for RNA coding potential prediction

The development of deep sequencing technologies has led to the discovery of novel transcripts. Many in silico methods have been developed to assess the coding potential of these transcripts to further investigate their functions. Existing methods perform well on distinguishing majority long noncoding RNAs (lncRNAs) and coding RNAs (mRNAs) but poorly on RNAs with small open reading frames (sORFs). Here, we present DeepCPP (deep neural network for coding potential prediction), a deep learning method for RNA coding potential prediction. Extensive evaluations on four previous datasets and six new datasets constructed in different species show that DeepCPP outperforms other state-of-the-art methods, especially on sORF type data, which overcomes the bottleneck of sORF mRNA identification by improving more than 4.31, 37.24 and 5.89% on its accuracy for newly discovered human, vertebrate and insect data, respectively. Additionally, we also revealed that discontinuous k-mer, and our newly proposed nucleotide bias and minimal distribution similarity feature selection method play crucial roles in this classification problem. Taken together, DeepCPP is an effective method for RNA coding potential prediction.

ORF57 Overcomes the Detrimental Sequence Bias of Kaposi's Sarcoma-Associated Herpesvirus Lytic Genes

ABSTRACTKaposi's sarcoma-associated herpesvirus (KSHV) encodes ORF57, which enhances the expression of intronless KSHV genes on multiple posttranscriptional levels. However, it remains elusive how ORF57 recognizes viral RNAs. Here, we demonstrate that ORF57 also increases the expression of the multiple intron-containing K15 gene. The nucleotide bias of the K15 cDNA revealed an unusual high AT content. Thus, we optimized the K15 cDNA by raising the frequency of GC nucleotides, yielding an ORF57-independent version. To further prove the importance of the sequence bias of ORF57-dependent RNAs, we grouped KSHV mRNAs according to their AT content and found a correlation between AT-richness and ORF57 dependency. More importantly, latent genes, which have to be expressed in the absence of ORF57, have a low AT content and are indeed ORF57 independent. The nucleotide composition of K15 resembles that of HIVgag, which cannot be expressed unless RNA export is facilitated by the HIV Rev protein. Interestingly, ORF57 can partially rescue HIV Gag expression. Thus, the KSHV target RNAs of ORF57 and HIVgagRNA may share certain motifs based on the nucleotide bias. A bioinformatic comparison between wild-type and sequence-optimized K15 revealed a higher density for hnRNP-binding motifs in the former. We speculate that binding of particular hnRNPs to KSHV lytic transcripts is the prerequisite for ORF57 to enhance their expression.IMPORTANCEThe mostly intronless genes of KSHV are only expressed in the presence of the viral regulator protein ORF57, but how ORF57 recognizes viral RNAs remains elusive. We focused on the multiple intron-containing KSHV gene K15 and revealed that its expression is also increased by ORF57. Moreover, sequences in the K15 cDNA mediate this enhancement. The quest for a target sequence or a response element for ORF57 in the lytic genes was not successful. Instead, we found the nucleotide bias to be the critical determinant of ORF57 dependency. Based on the fact that ORF57 has only a weak affinity for nucleic acids, we speculate that a cellular RNA-binding protein provides the sequence preference for ORF57. This study provides evidence that herpesviral RNA regulator proteins use the sequence bias of lytic genes and the resulting composition of the viral mRNP to distinguish between viral and cellular mRNAs.