The Role of Male Reproductive Organs in the Transmission of African Swine Fever—Implications for Transmission

African swine fever (ASF) has evolved from an exotic animal disease to a threat to global pig production. An important avenue for the wide-spread transmission of animal diseases is their dissemination through boar semen used for artificial insemination. In this context, we investigated the role of male reproductive organs in the transmission of ASF. Mature domestic boars and adolescent wild boars, inoculated with different ASF virus strains, were investigated by means of virological and pathological methods. Additionally, electron microscopy was employed to investigate in vitro inoculated sperm. The viral genome, antigens and the infectious virus could be found in all gonadal tissues and accessory sex glands. The viral antigen and viral mRNAs were mainly found in mononuclear cells of the respective tissues. However, some other cell types, including Leydig, endothelial and stromal cells, were also found positive. Using RNAScope, p72 mRNA could be found in scattered halo cells of the epididymal duct epithelium, which could point to the disruption of the barrier. No direct infection of spermatozoa was observed by immunohistochemistry, or electron microscopy. Taken together, our results strengthen the assumption that ASFV can be transmitted via boar semen. Future studies are needed to explore the excretion dynamics and transmission efficiency.

HSV-1 and influenza infection induce linear and circular splicing of the long NEAT1 isoform

The herpes simplex virus 1 (HSV-1) virion host shut-off (vhs) protein cleaves both cellular and viral mRNAs but not circular RNAs (circRNAs) without an internal ribosome entry site. Here, we show that vhs activity leads to an accumulation of circRNAs relative to linear mRNAs during HSV-1 infection. Strikingly, we found that circular splicing of the long isoform (NEAT1_2) of the nuclear paraspeckle assembly transcript 1 (NEAT1) was massively induced during HSV-1 infection in a vhs-independent manner while NEAT1_2 was still bound to the chromatin. This was associated with induction of linear splicing of NEAT1_2 both within and downstream of the circRNA. NEAT1_2 splicing was absent in uninfected cells but can be induced by ectopic co-expression of the HSV-1 immediate-early proteins ICP22 and ICP27. Interestingly, NEAT1_2 circular and linear splicing was also up-regulated in influenza infection but absent in stress conditions, which disrupt transcription termination similar to but not in the same manner as HSV-1 and influenza infection. Finally, large-scale analysis of published RNA-seq data uncovered induction of NEAT1_2 splicing in cancer cells upon inhibition or knockdown of cyclin-dependent kinase 7 (CDK7) or the MED1 subunit of the Mediator complex phosphorylated by CDK7. Interestingly, CDK7 inhibition also disrupted transcription termination, highlighting a possible link between disruption of transcription termination and NEAT1_2 splicing.

Hepatitis B virus X (HBx) Protein Expression is Tightly Regulated by N6-methyladenosine Modification of its mRNA

Hepatitis B virus (HBV) encodes a regulatory protein termed HBx, that has been intensely studied in the past and shown to play a key role(s) in viral transcription and replication. In addition, a huge body of work exists in the literature related to signal transduction and possible mechanism(s) leading to hepatocarcinogenesis associated with infection. We have previously reported that HBV transcripts are modified by N6-methyladenosine (m6A) at the single consensus DRACH motif at 1905-1909 nucleotide (nt) in the epsilon structural element and this m6A modification affects the HBV life cycle. In this study, we present evidence that additional variants of m6A (DRACH) motifs are located within 1606 to 1809 nt correspond on the coding region of HBx mRNA and 3′ untranslated region (UTR) of other viral mRNAs. Using the mutants of additional m6A site in 1606 to 1809 nt and a depletion strategy of m6A methyltransferases (METTL3/14) and reader proteins (YTHDFs), we show that m6A modification at 1616 nt, located in HBx coding region, regulates HBx protein expression. The HBx RNA and protein expressions were notably increased by the silencing of m6A reader YTHDF2 and methyltransferases as well as the mutation of m6A sites in the HBx coding region. However, other viral protein expressions were not affected by the m6A modification at 1616 nt. Thus, m6A modifications in the HBx open reading frame (ORF), downregulate HBx protein expression, commonly seen during HBV transfections, transgenic mice, and natural infections of human hepatocytes. These studies identify the functional role of m6A modification in the subtle regulation of HBx protein expression consistent with its possible role in establishing chronic hepatitis. Importance N6-methyladenosien (m6A) modifications have been recently implicated in the HBV life cycle. Previously, we observed that m6A modification occurs in the adenosine at 1907 nt of HBV genome and this modification regulates the viral life cycle. Here, we identified an additional m6A site located in 1616 nt of the HBV genome. This modification negatively affects HBx RNA and protein expression. In the absence of m6A methyltransferases (METTL3/14) and reader protein (YTHDF2), the HBx RNA and protein expression were increased. Using HBV mutants that lack m6A in the HBx coding region, we present the unique positional effects of m 6 A in the regulation of HBx protein expression.

The Male Reproductive Organs in African Swine Fever–Implications for Transmission

African swine fever (ASF) has evolved from an exotic animal disease to a threat to global pig production. An important avenue for wide-spread transmission of animal diseases is the dissemination of viruses through boar semen used for artificial insemination (AI). In this context, we investigated the role of male reproductive organs in ASF. Mature domestic boars and adolescent wild boar inoculated with different ASF virus strains were investigated by means of virological and pathological methods. Additionally, electron microscopy was employed to investigate in vitro inoculated sperm. Viral genome, antigen and infectious virus could be found in all gonadal tissues and accessory sex glands. The viral antigen and viral mRNAs were mainly found in mononuclear cells of the respective tissues. However, some other cell types, including Leydig, endothelial and stromal cells were also found positive. Using RNAScope, p72 mRNA could be found in scattered halo cells of the epididymal duct epithelium which could point to disruption of the barrier. No direct infection of spermatozoa was observed by immunohistochemistry or electron microscopy. Taken together, our results strengthen the assumption that ASFV can be transmitted via boar semen. Future studies are needed to explore excretion dynamics and transmission efficiency.

Transcriptomic Characterization Reveals Attributes of High Influenza Virus Productivity in MDCK Cells

The Madin–Darby Canine Kidney (MDCK) cell line is among the most commonly used cell lines for the production of influenza virus vaccines. As cell culture-based manufacturing is poised to replace egg-based processes, increasing virus production is of paramount importance. To shed light on factors affecting virus productivity, we isolated a subline, H1, which had twice the influenza virus A (IAV) productivity of the parent (P) through cell cloning, and characterized H1 and P in detail on both physical and molecular levels. Transcriptome analysis revealed that within a few hours after IAV infection, viral mRNAs constituted over one fifth of total mRNA, with several viral genes more highly expressed in H1 than P. Functional analysis of the transcriptome dynamics showed that H1 and P responded similarly to IAV infection, and were both subjected to host shutoff and inflammatory responses. Importantly, H1 was more active in translation and RNA processing intrinsically and after infection. Furthermore, H1 had more subdued inflammatory and antiviral responses. Taken together, we postulate that the high productivity of IAV hinges on the balance between suppression of host functions to divert cellular resources and the sustaining of sufficient activities for virus replication. Mechanistic insights into virus productivity can facilitate the process optimization and cell line engineering for advancing influenza vaccine manufacturing.

Structure of the poxvirus decapping enzyme D9 reveals its mechanism of cap recognition and catalysis

SUMMARYPoxviruses encode decapping enzymes that remove the protective 5’ cap from both host and viral mRNAs to commit transcripts for decay by the cellular exonuclease Xrn1. Decapping by these enzymes is critical for poxvirus pathogenicity by means of simultaneously suppressing host protein synthesis and limiting the accumulation of viral dsRNA, a trigger for antiviral responses. Here we present the first high resolution structural view of the vaccinia virus decapping enzyme D9. This Nudix enzyme contains a novel domain organization in which a three-helix bundle is inserted into the catalytic Nudix domain. The 5’ mRNA cap is positioned in a bipartite active site at the interface of the two domains. Specificity for the methylated guanosine cap is achieved by stacking between conserved aromatic residues in a manner similar to that observed in canonical cap binding proteins VP39, eIF4E, and CBP20 and distinct from eukaryotic decapping enzyme Dcp2.

Influenza A virus segments five and six can harbor artificial introns allowing expanded coding capacity

Influenza A viruses encode their genomes across eight, negative sense RNA segments. The six largest segments produce mRNA transcripts that do not generally splice; however, the two smallest segments are actively spliced to produce the essential viral proteins NEP and M2. Thus, viral utilization of RNA splicing effectively expands the viral coding capacity without increasing the number of genomic segments. As a first step towards understanding why splicing is not more broadly utilized across genomic segments, we designed and inserted an artificial intron into the normally nonsplicing NA segment. This insertion was tolerated and, although viral mRNAs were incompletely spliced, we observed only minor effects on viral fitness. To take advantage of the unspliced viral RNAs, we encoded a reporter luciferase gene in frame with the viral ORF such that when the intron was not removed the reporter protein would be produced. This approach, which we also show can be applied to the NP encoding segment and in different viral genetic backgrounds, led to high levels of reporter protein expression with minimal effects on the kinetics of viral replication or the ability to cause disease in experimentally infected animals. These data together show that the influenza viral genome is more tolerant of splicing than previously appreciated and this knowledge can be leveraged to develop viral genetic platforms with utility for biotechnology applications.

The key features of SARS-CoV-2 leader and NSP1 required for viral escape of NSP1-mediated repression

SARS-CoV-2, responsible for the ongoing global pandemic, must overcome a conundrum faced by all viruses. To achieve its own replication and spread, it simultaneously depends on and subverts cellular mechanisms. At the early stage of infection, SARS-CoV-2 expresses the viral nonstructural protein 1 (NSP1), which inhibits host translation by blocking the mRNA entry tunnel on the ribosome; this interferes with the binding of cellular mRNAs to the ribosome. Viral mRNAs, on the other hand, overcome this blockade. We show that NSP1 enhances expression of mRNAs containing the SARS-CoV-2 leader. The first stem-loop (SL1) in viral leader is both necessary and sufficient for this enhancement mechanism. Our analysis pinpoints specific residues within SL1 (three cytosine residues at the positions 15, 19 and 20) and another within NSP1 (R124) which are required for viral evasion, and thus might present promising drug targets. Additionally, we carried out analysis of a functional interactome of NSP1 using BioID and identified components of anti-viral defense pathways. Our analysis therefore suggests a mechanism by which NSP1 inhibits the expression of host genes while enhancing that of viral RNA. This analysis helps reconcile conflicting reports in the literature regarding the mechanisms by which the virus avoids NSP1 silencing.

Cap-independent translation and a precisely localized RNA sequence enable SARS-CoV-2 to control host translation and escape anti-viral response

Translation of SARS-CoV-2-encoded mRNAs by the host ribosomes is essential for its propagation. Following infection, the early expressed viral protein NSP1 binds the ribosome, represses translation and induces mRNA degradation, while the host elicits an anti-viral response. The mechanisms enabling viral mRNAs to escape this multifaceted repression remain obscure. Here we show that expression of NSP1 leads to destabilization of multi-exon cellular mRNAs, while intron-less transcripts, such as viral mRNAs and anti-viral interferon genes, remain relatively stable. We identified a conserved and precisely located cap-proximal RNA element devoid of guanosines that confers resistance to NSP1-mediated translation inhibition. Importantly, the primary sequence rather than the secondary structure is critical for protection. We further show that the genomic 5'UTR of SARS-CoV-2 exhibits an IRES-like activity and promotes expression of NSP1 in an eIF4E-independent and Torin-1 resistant manner. Upon expression, NSP1 enhances cap-independent translation. However, the sub-genomic 5'UTRs are highly sensitive to eIF4E availability, rendering viral propagation partially sensitive to Torin-1. The combined NSP1-mediated degradation of spliced mRNAs and translation inhibition of single-exon genes, along with the unique features present in the viral 5'UTRs, ensure robust expression of viral mRNAs. These features can be exploited as potential therapeutic targets.