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Abstract Nuclear export of influenza A virus (IAV) mRNAs occurs through the nuclear pore complex (NPC). Using the Auxin-Induced Degron (AID) system to rapidly degrade proteins, we show that among the nucleoporins localized at the nucleoplasmic side of the NPC, TPR is the key nucleoporin required for nuclear export of influenza virus mRNAs. TPR recruits the TRanscription and EXport complex (TREX)−2 to the NPC for exporting a subset of cellular mRNAs. By degrading components of the TREX-2 complex (GANP, Germinal-center Associated Nuclear Protein; PCID2, PCI domain containing 2), we show that influenza mRNAs require the TREX-2 complex for nuclear export and replication. Furthermore, we found that cellular mRNAs whose export is dependent on GANP have a small number of exons, a high mean exon length, long 3’ UTR, and low GC content. Some of these features are shared by influenza virus mRNAs. Additionally, we identified a 45 nucleotide RNA signal from influenza virus HA mRNA that is sufficient to mediate GANP-dependent mRNA export. Thus, we report a role for the TREX-2 complex in nuclear export of influenza mRNAs and identified RNA determinants associated with the TREX-2-dependent mRNA export.

Summary: mRNA in eukaryotic cells is packaged into highly compacted ribonucleoprotein particles (mRNPs) in the nucleus and exported to the cytoplasm for translation. mRNP packaging and export require the evolutionarily conserved transcription-export (TREX) complex. TREX facilitates loading of various RNA-binding proteins on mRNA through the action of its DDX39B subunit. SARNP (Tho1 [transcriptional defect of Hpr1 by overexpression 1] in yeast) is shown to interact with DDX39B and affect mRNA export. The molecular mechanism of how SARNP recognizes DDX39B and functions in mRNP assembly is unclear. Here, we determine the crystal structure of a Tho1/DDX39B/RNA complex, revealing a multivalent interaction mediated by tandem DDX39B interacting motifs in SARNP/Tho1. The high-order complex of SARNP and DDX39B is evolutionarily conserved, and human SARNP can engage with five DDX39B molecules. RNA sequencing (RNA-seq) from SARNP knockdown cells shows the most affected RNAs in export are GC rich. Our work suggests the role of the high-order SARNP/DDX39B/RNA complex in mRNP assembly and export.

mRNAs export from the nucleus is thought to be regulated in part by three nucleoporins that comprise the nuclear basket, but whether and how distinct basket nucleoporins interact with the RNA export machinery is unclear. Here, the authors use rapid auxin-mediated degradation of basket nucleoporins Nup153, Nup50, and Tpr, and see that Tpr interacts with the TREX-2 mRNA export complex.

Viral infection induces the expression of numerous host genes that impact the outcome of infection. Here, we show that infection of human lung epithelial cells with influenza A virus (IAV) also induces a broad program of alternative splicing of host genes. Although these splicing-regulated genes are not enriched for canonical regulators of viral infection, we find that many of these genes do impact replication of IAV. Moreover, in several cases, specific inhibition of the IAV-induced splicing pattern also attenuates viral infection. We further show that approximately a quarter of the IAV-induced splicing events are regulated by hnRNP K, a host protein required for efficient splicing of the IAV M transcript in nuclear speckles. Finally, we find an increase in hnRNP K in nuclear speckles upon IAV infection, which may alter accessibility of hnRNP K for host transcripts thereby leading to a program of host splicing changes that promote IAV replication.

Alternative splicing of influenza A virus (IAV) M transcript is regulated by hnRNP K and NS1-BP, but mechanistic details are unknown. Here, Thompson et al. show how hnRNP K and NS1-BP bind M mRNA and that these proteins regulate splicing of host transcripts in both the absence and presence of IAV infection.

Introduction: Gram staining of smears prepared from positively flagged blood culture bottles plays a vital role because its accurate interpretation helps the clinicians to modify empirical therapy as well as to perform the direct susceptibility test, which reduces the turnaround time by 24 h to initiate pathogen-directed antimicrobial therapy. However, the interpretation of direct Gram stain from blood culture bottles has always been at risk of errors in interpreting. Aim: The present study was conducted to find the agreement between the blood culture broth Gram stain finding of four different methods (conventional, water wash, blood film, and drop and rest) of smear preparation with the culture smear Gram stain result and also to compare the quantity of charcoal and resin particles present in the Gram stained smears made from four different smear preparation methods. Method and Result: Smears were made from 500 positively flagged automated blood culture bottles using the four different methods and then compared for agreement with the culture smear Gram stain using kappa statistics and were found to be maximum for the blood film method (0.637), followed by drop and rest (0.570), water-wash (0.496), and conventional method (0.475). The 3+ grading for resin/charcoal was found in 184 (36.80%) smears, 169 (33.80%), 93 (18.60%), and 26 (5.20%) smears prepared by water-wash, conventional, drop and rest, and blood film method, respectively. The interference caused by resin/charcoal was maximum when smears were prepared by the conventional method resulting in high inaccurate interpretation. Conclusion: In this study, the agreement between direct smear preparation methods from positively flagged blood culture broth with the culture smear Gram stain was found to be maximum for the blood film method, followed by drop and rest, waterwash, and conventional method.It was also observed that the least amount of resin/charcoal was encountered when the direct smears were prepared using the blood film method.Hence, we found that the blood film method helps in interpreting the Gram stained smears from positively flagged blood culture broth more accurately compared to other methods.

Introduction: Hepatitis B Virus (HBV) causes acute and chronic liver disease which can progress to cirrhosis and hepatocellular carcinoma. The seroprevalence in India is about 2% to 8%. When compared to general population, seroprevalence in HCWs is 2-4 times higher. A 40%-65% of HBV in Health Care Workers (HCWs) is due to occupational exposures. Housekeeping staff are also exposed to risk, as they are involved in handling of inappropriately discarded bio medical waste, low vaccination coverage due to low educational and socio-economic status. This is the first study conducted in India, to do Knowledge Attitude Practice (KAP) assessment using Turning point technology, an innovative method using clickers. Aim: To evaluate the role of educational intervention, to increase hepatitis B vaccination among housekeeping staffs of our hospital. Materials and Methods: This study was conducted for six months, between April-September 2017 in a tertiary care hospital in southern India. The sample size for the given study was 656. Knowledge was assessed in two phases, at baseline and one month after education intervention. Results: The results at baseline showed that average correct response to knowledge on HBV disease, transmission, prevention and post exposure prophylaxis was 22%, 45%, 36% and 26% respectively which improved to 76%, 82%, 90% and 70% respectively post intervention. Attitude and practice towards preventing HBV was analysed at baseline and 60% opined that they were not vaccinated against HBV because of lack of awareness. Conclusion: In the present study percentage of HCWs protected from HBV increased from 26% to 39%. We consider that all cadre of HCWs have right to be protected against hepatitis B in view of their increased risk from occupational exposure. Having institutional protocol, availability of vaccine and test for anti HBs titre will largely influence the vaccination compliance.