ned three key stem cell subsets with differentially regulated latent and replicative states, which provide cellular candidates for isolation and treatment of transplant-mediated disease. This work provides a direction toward developing strategies to control the switch between latency and reactivation.Neuraminidase (NA) has multiple functions in the life cycle of influenza virus, especially in the late stage of virus replication. Both of hemagglutinin (HA) and NA are highly glycosylated proteins. N-linked glycosylation (NLG) of HA has been reported to contribute to immune escape and virulence of influenza viruses. However, the function of NLG of NA remains largely unclear. In this study, we found that NLG is critical for budding ability of NA. Tunicamycin treatment or NLG knockout significantly inhibited the budding of NA. Further studies showed that the NLG knockout caused attenuation of virus in vitro and in vivo Notably, the NLG at 219 position plays an important role in the budding, replication, and virulence of H1N1 influenza virus. To explore the underlying mechanism, the unfolded protein response (UPR) was determined in NLG knockout NA overexpressed cells, which showed that the mutant NA was mainly located in the endoplasmic reticulum (ER), the UPR markers BIP and p-eIF2α were upregulated, and XBP1 was downregulated. All the results indicated that NLG knockout NA was stacked in the ER and triggered UPR, which might shut down the budding process of NA. Overall, the study shed light on the function of NLG of NA in virus replication and budding.IMPORTANCE NA is a highly glycosylated protein. Nevertheless, how the NLG affects the function of NA protein remains largely unclear. In this study, we found that NLG plays important roles in budding and Neuraminidase activity of NA protein. Loss of NLG attenuated viral budding and replication. In particular, the 219 NLG site mutation significantly attenuated the replication and virulence of H1N1 influenza virus in vitro and in vivo, which suggested that NLG of NA protein is a novel virulence marker for influenza viruses.PP7 is a leviphage, with a single-stranded RNA genome, that infects Pseudomonas aeruginosa PAO1. A reverse genetic system for PP7 was previously created by using reverse-transcribed cDNA (PP7O) from a virion-derived RNA genome. Here, we have found that the PP7O cDNA contained 20 nucleotide differences from the PP7 genome sequence deposited in the database. We created another reverse genetic system exploiting chemically synthesized cDNA (PP7S) based on the database sequence. Unlike PP7O, which yielded infectious PP7 virions, PP7S-derived particles were incapable of plaque formation on PAO1 cells, which was restored in the PAO1 cells expressing the maturation protein (MP) from PP7O Using this reverse genetic system, we revealed two amino acid residues involved in the known roles of MP (i.e., adsorption and genome replication), fortuitously providing a lesson that the viral RNA genome sequencing needs functional verification, possibly by a reverse genetic system.IMPORTANCE The biological significance of RNA phages has been largely ignored, ironically, because few studies have focused on RNA phages. As an initial attempt to properly represent RNA phages in the phageome, we previously created, by using reverse-transcribed cDNA, a reverse genetic system for the small RNA phage PP7, which infects the opportunistic human pathogen Pseudomonas aeruginosa We report another system by using chemically synthesized cDNA based on the database genome that has 20 nucleotide differences from the previous cDNA.  https://www.selleckchem.com/products/pepstatin-a.html  Investigation of those cDNA-derived phage virions revealed that two amino acids of the maturation protein are crucial for the normal phage lifecycle at different steps. Our study provides insight into the molecular basis for the RNA phage lifecycle and a lesson that the RNA genome sequencing needs to be carefully validated by cDNA-based phage assembly systems.Torovirus (ToV) has recently been classified into the new family Tobaniviridae, although historically, it belonged to the Coronavirus (CoV) family. The nucleocapsid (N) proteins of CoVs are predominantly localized in the cytoplasm, where the viruses replicate, but in some cases the proteins are partially located in the nucleolus. Many studies have investigated the subcellular localization and nucleocytoplasmic trafficking signals of the CoV N proteins, but little is known about ToV N proteins. Here, we studied the subcellular localization of the bovine ToV (BToV) N protein (BToN) and characterized its nucleocytoplasmic trafficking signals. Unlike other CoVs, BToN in infected cells was transported mainly to the nucleolus during early infection but was distributed predominantly in the nucleoplasm rather than in the nucleolus during late infection. Interestingly, a small quantity of BToN was detected in the cytoplasm during infection. Examination of a comprehensive set of substitution or deletion mutants of BToNd RNA viruses, including ToVs, replicate in the cytoplasm, and their structural proteins generally accumulate in the cytoplasm. Interestingly, BToN accumulated predominantly in the nucleus/nucleolus during all infectious processes, with only a small fraction accumulating in the cytoplasm despite being a major structural protein. Furthermore, we identified unique nucleocytoplasmic trafficking signals and demonstrated the importance of NLS/NoLS for virus growth. This study is the first to undertake an in-depth investigation of the subcellular localization and intracellular trafficking signals of BToN. Our findings additionally suggest that the NLS/NoLS-mediated nuclear accumulation of BToN is important for virus replication. An understanding of the unique features of BToV may provide novel insights into the assembly mechanisms of not only ToVs but also other positive-stranded RNA viruses.Daily burden and clinical toxicities associated with antiretroviral therapy (ART) emphasize the need for alternative strategies to induce long-term human immunodeficiency virus (HIV) remission upon ART cessation. Broadly neutralizing antibodies (bNAbs) can both neutralize free virions and mediate effector functions against infected cells and therefore represent a leading immunotherapeutic approach. To increase potency and breadth, as well as to limit the development of resistant virus strains, it is likely that bNAbs will need to be administered in combination. It is therefore critical to identify bNAb combinations that can achieve robust polyfunctional antiviral activity against a high number of HIV strains. In this study, we systematically assessed the abilities of single bNAbs and triple bNAb combinations to mediate robust polyfunctional antiviral activity against a large panel of cross-clade simian-human immunodeficiency viruses (SHIVs), which are commonly used as tools for validation of therapeutic strategies targeting the HIV envelope in nonhuman primate models.