N-terminal acetylation separately promotes nuclear localization and host shutoff activity of the influenza A virus ribonuclease PA-X.

To counteract host antiviral responses, influenza A virus triggers a global reduction of cellular gene expression, a process termed "host shutoff." A key effector of influenza A virus host shutoff is the viral endoribonuclease PA-X, which degrades host mRNAs. While many of the molecular determinants of PA-X activity remain unknown, a previous study found that N-terminal acetylation of PA-X is required for its host shutoff activity. However, it remains unclear how this co-translational modification promotes PA-X activity. Here, we report that PA-X N-terminal acetylation has two functions that can be separated based on the position of the acetylation, i.e. on the first amino acid, the initiator methionine, or the second amino acid following initiator methionine excision. Modification at either site is sufficient to ensure PA-X localization to the nucleus. However, modification of the second amino acid is not sufficient for host shutoff activity of ectopically expressed PA-X, which specifically requires N-terminal acetylation of the initiator methionine. Interestingly, during infection N-terminal acetylation of PA-X at any position results in host shutoff activity, which is in part due to a functional interaction with the influenza protein NS1. This result reveals an unexpected role for another viral protein in PA-X activity. Our studies uncover a multifaceted role for PA-X N-terminal acetylation in regulation of this important immunomodulatory factor.

Targeted Transcriptomic Screen of Pneumococcal Genes Expressed during Murine and Human Infection.

The advent of pneumococcal conjugate vaccines led to the near disappearance of most of the included serotypes in high-income settings but also the rise of nonvaccine-type colonization and disease. Alternative strategies, using genetically conserved proteins as antigens, have been evaluated preclinically and clinically for years, so far unsuccessfully. One possible explanation for the failure of these efforts is that the choice of antigens may not have been sufficiently guided by an understanding of the gene expression pattern in the context of infection. Here, we present a targeted transcriptomic analysis of 160 pneumococcal genes encoding bacterial surface-exposed proteins in mouse models, human colonization, and human meningitis. We present the overlap of these different transcriptomic profiles. We identify two bacterial genes that are highly expressed in the context of mouse and human infection: SP_0282, an IID component of a mannose phosphotransferase system (PTS), and SP_1739, encoding RNase Y. We show that these two proteins can confer protection against pneumococcal nasopharyngeal colonization and intraperitoneal challenge in a murine model and generate opsonophagocytic antibodies. This study emphasizes and confirms the importance of studies of pneumococcal gene expression of bacterial surface proteins during human infection and colonization and may pave the way for the selection of a protein-based vaccine candidate.

Contributions of the Four Essential Entry Glycoproteins to HSV-1 Tropism and the Selection of Entry Routes.

Herpes simplex viruses (HSV-1 and HSV-2) encode up to 16 envelope proteins, four of which are essential for entry. However, whether these four proteins alone are sufficient to dictate the broad cellular tropism of HSV-1 and the selection of different cell type-dependent entry routes is unknown. To begin addressing this, we previously pseudotyped vesicular stomatitis virus (VSV), lacking its native glycoprotein G, with only the four essential entry glycoproteins of HSV-1: gB, gH, gL, and gD. This novel VSVΔG-BHLD pseudotype recapitulated several important features of HSV-1 entry: the requirement for gB, gH, gL, gD, and a cellular receptor and sensitivity to anti-gB and anti-gH/gL neutralizing antibodies. However, due to the use of a single cell type in that study, the tropism of the VSVΔG-BHLD pseudotype was not investigated. Here, we show that the cellular tropism of the pseudotype is severely limited compared to that of wild-type HSV-1 and that its entry pathways differ from the native HSV-1 entry pathways. To test the hypothesis that other HSV-1 envelope proteins may contribute to HSV-1 tropism, we generated a derivative pseudotype containing the HSV-1 glycoprotein C (VSVΔG-BHLD-gC) and observed a gC-dependent increase in entry efficiency in two cell types. We propose that the pseudotyping platform developed here has the potential to uncover functional contributions of HSV-1 envelope proteins to entry in a gain-of-function manner.IMPORTANCE Herpes simplex viruses (HSV-1 and HSV-2) contain up to 16 different proteins in their envelopes. Four of these, glycoproteins gB, gD, gH, and gL, are termed essential with regard to entry, whereas the rest are typically referred to as nonessential based on the entry phenotypes of the respective single genetic deletions. However, the single-gene deletion approach, which relies on robust loss-of-function phenotypes, may be confounded by functional redundancies among the many HSV-1 envelope proteins. We have developed a pseudotyping platform in which the essential four entry glycoproteins are isolated from the rest, which can be added back individually for systematic gain-of-function entry experiments. Here, we show the utility of this platform for dissecting the contributions of HSV envelope proteins, both the essential four and the remaining dozen (using gC as an example), to HSV entry.

The Pneumococcal Type 1 Pilus Genes Are Thermoregulated and Are Repressed by a Member of the Snf2 Protein Family.

In Streptococcus pneumoniae, the type 1 pilus is involved in many steps of pathogenesis, including adherence to epithelial cells, mediation of inflammation, escape from macrophages, and the formation of biofilms. The type 1 pilus genes are expressed in a bistable fashion with cells switching between "on" and "off" expression states. Bistable expression of these genes is due to their control by RlrA, a positive regulator subject to control by a positive-feedback loop. The type 1 pilus genes are also thought to be negatively regulated by a large number of repressors. Here we show that expression of the type 1 pilus genes is thermosensitive and switched off at growth temperatures below 31°C. We also report that the on expression state of the type 1 pilus genes is highly stable, a phenomenon which we show likely contributed to the erroneous identification of many proteins as negative regulators of these genes. Finally, we exploited the effect of low temperature on pilus gene expression to help identify SP_1523, an Snf2-type protein, as a novel negative regulator of the pilus genes. Our findings establish that the type 1 pilus genes are thermoregulated and are repressed by a member of the Snf2 protein family. They also refute the notion that these genes are controlled by 8 previously described negative regulators.IMPORTANCEStreptococcus pneumoniae is the leading cause of death from respiratory infections in children. Many bacterial factors contribute to pneumococcal virulence and nasopharyngeal colonization. The type 1 pneumococcal pilus plays an important role in mouse models and in epithelial adherence and is expressed in a bistable fashion. Here we show that the "on" state is highly stable, which may explain the prior misidentification of negative regulators of pilus expression. We also show that expression of pilus genes is thermosensitive: virtually no expression can be detected at temperatures found in the anterior nares of humans. We took advantage of this property to identify a negative regulator of pilus expression, a member of a family of proteins widely conserved across Gram-positive bacteria.