Analysis of the Relationship between Cellular Thymidine Kinase Activity and Virulence of Thymidine Kinase-Negative Herpes Simplex Virus Types 1 and 2

The virulence of thymidine kinase-negative herpes simplex virus type 1 (HSV-1; VRTK^? strain) and type 2 (HSV-2; UWTK^? strain) was studied in comparison with that of their parental strains (VR-3 and UW-268, respectively) in an encephalitis model of adult (4-week-old) and newborn (3-day-old) mice. Viral thymidine kinase (TK) activity was essential for the maximum expression of virulence of HSV-1, because the 50% lethal dose (LD₅₀) of VRTK^? was 60 times higher than that of VR-3 in the brains of newborn mice expressing high levels of cellular TK activity. However, the UWTK^? strain showed the same virulence as the parental strain in newborn mice, despite the lack virulence in adults, suggesting that replication of the UWTK^? strain was completely supported by cellular TK activity. This difference in the role of viral and cellular TKs for virus growth between HSV-1 and HSV-2 was confirmed with the one-step growth of virus strains in L-M and L-M(TK^?) cells.     

HSV‐1 ICP27 targets the TBK1‐activated STING signalsome to inhibit virus‐induced type I IFN expression

Herpes simplex virus (HSV) 1 stimulates type I IFN expression through the cGAS–STING–TBK1 signaling axis. Macrophages have recently been proposed to be an essential source of IFN during viral infection. However, it is not known how HSV‐1 inhibits IFN expression in this cell type. Here, we show that HSV‐1 inhibits type I IFN induction through the cGAS–STING–TBK1 pathway in human macrophages, in a manner dependent on the conserved herpesvirus protein ICP27. This viral protein was expressed de novo in macrophages with early nuclear localization followed by later translocation to the cytoplasm where ICP27 prevented activation of IRF3. ICP27 interacted with TBK1 and STING in a manner that was dependent on TBK1 activity and the RGG motif in ICP27. Thus, HSV‐1 inhibits expression of type I IFN in human macrophages through ICP27‐dependent targeting of the TBK1‐activated STING signalsome.     

A single‐molecule approach to DNA replication in Escherichia coli cells demonstrated that DNA polymerase III is a major determinant of fork speed

The replisome catalyses DNA synthesis at a DNA replication fork. The molecular behaviour of the individual replisomes, and therefore the dynamics of replication fork movements, in growing Escherichia coli cells remains unknown. DNA combing enables a single‐molecule approach to measuring the speed of replication fork progression in cells pulse‐labelled with thymidine analogues. We constructed a new thymidine‐requiring strain, eCOMB (E. coli for combing), that rapidly and sufficiently incorporates the analogues into newly synthesized DNA chains for the DNA‐combing method. In combing experiments with eCOMB, we found the speed of most replication forks in the cells to be within the narrow range of 550–750 nt s^?1 and the average speed to be 653 ± 9 nt s^?1 (± SEM). We also found the average speed of the replication fork to be only 264 ± 9 nt s^?1 in a dnaE173‐eCOMB strain producing a mutant‐type of the replicative DNA polymerase III (Pol III) with a chain elongation rate (300 nt s^?1) much lower than that of the wild‐type Pol III (900 nt s^?1). This indicates that the speed of chain elongation by Pol III is a major determinant of replication fork speed in E. coli cells.     

Selective ablation of virion host shutoff protein RNase activity attenuates herpes simplex virus 2 in mice

The virion host shutoff (vhs) protein of herpes simplex virus (HSV) has endoribonuclease activity and rapidly reduces protein synthesis in infected cells through mRNA degradation. Herpes simplex virus 1 (HSV-1) and HSV-2 vhs mutants are highly attenuated in vivo, but replication and virulence are largely restored to HSV-2 vhs mutants in the absence of a type I interferon (IFN) response. The role of vhs in pathogenesis and the hindrance of the type I IFN response have classically been examined with viruses that completely lack vhs or express a truncated vhs protein. To determine whether RNase activity is the principal mechanism of vhs-mediated type I IFN resistance and virulence, we constructed a HSV-2 point mutant that synthesizes full-length vhs protein lacking RNase activity (RNase(-) virus). Wild-type and mutant HSV-2 vhs proteins coimmunoprecipitated with VP16 and VP22. vhs protein bearing the point mutation was packaged into the virion as efficiently as the wild-type vhs protein. Like a mutant encoding truncated vhs, the RNase(-) virus showed IFN-dependent replication that was restricted compared with that of the wild-type virus. The RNase(-) virus was highly attenuated in wild-type mice infected intravaginally, with reduced mucosal replication, disease severity, and spread to the nervous system comparable to those of the vhs truncation mutant. Surprisingly, in alpha/beta interferon (IFN-alpha/beta) receptor knockout mice, the vhs RNase mutant was more attenuated than the vhs truncation mutant in terms of disease severity and virus titer in vaginal swabs and central nervous system samples, suggesting that non-enzymatically active vhs protein interferes with efficient virus replication. Our results indicate that vhs enzymatic activity plays a complex role in vhs-mediated type I IFN resistance during HSV-2 infection.     

Replication of herpes simplex virus type 1 within trigeminal ganglia is required for high frequency but not high viral genome copy number latency

The replication properties of a thymidine kinase-negative (TK(-)) mutant of herpes simplex virus type 1 (HSV-1) were exploited to examine the relative contributions of replication at the body surface and within trigeminal ganglia (TG) on the establishment of latent infections. The replication of a TK(-) mutant, 17/tBTK(-), was reduced by approximately 12-fold on the mouse cornea compared to the rescued isolate 17/tBRTK(+), and no replication of 17/tBTK(-) in the TG of these mice was detected. About 1.8% of the TG neurons of mice infected with 17/tBTK(-) harbored the latent viral genome compared to 23% of those infected with 17/tBRTK(+). In addition, the latent sites established by the TK(-) mutant contained fewer copies of the HSV-1 genome (average, 2.3/neuron versus 28/neuron). On the snout, sustained robust replication of 17tBTK(-) in the absence of significant replication within the TG resulted in a modest increase in the number of latent sites. Importantly, these latently infected neurons displayed a wild-type latent-genome copy number profile, with some neurons containing hundreds of copies of the TK(-) mutant genome. As expected, the replication of the TK(-) mutant appeared to be blocked prior to DNA replication in most ganglionic neurons in that (i) virus replication was severely restricted in ganglia, (ii) the number of neurons expressing HSV proteins was reduced 30-fold compared to the rescued isolate, (iii) cell-to-cell spread of virus was not detected within ganglia, and (iv) the proportion of infected neurons expressing late proteins was reduced by 89% compared to the rescued strain. These results demonstrate that the viral TK gene is required for the efficient establishment of latency. This requirement appears to be primarily for efficient replication within the ganglion, which leads to a sixfold increase in the number of latent sites established. Further, latent sites with high genome copy number can be established in the absence of significant virus genome replication in neurons. This suggests that neurons can be infected by many HSV virions and still enter the latent state.     

Neisseria gonorrhoeae effectively blocks HIV‐1 replication by eliciting a potent TLR9‐dependent interferon‐α response from plasmacytoid dendritic cells

Clinical and epidemiological research provides evidence for a positive correlation between Neisseria gonorrhoeae infection and HIV transmission; however, mechanistic studies examining this relationship have yielded conflicting results. To explore this interaction, we exposed ex vivo cultured peripheral blood cells from acute HIV⁺ individuals to N. gonorrhoeae. Unexpectedly, we observed a profound inhibition in HIV‐1 replication in the ex vivo cultures, and this was recapitulated when peripheral blood mononuclear cells (PBMCs) from healthy donors were co‐infected with HIV‐1 and N. gonorrhoeae. Next, we established that gonococcal‐infected PBMCs liberated a soluble factor that effectively blocked HIV‐1 replication. Cytokine analyses and antibody blocking experiments revealed that the type I interferon, interferon‐α (IFNα), was expressed upon exposure to N. gonorrhoeae and was responsible for the inhibition of HIV‐1. Intracellular staining, TLR9‐blocking and cell depletion‐based studies demonstrated that the IFNα was elicited by plasmacytoid dendritic cells (pDCs) in a TLR9‐dependent manner. The pDC response to N. gonorrhoeae was unexpected given pDCs more established role in innate defence against intracellular pathogens, suggesting this may be a bacterial immune evasion strategy. In the context of HIV, this overcomes the virus's otherwise effective avoidance of the interferon response and represents a previously unrecognized intersection between these two sexually transmitted pathogens.     

Type I interferon production during herpes simplex virus infection is controlled by cell-type-specific viral recognition through Toll-like receptor 9, the mitochondrial antiviral signaling protein pathway, and novel recognition systems

Recognition of viruses by germ line-encoded pattern recognition receptors of the innate immune system is essential for rapid production of type I interferon (IFN) and early antiviral defense. We investigated the mechanisms of viral recognition governing production of type I IFN during herpes simplex virus (HSV) infection. We show that early production of IFN in vivo is mediated through Toll-like receptor 9 (TLR9) and plasmacytoid dendritic cells, whereas the subsequent alpha/beta IFN (IFN-α/β) response is derived from several cell types and induced independently of TLR9. In conventional DCs, the IFN response occurred independently of viral replication but was dependent on viral entry. Moreover, using a HSV-1 UL15 mutant, which fails to package viral DNA into the virion, we found that entry-dependent IFN induction also required the presence of viral genomic DNA. In macrophages and fibroblasts, where the virus was able to replicate, HSV-induced IFN-α/β production was dependent on both viral entry and replication, and ablated in cells unable to signal through the mitochondrial antiviral signaling protein pathway. Thus, during an HSV infection in vivo, multiple mechanisms of pathogen recognition are active, which operate in cell-type- and time-dependent manners to trigger expression of type I IFN and coordinate the antiviral response.     

H‐NOX regulation of c‐di‐GMP metabolism and biofilm formation in Legionella pneumophila

Haem Nitric oxide/OXygen (H‐NOX) binding domains are a family of haemoprotein sensors that are widespread in bacterial genomes, but limited information is available on their function. Legionella pneumophila is the only prokaryote found, thus far, to encode two H‐NOX proteins. This paper presents data supporting a role for one of the L. pneumophila H‐NOXs in the regulation of biofilm formation. In summary: (i) unmarked deletions in the hnox1 gene do not affect growth rate in liquid culture or replication in permissive macrophages; (ii) the Δhnox1 strain displays a hyper‐biofilm phenotype; (iii) the gene adjacent to hnox1 is a GGDEF‐EAL protein, lpg1057, and overexpression in L. pneumophila of this protein, or the well‐studied diguanylate cyclase, vca0956, results in a hyper‐biofilm phenotype; (iv) the Lpg1057 protein displays diguanylate cyclase activity in vitro and this activity is inhibited by the Hnox1 protein in the Fe(II)‐NO ligation state, but not the Fe(II) unligated state; and (v) consistent with the Hnox1 regulation of Lpg1057, unmarked deletions of lpg1057 in the Δhnox1 background results in reversion of the hyper‐biofilm phenotype back to wild‐type biofilm levels. Taken together, these results suggest a role for hnox1 in regulating c‐di‐GMP production by lpg1057 and biofilm formation in response to NO.     

ESX‐1 exploits type I IFN‐signalling to promote a regulatory macrophage phenotype refractory to IFNγ‐mediated autophagy and growth restriction of intracellular mycobacteria

The ability of macrophages to eradicate intracellular pathogens is normally greatly enhanced by IFNγ, a cytokine produced mainly after onset of adaptive immunity. However, adaptive immunity is unable to provide sterilizing immunity against mycobacteria, suggesting that mycobacteria have evolved virulence strategies to inhibit the bactericidal effect of IFNγ‐signalling in macrophages. Still, the host–pathogen interactions and cellular mechanisms responsible for this feature have remained elusive. We demonstrate that the ESX‐1 type VII secretion systems of Mycobacterium tuberculosis and Mycobacterium marinum exploit type I IFN‐signalling to promote an IL‐12^low/IL‐10^high regulatory macrophage phenotype characterized by secretion of IL‐10, IL‐27 and IL‐6. This mechanism had no impact on intracellular growth in the absence of IFNγ but suppressed IFNγ‐mediated autophagy and growth restriction, indicating that the regulatory phenotype extends to function. The IFNγ‐refractory phenotype was partly mediated by IL‐27‐signalling, establishing functional relevance for this downstream cytokine. These findings identify a novel macrophage‐modulating function for the ESX‐1 secretion system that may contribute to suppress the efficacy of adaptive immunity and provide mechanistic insight into the antagonistic cross talk between type I IFNs and IFNγ in mycobacterial infection.     

Differential activation of hybrid genes containing herpes simplex virus immediate-early or delayed-early promoters after superinfection of stable DNA-transfected cell lines

We compared the levels of gene expression obtained after herpes simplex virus (HSV) superinfection of cell lines containing integrated human beta-interferon (IFN) or chloramphenicol acetyltransferase (CAT) genes under the control of HSV immediate-early (IE) or delayed-early class promoters. DNA-transfected mouse Ltk+ cell lines harboring coselected IE175-IFN or thymidine kinase (TK)-IFN hybrid genes gave only low basal expression of human IFN. However, infection of both cell types with HSV type 1 or HSV type 2 produced abundant synthesis of IFN-specific RNA and biologically active IFN protein product. The IE175-IFN cell lines consistently gave 20- to 150-fold increases in IFN titers, and several TK-IFN cell lines yielded 100- to 500-fold induction. In the IE175-IFN cells, expression of IFN RNA also increased up to 200-fold and was detectable within 30 to 60 min after virus infection. Qualitatively similar results were obtained with hybrid G418-resistant Ltk- or Vero cell lines containing coselected IE175-CAT and TK-CAT constructs, except that there was relatively high basal expression of IE175-CAT. All three sets of IE cell lines (but not the delayed-early cell lines) responded to virus infection both in the presence of cycloheximide and with mutants defective in IE gene expression, demonstrating specific trans-activation by the pre-IE virion factor. In contrast, activation in the TK hybrid cell types required viral gene expression and the presence of a functional IE175 gene product. Up to 30-fold amplification in the copy number of the resident IFN or CAT DNA sequences also occurred within 20 h after HSV infection in IE175 hybrid cells but not in TK hybrid cells. Amplification was abolished either by treatment with phosphonacetate or by superinfection with a ts mutant unable to synthesize viral DNA, demonstrating specific HSV activation of the viral DNA replication origin (oriS) present in the IE hybrid constructs.     

Zika virus elicits inflammation to evade antiviral response by cleaving cGAS via NS1‐caspase‐1 axis

Viral infection triggers host innate immune responses, which primarily include the activation of type I interferon (IFN) signaling and inflammasomes. Here, we report that Zika virus (ZIKV) infection triggers NLRP3 inflammasome activation, which is further enhanced by viral non‐structural protein NS1 to benefit its replication. NS1 recruits the host deubiquitinase USP8 to cleave K11‐linked poly‐ubiquitin chains from caspase‐1 at Lys134, thus inhibiting the proteasomal degradation of caspase‐1. The enhanced stabilization of caspase‐1 by NS1 promotes the cleavage of cGAS, which recognizes mitochondrial DNA release and initiates type I IFN signaling during ZIKV infection. NLRP3 deficiency increases type I IFN production and strengthens host resistance to ZIKVin vitro and in vivo. Taken together, our work unravels a novel antagonistic mechanism employed by ZIKV to suppress host immune response by manipulating the interplay between inflammasome and type I IFN signaling, which might guide the rational design of therapeutics in the future.     

ISOLATION AND CHARACTERIZATION OF A CELL WALL‐DEFECTIVE MUTANT OF CHLAMYDOMONAS MONOICA (CHLOROPHYTA)1

Cell wall–defective strains of Chlamydomonas have played an important role in the development of transformation protocols for introducing exogenous DNA (foreign genes or cloned Chlamydomonas genes) into C. reinhardtii. To promote the development of similar protocols for transformation of the distantly related homothallic species, C. monoica, we used UV mutagenesis to obtain a mutant strain with a defective cell wall. The mutant, cw‐1, was first identified on the basis of irregular colony shape and was subsequently shown to have reduced plating efficiency and increased sensitivity to lysis by a non‐ionic detergent as compared with wild‐type cells. Tetrad analysis of crosses involving the cw‐1 mutant confirmed 2:2 segregation of the cw:cw⁺ phenotypes, indicating that the wall defect resulted from mutation of a single nuclear gene. The phenotype showed incomplete penetrance and variable expressivity. Although some cells had apparently normal cell walls as viewed by TEM, many cells of the cw‐1 strain had broken cell walls and others were protoplasts completely devoid of a cell wall. Several cw‐1 isolates obtained from crosses involving the original mutant strain showed a marked enhancement of the mutant phenotype and may prove especially useful for future work involving somatic cell fusions or development of transformation protocols.     

Inhibition of herpes simplex virus type 1 replication in fibroblast cultures by human blood mononuclear cells.

An assay was developed to test the effect of human blood mononuclear cells (MNCs) on herpes simplex virus (HSV) replication. In this assay, human fibroblast monolayers were inoculated with HSV and then cultured with or without blood MNCs. Fewer HSV-infected cells were recovered from human fibroblasts cultured in the presence than in the absence of blood MNCs. This inhibition of viral replication by MNCs was independent of HLA matching between the MNCs and fibroblasts and persisted even when T cells were depleted by antibody and complement. However, depletion of Leu11+ MNCs either by panning or with antibody and complement reduced the ability of the cells to suppress HSV infection, whereas enrichment of Leu11+ cells by fluorescence-activated cell sorting increased the viral suppression. Depletion of OKM1+ MNCs also reduced the viral suppression. After coculturing of MNCs and HSV-infected fibroblasts for 3 days, alpha interferon (IFN) and gamma IFN were detected in the supernatants. Predepletion of Leu11+ MNCs reduced the amount of gamma IFN produced in these cultures. Incubation of the MNCs and HSV-infected fibroblasts with antibody specific for either alpha or gamma IFN resulted in reduced viral suppression. Preincubation of MNCs for 18 h with either interleukin 2 or alpha IFN or for 7 days with antigen increased the suppression of HSV infection. These results suggest that natural killer cells with the Leu11+ phenotype participate in the recognition of HSV-infected cells at a point sufficiently early to interfere with the spread of infection in vitro and may inhibit viral replication by natural killer cell cytotoxicity, by generation of interferon, and by lymphokine-activated killing.     

Shp‐2 contributes to anti‐RSV activity in human pulmonary alveolar epithelial cells by interfering with the IFN‐α‐induced Jak/Stat1 pathway

Src homology phosphotyrosyl phosphatase 2 (Shp‐2) is a ubiquitously expressed protein that is involved in a variety of cellular processes, including antiviral interferon signalling pathways. In this study, we investigated the role of Shp‐2 in the host cell interactions of human respiratory syncytial virus (RSV). We report significant changes in the expression of Shp‐2 in human pulmonary alveolar epithelial cells (A549) upon RSV infection. We also report that blocking Shp‐2 does not affect viral replication or virus‐induced interferon‐alpha (IFN‐α) production. Interestingly, whereas A549 cells were activated by IFN‐α, the blocking of Shp‐2 resulted in increased viral replication that was associated with the reduced expression of the IFN‐stimulated genes of 2′,5′‐oligoadenylate synthetases and Mx1, and the concomitant inhibition of Stat1 tyrosine phosphorylation. Our findings suggest that Shp‐2 contributes to the control of RSV replication and progeny production in pulmonary alveolar epithelial cells by interfering with IFN‐α‐induced Jak/Stat1 pathway activation rather than by affecting the production of IFN‐α itself.     

Human alpha‐ and beta‐NRXN1 isoforms rescue behavioral impairments of Caenorhabditis elegans neurexin‐deficient mutants

Neurexins are cell adhesion proteins that interact with neuroligin and other ligands at the synapse. In humans, mutations in neurexin or neuroligin genes have been associated with autism and other mental disorders. The human neurexin and neuroligin genes are orthologous to the Caenorhabditis elegans genes nrx‐1 and nlg‐1, respectively. Here we show that nrx‐1‐deficient mutants are defective in exploratory capacity, sinusoidal postural movements and gentle touch response. Interestingly, the exploratory behavioral phenotype observed in nrx‐1 mutants was markedly different to nlg‐1‐deficient mutants; thus, while the former had a ‘hyper‐reversal’ phenotype increasing the number of changes of direction with respect to the wild‐type strain, the nlg‐1 mutants presented a ‘hypo‐reversal’ phenotype. On the other hand, the nrx‐1‐ and nlg‐1‐defective mutants showed similar abnormal sinusoidal postural movement phenotypes. The response of these mutant strains to aldicarb (acetylcholinesterase inhibitor), levamisole (ACh agonist) and pentylenetetrazole [gamma‐aminobutyric (GABA) receptor antagonist], suggested that the varying behavioral phenotypes were caused by defects in ACh and/or GABA inputs. The defective behavioral phenotypes of nrx‐1‐deficient mutants were rescued in transgenic strains expressing either human alpha‐ or beta‐NRXN‐1 isoforms under the worm nrx‐1 promoter. A previous report had shown that human and rat neuroligins were functional in C. elegans. Together, these results suggest that the functional mechanism underpinning both neuroligin and neurexin in the nematode are comparable to human. In this sense the nematode might constitute a simple in vivo model for understanding basic mechanisms involved in neurological diseases for which neuroligin and neurexin are implicated in having a role.     

Proteomics analysis of a long‐term survival strain of Escherichia coli K‐12 exhibiting a growth advantage in stationary‐phase (GASP) phenotype

The aim of this work was the functional and proteomic analysis of a mutant, W3110 Bgl⁺/10, isolated from a batch culture of an Escherichia coli K‐12 strain maintained at room temperature without addition of nutrients for 10 years. When the mutant was evaluated in competition experiments in co‐culture with the wild‐type, it exhibited the growth advantage in stationary phase (GASP) phenotype. Proteomes of the GASP mutant and its parental strain were compared by using a 2DE coupled with MS approach. Several differentially expressed proteins were detected and many of them were successful identified by mass spectrometry. Identified expression‐changing proteins were grouped into three functional categories: metabolism, protein synthesis, chaperone and stress responsive proteins. Among them, the prevalence was ascribable to the “metabolism” group (72%) for the GASP mutant, and to “chaperones and stress responsive proteins” group for the parental strain (48%).     

Inhibition of TANK binding kinase 1 by herpes simplex virus 1 facilitates productive infection

The γ(1)34.5 protein of herpes simplex viruses (HSV) is essential for viral pathogenesis, where it precludes translational arrest mediated by double-stranded-RNA-dependent protein kinase (PKR). Paradoxically, inhibition of PKR alone is not sufficient for HSV to exhibit viral virulence. Here we report that γ(1)34.5 inhibits TANK binding kinase 1 (TBK1) through its amino-terminal sequences, which facilitates viral replication and neuroinvasion. Compared to wild-type virus, the γ(1)34.5 mutant lacking the amino terminus induces stronger antiviral immunity. This parallels a defect of γ(1)34.5 for interacting with TBK1 and reducing phosphorylation of interferon (IFN) regulatory factor 3. This activity is independent of PKR. Although resistant to IFN treatment, the γ(1)34.5 amino-terminal deletion mutant replicates at an intermediate level between replication of wild-type virus and that of the γ(1)34.5 null mutant in TBK1(+/+) cells. However, such impaired viral growth is not observed in TBK1(-/-) cells, indicating that the interaction of γ(1)34.5 with TBK1 dictates HSV infection. Upon corneal infection, this mutant replicates transiently but barely invades the trigeminal ganglia or brain, which is a difference from wild-type virus and the γ(1)34.5 null mutant. Therefore, in addition to PKR, γ(1)34.5 negatively regulates TBK1, which contributes viral replication and spread in vivo.