Infected HeLa Cells Research Articles

The alkylation response protein AidB is localized at the new poles and constriction sites in Brucella abortus

BackgroundBrucella abortus is the etiological agent of a worldwide zoonosis called brucellosis. This alpha-proteobacterium is dividing asymmetrically, and PdhS, an essential histidine kinase, was reported to be an old pole marker.ResultsWe were interested to identify functions that could be recruited to bacterial poles. The Brucella ORFeome, a collection of cloned predicted coding sequences, was placed in fusion with yellow fluorescent protein (YFP) coding sequence and screened for polar localizations in B. abortus. We report that AidB-YFP was systematically localized to the new poles and at constrictions sites in B. abortus, either in culture or inside infected HeLa cells or RAW264.7 macrophages. AidB is an acyl-CoA dehydrogenase (ACAD) homolog, similar to E. coli AidB, an enzyme putatively involved in destroying alkylating agents. Accordingly, a B. abortus aidB mutant is more sensitive than the wild-type strain to the lethality induced by methanesulphonic acid ethyl ester (EMS). The exposure to EMS led to a very low frequency of constriction events, suggesting that cell cycle is blocked during alkylation damage. The localization of AidB-YFP at the new poles and at constriction sites seems to be specific for this ACAD homolog since two other ACAD homologs fused to YFP did not show specific localization. The overexpression of aidB, but not the two other ACAD coding sequences, leads to multiple morphological defects.ConclusionsData reported here suggest that AidB is a marker of new poles and constriction sites, that could be considered as sites of preparation of new poles in the sibling cells originating from cell division. The possible role of AidB in the generation or the function of new poles needs further investigation.

Mitogen-Activated Protein Kinase-Dependent Interleukin-1α Intracrine Signaling Is Modulated by YopP during Yersinia enterocolitica Infection

Yersinia enterocolitica is a food-borne pathogen that preferentially infects the Peyer's patches and mesenteric lymph nodes, causing an acute inflammatory reaction. Even though Y. enterocolitica induces a robust inflammatory response during infection, the bacterium has evolved a number of virulence factors to limit the extent of this response. We previously demonstrated that interleukin-1α (IL-1α) was critical for the induction of gut inflammation characteristic of Y. enterocolitica infection. More recently, the known actions of IL-1α are becoming more complex because IL-1α can function both as a proinflammatory cytokine and as a nuclear factor. In this study, we tested the ability of Y. enterocolitica to modulate intracellular IL-1α-dependent IL-8 production in epithelial cells. Nuclear translocation of pre-IL-1α protein and IL-1α-dependent secretion of IL-8 into the culture supernatant were increased during infection with a strain lacking the 70-kDa virulence plasmid compared to the case during infection with the wild type, suggesting that Yersinia outer proteins (Yops) might be involved in modulating intracellular IL-1α signaling. Infection of HeLa cells with a strain lacking the yopP gene resulted in increased nuclear translocation of pre-IL-1α and IL-1α-dependent secretion of IL-8 similar to what is observed with bacteria lacking the virulence plasmid. YopP is a protein acetylase that inhibits mitogen-activated protein kinase (MAP kinase)- and NF-κB-dependent signal transduction pathways. Nuclear translocation of pre-IL-1α and IL-1α-dependent secretion of IL-8 in response to Yersinia enterocolitica infection were dependent on extracellular signal-regulated kinase (ERK) and p38 MAP kinase signaling but independent of NF-κB. These data suggest that Y. enterocolitica inhibits intracellular pre-IL-1α signaling and subsequent proinflammatory responses through inhibition of MAP kinase pathways.

Construction of recombinant lentiviral vectors for mouse NMNAT1 gene expression and its interference RNA

To construct two recombinant lentiviral vectors carrying mouse NMNAT1 gene and RNAi targeting NMNAT1. According to GenBank, the full-length cDNA sequence of mouse NMNAT1, an interfering sequence targeting NMNAT1 and a negative sequence were designed, synthesized and inserted into plasmid pLenti6 lentiviral vector. The viral stock was prepared by cotransfection of plasmids and the packaging plasmid mix to 293T cells. The virus titer was tested by qPCR methods. After infection of Hela cells with these lentiviruses, the expression of NMNAT1 was detected by qPCR and Western blot. All the recombinant plasmids were confirmed by sequencing. The titer of virus was over 2 X10(8) TU/mL. Hela cells infected with lentiviral vector carrying full length NMNAT1 gene successfully expressed high-level NMNAT1. The expression of NMNAT1 reduced to less than 30% after delivery of lentiviral vector carrying RNAi sequence. The lentiviral vectors carrying full length NMNAT1 gene and RNAi sequence targeting NMNAT1 have been successfully constructed.

Persistent Chlamydia trachomatis Infection of HeLa Cells Mediates Apoptosis Resistance through a Chlamydia Protease-Like Activity Factor-Independent Mechanism and Induces High Mobility Group Box 1 Release

Intracellular persistence of Chlamydia trachomatis has been implicated in the development of chronic infection that can result in pelvic inflammatory disease and tubal sterility. By inhibition of host cell apoptosis, chlamydiae have evolved a strategy to maintain the intracellular environment for replication and persistence. Both antiapoptotic host cell-derived factors and the chlamydial protease-like activity factor (CPAF) are involved in Chlamydia-mediated apoptosis resistance. Here, we show that in HeLa cells infected with gamma interferon (IFN-γ)-induced persistent C. trachomatis serovar D, the expression of CPAF is downregulated, and proapoptotic protease substrates are not cleaved. Persistent infection protected HeLa cells from apoptosis when they were exposed to staurosporine. Small-interfering RNA-mediated inhibition of myeloid cell leukemia 1 (Mcl-1) protein upregulation sensitized persistently infected cells for apoptosis. The inhibitor of apoptosis protein 2 (IAP-2) seems not to be relevant in this context because IAP-2 protein was not induced in response to IFN-γ treatment. Although apoptosis was inhibited, persistent infection caused cell membrane disintegration, as measured by the increased release of cytokeratin 18 from HeLa cells. Moreover, persistently infected cells released significantly increased amounts of high mobility group box 1 (HMGB1) protein which represents a proinflammatory damage-associated pattern molecule. The data of this study suggest that cells infected with persistent C. trachomatis are protected from apoptosis independently of CPAF but may promote chronic inflammation through HMGB1 release.

Inhibition of Indoleamine 2,3-Dioxygenase Activity by Levo-1-Methyl Tryptophan Blocks Gamma Interferon-Induced Chlamydia trachomatis Persistence in Human Epithelial Cells

Gamma interferon (IFN-γ) induces expression of the tryptophan-catabolizing enzyme indoleamine 2,3-dioxygenase (IDO1) in human epithelial cells, the permissive cells for the obligate intracellular bacterium Chlamydia trachomatis. IDO1 depletes tryptophan by catabolizing it to kynurenine with consequences for C. trachomatis, which is a tryptophan auxotroph. In vitro studies reveal that tryptophan depletion can result in the formation of persistent (viable but noncultivable) chlamydial forms. Here, we tested the effects of the IDO1 inhibitor, levo-1-methyl-tryptophan (L-1MT), on IFN-γ-induced C. trachomatis persistence. We found that addition of 0.2 mM L-1MT to IFN-γ-exposed infected HeLa cell cultures restricted IDO1 activity at the mid-stage (20 h postinfection [hpi]) of the chlamydial developmental cycle. This delayed tryptophan depletion until the late stage (38 hpi) of the cycle. Parallel morphological and gene expression studies indicated a consequence of the delay was a block in the induction of C. trachomatis persistence by IFN-γ. Furthermore, L-1MT addition allowed C. trachomatis to undergo secondary differentiation, albeit with limited productive multiplication of the bacterium. IFN-γ-induced persistent infections in epithelial cells have been previously reported to be more resistant to doxycycline than normal productive infections in vitro. Pertinent to this observation, we found that L-1MT significantly improved the efficacy of doxycycline in clearing persistent C. trachomatis forms. It has been postulated that persistent forms of C. trachomatis may contribute to chronic chlamydial disease. Our findings suggest that IDO1 inhibitors such as L-1MT might provide a novel means to investigate, and potentially target, persistent chlamydial forms, particularly in conjunction with conventional therapeutics.

High-Throughput-Screening-Based Identification and Structure–Activity Relationship Characterization Defined (S)-2-(1-Aminoisobutyl)-1-(3-chlorobenzyl)benzimidazole as a Highly Antimycotic Agent Nontoxic to Cell Lines

Novel nontoxic (S)-2-aminoalkylbenzimidazole derivatives were found to be effective against Candida spp. at low micromolar concentrations using high-throughput screening with infected HeLa cells. A collection of analogues defined the chemical groups relevant for activity. The most active compound was characterized by transcriptional analysis of the response of C. albicans Sc5314. (S)-2-(1-Aminoisobutyl)-1-(3-chlorobenzyl)benzimidazole had a strong impact on membrane biosynthesis. Testing different clinically relevant pathogenic fungi showed the selectivity of the antimycotic activity against Candida species.

HIV-1 Infection Suppresses Expression of Host Cell Cycle-Associated Gene PDS5A

Objective: To unravel the interplay between HIV-1 and its host cell, the effect of HIV-1 infection on cellular gene expression was investigated. Methods: HIV-1_SF33-infected and uninfected H9 T cells were screened by differential display and RNase protection assay. The finding (PDS5A) was confirmed in HIV-1_Lai-infected P4-CCR5 HeLa cells, which were also examined after PDS5A siRNA knockdown in regard to HIV-1 replication by quantitative RT-PCR, p24 ELISA and LTR-driven β-galactosidase expression. The PDS5A knockdown effect on cellular gene expressions was studied by microarray analysis. PDS5A tissue expression was determined by Northern blotting. Results: Regulator of cohesion maintenance, homolog A (PDS5A) was found to be down-regulated by HIV-1. When PDS5A was suppressed by siRNA, HIV-1 replication was unaffected. PDS5A was found to be highly expressed in skeletal muscle tissue, and to lesser degrees in pancreas, heart, placenta, lung, kidney, liver and brain. Microarray analysis of PDS5A knockdown revealed 91 differential gene products over-representing cell cycle, transport and protein stability regulation, including 4 genes (PP2A, RANTES, PCAF, TCF7L2) previously reported to interact with HIV-1. Conclusion: The data show a downregulation of proliferation-associated host gene PDS5A and suggest a role of PDS5A in HIV-1-induced cellular pathogenesis but not viral replication.

The matrix (M) protein of newcastle disease virus binds to human bax through its BH3 domain

The underlying mechanisms by which Newcastle disease virus (NDV) kills cancer cells are still unclear. Recent discoveries have shown that many viruses contain Bcl-2 homology-like domains which enabled their interaction with Bcl-2 family members, and thereby accounting for their virulence and pathogenicity. Alignment of the protein sequences of Malaysian strain of NDV, known as AF2240, with those from members of the human Bcl-2 family showed many similar regions; most notably we found that its matrix (AF2240-M) protein, large (AF2240-L) protein and fusion (AF2240-F) protein all contain BH3-like regions. In addition, there are BH1-like domains in these proteins, where AF2240-F and Mcl-1 share 55% identity within this region. To further investigate our hypothesis that the presence of the BH3-like domains in these proteins may convey cytotoxicity, AF2240-M and AF2240-F genes were cloned into pFLAG and pEGFP.N2 vectors and transfected into HeLa cells. The expression of these constructs promoted cell death. As shown by flow cytometry, AF2240-M protein with deleted BH3-like region showed five-fold decrease in apoptosis. Moreover, the construct containing the N-terminal of AF2240-M showed nearly the same cell death rate as to that of the full-length protein, strongly suggesting that the BH3-like domain within this protein participates in promoting cell death. Moreover, AF2240-M transfection promoted Bax redistribution to mitochondria. Therefore, to determine whether there is any direct interaction between NDV viral proteins with some members of the Bcl-2 family, various constructs were co-transfected into HeLa cells. Co-immunoprecipitation trials showed that the AF2240-M indeed directly interacted with Bax protein via its BH3-domain, as the mutant proteins failed to interact with Bax. AF2240-F failed to interact with any of the tested proteins, although Bcl-XL slowed down the rate of cell death caused by this construct by nearly five-fold. In a parallel experiment, the level of expression of endogenous Bax and Bcl-2 after infection of HeLa cells with NDV was assessed by qRT-PCR, but no statistically significant change was observed. Consequently, the Bax/Bcl-2 ratio at the mRNA level did not alter. Overall, our study has shed additional light into the mechanisms by which NDV induces apoptosis.

Improved Expression Systems for Regulated Expression in Salmonella Infecting Eukaryotic Cells

In this work we describe a series of improvements to the Salmonella-based salicylate-inducible cascade expression system comprised of a plasmid-borne expression module, where target gene expression is driven by the Pm promoter governed by the XylS2 regulator, and a genome-integrated regulatory module controlled by the nahR/Psal system. We have constructed a set of high and low-copy number plasmids bearing modified versions of the expression module with a more versatile multiple cloning site and different combinations of the following elements: (i) the nasF transcriptional attenuator, which reduces basal expression levels, (ii) a strong ribosome binding site, and (iii) the Type III Secretion System (TTSS) signal peptide from the effector protein SspH2 to deliver proteins directly to the eukaryotic cytosol following bacterial infection of animal cells. We show that different expression module versions can be used to direct a broad range of protein production levels. Furthermore, we demonstrate that the efficient reduction of basal expression by the nasF attenuator allows the cloning of genes encoding highly cytotoxic proteins such as colicin E3 even in the absence of its immunity protein. Additionally, we show that the Salmonella TTSS is able to translocate most of the protein produced by this regulatory cascade to the cytoplasm of infected HeLa cells. Our results indicate that these vectors represent useful tools for the regulated overproduction of heterologous proteins in bacterial culture or in animal cells, for the cloning and expression of genes encoding toxic proteins and for pathogenesis studies.

Vaccinia extracellular virions enter cells by macropinocytosis and acid-activated membrane rupture

Vaccinia virus (VACV), the model poxvirus, produces two types of infectious particles: mature virions (MVs) and extracellular virions (EVs). EV particles possess two membranes and therefore require an unusual cellular entry mechanism. By a combination of fluorescence and electron microscopy as well as flow cytometry, we investigated the cellular processes that EVs required to infect HeLa cells. We found that EV particles were endocytosed, and that internalization and infection depended on actin rearrangements, activity of Na(+)/H(+) exchangers, and signalling events typical for the macropinocytic mechanism of endocytosis. To promote their internalization, EVs were capable of actively triggering macropinocytosis. EV infection also required vacuolar acidification, and acid exposure in endocytic vacuoles was needed to disrupt the outer EV membrane. Once exposed, the underlying MV-like particle presumably fused its single membrane with the limiting vacuolar membrane. Release of the viral core into the host cell cytosol allowed for productive infection.

Activation of Akt by the bacterial inositol phosphatase, SopB, is wortmannin insensitive.

Salmonella enterica uses effector proteins translocated by a Type III Secretion System to invade epithelial cells. One of the invasion-associated effectors, SopB, is an inositol phosphatase that mediates sustained activation of the pro-survival kinase Akt in infected cells. Canonical activation of Akt involves membrane translocation and phosphorylation and is dependent on phosphatidyl inositide 3 kinase (PI3K). Here we have investigated these two distinct processes in Salmonella infected HeLa cells. Firstly, we found that SopB-dependent membrane translocation and phosphorylation of Akt are insensitive to the PI3K inhibitor wortmannin. Similarly, depletion of the PI3K regulatory subunits p85α and p85ß by RNAi had no inhibitory effect on SopB-dependent Akt phosphorylation. Nevertheless, SopB-dependent phosphorylation does depend on the Akt kinases, PDK1 and rictor-mTOR. Membrane translocation assays revealed a dependence on SopB for Akt recruitment to Salmonella ruffles and suggest that this is mediated by phosphoinositide (3,4) P2 rather than phosphoinositide (3,4,5) P3. Altogether these data demonstrate that Salmonella activates Akt via a wortmannin insensitive mechanism that is likely a class I PI3K-independent process that incorporates some essential elements of the canonical pathway.

Comparison of rhinovirus A infection in human primary epithelial and HeLa cells

HeLa cells are used to study the life cycles of many different viruses, including the human rhinoviruses (HRV) in the family Picornaviridae. Although the natural targets of HRV are human bronchial epithelial cells (hBE), it is generally more difficult to obtain and maintain the relevant primary cell cultures, relative to HeLa cells. Given that the HRV are now identified as a major cause of human asthma exacerbations, it becomes important to document how much of the virus biology learned from HeLa cells is common also to natural primary cells. When compared directly in matched infections using A01a virus, the kinetics of RNA replication, the synthesis and processing of viral proteins and the general subcellular localization of key non-structural proteins were resembled in hBE and HeLa cells. Viral-induced shutoff of host cell processes (e.g. nucleo-cytoplasmic trafficking) was also comparable.

TRAF7 Protein Promotes Lys-29-linked Polyubiquitination of IκB Kinase (IKKγ)/NF-κB Essential Modulator (NEMO) and p65/RelA Protein and Represses NF-κB Activation

TRAF7 Protein Promotes Lys-29-linked Polyubiquitination of IκB Kinase (IKKγ)/NF-κB Essential Modulator (NEMO) and p65/RelA Protein and Represses NF-κB Activation

Canine distemper virus induces apoptosis in cervical tumor derived cell lines

Apoptosis can be induced or inhibited by viral proteins, it can form part of the host defense against virus infection, or it can be a mechanism for viral spread to neighboring cells. Canine distemper virus (CDV) induces apoptotic cells in lymphoid tissues and in the cerebellum of dogs naturally infected. CDV also produces a cytopathologic effect, leading to apoptosis in Vero cells in tissue culture. We tested canine distemper virus, a member of the Paramyxoviridae family, for the ability to trigger apoptosis in HeLa cells, derived from cervical cancer cells resistant to apoptosis. To study the effect of CDV infection in HeLa cells, we examined apoptotic markers 24 h post infection (pi), by flow cytometry assay for DNA fragmentation, real-time PCR assay for caspase-3 and caspase-8 mRNA expression, and by caspase-3 and -8 immunocytochemistry. Flow cytometry showed that DNA fragmentation was induced in HeLa cells infected by CDV, and immunocytochemistry revealed a significant increase in the levels of the cleaved active form of caspase-3 protein, but did not show any difference in expression of caspase-8, indicating an intrinsic apoptotic pathway. Confirming this observation, expression of caspase-3 mRNA was higher in CDV infected HeLa cells than control cells; however, there was no statistically significant change in caspase-8 mRNA expression profile. Our data suggest that canine distemper virus induced apoptosis in HeLa cells, triggering apoptosis by the intrinsic pathway, with no participation of the initiator caspase -8 from the extrinsic pathway. In conclusion, the cellular stress caused by CDV infection of HeLa cells, leading to apoptosis, can be used as a tool in future research for cervical cancer treatment and control.

Flavivirus NS4A-induced Autophagy Protects Cells against Death and Enhances Virus Replication

Flaviviruses include the most prevalent and medically challenging viruses. Persistent infection with flaviviruses of epithelial cells and hepatocytes that do not undergo cell death is common. Here, we report that, in epithelial cells, up-regulation of autophagy following flavivirus infection markedly enhances virus replication and that one flavivirus gene, NS4A, uniquely determines the up-regulation of autophagy. Dengue-2 and Modoc (a murine flavivirus) kill primary murine macrophages but protect epithelial cells and fibroblasts against death provoked by several insults. The flavivirus-induced protection derives from the up-regulation of autophagy, as up-regulation of autophagy by starvation or inactivation of mammalian target of rapamycin also protects the cells against insult, whereas inhibition of autophagy via inactivation of PI3K nullifies the protection conferred by flavivirus. Inhibition of autophagy also limits replication of both Dengue-2 and Modoc virus in epithelial cells. Expression of flavivirus NS4A is sufficient to induce PI3K-dependent autophagy and to protect cells against death; expression of other viral genes, including NS2A and NS4B, fails to protect cells against several stressors. Flavivirus NS4A protein induces autophagy in epithelial cells and thus protects them from death during infection. As autophagy is vital to flavivirus replication in these cells, NS4A is therefore also identified as a critical determinant of flavivirus replication.

Apoptotic and Antiapoptotic Activity of L Protein of Theiler's Murine Encephalomyelitis Virus

Cellular apoptosis induced by viral genes can play a critical role in determining virulence as well as viral persistence. This form of cell death has been of interest with respect to Theiler's murine encephalomyelitis virus (TMEV) because the GDVII strain and members of the GDVII subgroup are highly neurovirulent, while the DA strain and members of the TO subgroup induce a chronic progressive inflammatory demyelination with persistence of the virus in the central nervous system. The TMEV L protein has been identified as important in the pathogenesis of Theiler's virus-induced demyelinating disease (TMEV-IDD). We now show that DA L is apoptotic following transfection of L expression constructs or following DA virus infection of HeLa cells; the apoptotic activity depends on the presence of the serine/threonine domain of L, especially a serine at amino acid 57. In contrast, GDVII L has little apoptotic activity following transfection of L expression constructs in HeLa cells and is antiapoptotic following GDVII infection of HeLa cells. Of note, both DA and GDVII L cleave caspase-3 in BHK-21 cells, although neither implements the full apoptotic machinery in this cell type as manifested by the induction of terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) staining. The differences in apoptotic activities of DA and GDVII L in varied cell types may play an important role in TMEV subgroup-specific disease phenotypes.

HIV-1 clade C envelopes obtained from late stage symptomatic Indian patients varied in their ability towards relative CD4 usages and sensitivity to CCR5 antagonist TAK-779

The mechanism by which strictly CCR5 using HIV-1 clade C variants exacerbate disease progression in absence of coreceptor switch is not clearly known. We previously reported HIV-1 clade C envelopes (Env) obtained from late stage Indian patients with expanded coreceptor tropism. Here we compared such Envs (having expanded coreceptor tropism) with strictly CCR5 using Envs also obtained from late stage in their capacity to utilize CD4 and CCR5 for productive entry. We found that while envelopes with low CD4 dependence tend to infect primary CD4 + T cells better than those required optimum CD4 for entry, no significant association was found between low CD4 usage and infectivity of primary CD4 + T cells by Env-pseudotyped viruses and their sensitivity to CCR5 antagonist TAK-779. Interestingly, Envs that readily infected HeLa cells expressing low CD4 showed relative resistance to T20 indicating that conformational intermediates of these envelopes remained for a shorter period of time than is required for efficient inhibition by T20.

Molecular modeling, organ culture and reverse genetics for a newly identified human rhinovirus C.

A recently recognized human rhinovirus species C (HRV-C) is associated with up to half of HRV infections in young children. Here we propagated two HRV-C isolates ex vivo in organ culture of nasal epithelial cells, sequenced a new C15 isolate and developed the first, to our knowledge, reverse genetics system for HRV-C. Using contact points for the known HRV receptors, intercellular adhesion molecule-1 (ICAM-1) and low-density lipoprotein receptor (LDLR), inter- and intraspecies footprint analyses predicted a unique cell attachment site for HRV-Cs. Antibodies directed to binding sites for HRV-A and -B failed to inhibit HRV-C attachment, consistent with the alternative receptor footprint. HRV-A and HRV-B infected HeLa and WisL cells but HRV-C did not. However, HRV-C RNA synthesized in vitro and transfected into both cell types resulted in cytopathic effect and recovery of functional virus, indicating that the viral attachment mechanism is a primary distinguishing feature of HRV-C.

Engineered Picornavirus VPg-RNA Substrates: Analysis of a Tyrosyl-RNA Phosphodiesterase Activity

Using poliovirus, the prototypic member of Picornaviridae, we have further characterized a host cell enzymatic activity found in uninfected cells, termed “unlinkase,” that recognizes and cleaves the unique 5′ tyrosyl-RNA phosphodiester bond found at the 5′ end of picornavirus virion RNAs. This bond connects VPg, a viral-encoded protein primer essential for RNA replication, to the viral RNA; it is cleaved from virion RNA prior to its engaging in protein synthesis as mRNA. Due to VPg retention on nascent RNA strands and replication templates, but not on viral mRNA, we hypothesize that picornaviruses utilize unlinkase activity as a means of controlling the ratio of viral RNAs that are translated versus those that either serve as RNA replication templates or are encapsidated. To test our hypothesis and further characterize this enzyme, we have developed a novel assay to detect unlinkase activity. We demonstrate that unlinkase activity can be detected using this assay, that this unique activity remains unchanged over the course of a poliovirus infection in HeLa cells, and that unlinkase activity is unaffected by the presence of exogenous VPg or anti-VPg antibodies. Furthermore, we have determined that unlinkase recognizes and cleaves a human rhinovirus-poliovirus chimeric substrate with the same efficiency as the poliovirus substrate.

Viral Double-stranded RNAs from Vaccinia Virus Early or Intermediate Gene Transcripts Possess PKR Activating Function, Resulting in NF-κB Activation, When the K1 Protein Is Absent or Mutated

PKR is a potent antiviral molecule that can terminate infection by inhibiting protein synthesis and stimulating NF-κB activation and apoptosis. Originally, it was thought that only intermediate and late gene transcription produced double-stranded (ds) RNA to activate PKR during vaccinia virus (VACV) infection. The VACV E3 or K3 proteins squelch this effect by binding to either dsRNA or PKR. However, in the absence of the K1 protein, VACV infection activates PKR at very early times post-infection and despite the presence of E3 and K3. These data suggest that VACV infection induces PKR activation by a currently unknown mechanism. To determine this mechanism, cells were infected with K1L-containing or -deficient VACVs. By using conditions that limited the progression of the poxvirus replication cycle, we observed that early gene transcripts activated PKR in RK13 cells, identifying a new PKR-activating mechanism of poxvirus infection. Using a similar approach for HeLa cells, intermediate gene transcription was sufficient to activate PKR. RNA isolated from infected RK13 or HeLa cells maintained PKR-activating properties only when dsRNA was present. Moreover, viral dsRNA was directly detected in infected cells either by RT-PCR or immunofluorescent microscopy. Interestingly, dsRNA levels were higher in infected cells in which the K1 protein was nonfunctional. Only K1 proteins with PKR inhibitory function prevented downstream NF-κB activation. These results reveal a new PKR activation pathway during VACV infection, in which the K1 protein reduces dsRNA levels early in VACV infection to directly inhibit PKR and several of its downstream antiviral effects, thereby enhancing virus survival.