Stimulation of BK virus DNA replication by NFI family transcription factors

BK polyomavirus (BKV) establishes persistent, low-level, and asymptomatic infections in most humans and causes polyomavirus-associated nephropathy (PVAN) and other pathologies in some individuals. The activation of BKV replication following kidney transplantation, leading to viruria, viremia, and, ultimately, PVAN, is associated with immune suppression as well as inflammation and stress from ischemia-reperfusion injury of the allograft, but the stimuli and molecular mechanisms leading to these pathologies are not well defined. The replication of BKV DNA in cell cultures is regulated by the viral noncoding control region (NCCR) comprising the core origin and flanking sequences, to which BKV T antigen (Tag), cellular proteins, and small regulatory RNAs bind. Six nuclear factor I (NFI) binding sites occur in sequences flanking the late side of the core origin (the enhancer) of the archetype virus, and their mutation, either individually or in toto, reduces BKV DNA replication when placed in competition with templates containing intact BKV NCCRs. NFI family members interacted with the helicase domain of BKV Tag in pulldown assays, suggesting that NFI helps recruit Tag to the viral core origin and may modulate its function. However, Tag may not be the sole target of the replication-modulatory activities of NFI: the NFIC/CTF1 isotype stimulates BKV template replication in vitro at low concentrations of DNA polymerase-α primase (Pol-primase), and the p58 subunit of Pol-primase associates with NFIC/CTF1, suggesting that NFI also recruits Pol-primase to the NCCR. These results suggest that NFI proteins (and the signaling pathways that target them) activate BKV replication and contribute to the consequent pathologies caused by acute infection.     

BK virus large T antigen: interactions with the retinoblastoma family of tumor suppressor proteins and effects on cellular growth control.

BK virus (BKV) is a polyomavirus which infects a large percentage of the human population. It is a potent transforming agent and is tumorigenic in rodents. BKV DNA has also been found in human brain, pancreatic islet, and urinary tract tumors, implicating this virus in neoplastic processes. BKV T antigen (TAg) is highly homologous to simian virus 40 TAg, particularly in regions required for mitogenic stimulation and binding to tumor suppressor proteins, The experiments presented in this report show that BKV TAg can bind the tumor suppressor protein p53. BKV TAg also has the ability to bind to members of the retinoblastoma (pRb) family of tumor suppressor proteins both in vivo and in vitro. However, these interactions are detected only when large amounts of total protein are used, because the levels of BKV TAg normally produced from viral promoter-enhancer elements are too low to bind a significant amount of the pRb family proteins in the cell. The low levels of BKV TAg produced by the viral promoter elements are sufficient to affect the levels and the phosphorylation patterns of these proteins and to induce serum-independent growth in these cells. Additional events, however, are required for full transformation. These data further support the notion that BKV TAg can affect cellular growth control mechanisms and may in fact be involved in neoplastic processes.     

Host-Specific Replication of BK Virus DNA in Mouse Cell Extracts Is Independently Controlled by DNA Polymerase α-Primase and Inhibitory Activities

The activation of the human polyomavirus BK causes polyomavirus-associated nephropathy in immunocompromised humans. Studies of the virus have been restricted since the virus DNA replication is species specific. Cell-based and cell-free DNA replication systems, including the BK virus (BKV) monopolymerase DNA replication system using purified proteins, reproduce the species specificity (28). Therefore, the major host proteins comprising this assay, DNA polymerase α-primase (Pol-prim) and replication protein A (RPA), were intensively studied here. We demonstrate that Pol-prim plays a major role in the species specificity of BKV DNA replication. Both large subunits p180 and p68 of the enzyme complex have central functions in modulating the host specificity. Recently, an inhibitory activity of BKV DNA replication was described (C. Mahon, B. Liang, I. Tikhanovich, J. R. Abend, M. J. Imperiale, H. P. Nasheuer, and W. R. Folk, J. Virol. 83:5708-5717, 2009), but neither mouse Pol-prim nor mouse RPA diminishes cell-free BKV DNA replication. However, the inhibition of BKV DNA replication in mouse extracts depends on sequences flanking the core origin. In the presence of human Pol-prim, the inhibitory effect of mouse cell factors is abolished with plasmid DNAs containing the murine polyomavirus early promoter region, whereas the late enhancer region and the core origin are supplied from BKV. Thus, BKV replication is regulated by both Pol-prim, as a core origin species-specific factor, and inhibitory activities, as origin-flanking sequence-dependent factor(s).BK virus (BKV) is a human polyomavirus that was first isolated in the 1970s (15). Up to 90% of adults have serologic evidence of exposure to BKV, but in most humans the virus remains latent (25, 26). Almost all disease accompanied by BKV reactivation has been found in immunocompromised patients (22). In recent years, BKV has been associated with nephropathy (polyomavirus-associated nephropathy, or PVAN) in up to 10% of renal transplant patients. Once established, the disease results in allograft loss in 45 to 70% of the patients (18). Importantly, BKV preferentially replicates in human cells and less well in cells of other primates, and the virus is highly tumorigenic in rodents (21, 41, 44). This fact and the lack of sustainable viral replication in rodents or other convenient, experimental animal models have been an enormous setback to the study of PVAN.As with other members of the Polyomaviridae family, BKV virions are nonenveloped icosahedral particles with a diameter of 45 nm that contain a circular double-stranded DNA genome of 5.3 kb (1). In BKV and in other polyomaviruses, three genomic areas have been distinguished: (i) a noncoding control region including the origin of viral DNA replication, (ii) the early genes encoding large and small T antigens (TAgs), and (iii) the late genes which code for the capsid proteins VP-1, VP-2, and VP-3 and the agnoprotein (22).BKV DNA replication is similar to that of all other members of the Polyomaviridae family and requires only one viral protein, the multifunctional large TAg, whereas all other replication factors are supplied by the host (13, 14, 28, 39, 47). As the first step, TAg binds to the core origin, which contains the early palindrome, an AT-rich sequence, and the TAg binding site II, which consists of two pairs of G(G/A)GGC pentanucleotides. In the presence of ATP, TAg forms a double hexamer and partially melts the early palindrome (EP) and untwists the AT-rich sequence of the BKV core origin (5, 6, 14). Then the TAg double hexamers bidirectionally unwind the viral replication origin, which requires ATP hydrolysis. In the following process the two hexamers remain associated with each other, with the separated single-stranded DNA (ssDNA) threading through the hexameric channels (14). The viral core origin is sufficient to constitute a functional replication origin, but the presence of auxiliary domains increases its activity 5- to 100-fold in vivo (16, 30). After the viral TAg unwinds the core origin and its flanking sequences, replication protein A (RPA), the main eukaryotic ssDNA-binding protein, covers the resulting stretches of ssDNA, whereas topoisomerase I releases the resulting torsional stress and enhances initiation of DNA replication (5, 7, 43). Then, DNA polymerase α-primase (Pol-prim) is loaded onto this TAg-RPA-topoisomerase 1-DNA complex, yielding a functional initiation complex. In the following step, Pol-prim synthesizes short RNA primers at the origin, and these RNA primers are elongated by the DNA polymerase function of the enzyme complex (9, 35, 47). After a polymerase switch from Pol-prim to DNA polymerase δ (Pol δ) with the help of RPA, replication factor C (RFC), and proliferating cell nuclear antigen (PCNA), processive DNA synthesis is completed by Pol δ in association with PCNA, the sliding clamp, on the leading strand (38, 51, 54, 59). Lagging-strand synthesis is discontinuous, and multiple initiation events catalyzed by Pol-prim must take place. Again, after the elongation of the RNA primers by Pol-prim, DNA synthesis is switched to Pol δ, which then synthesizes the complete Okazaki fragments. The maturation of these Okazaki fragments requires the collaboration of RNase H, PCNA, flap endonuclease 1 (Fen-1), Pol δ, and DNA ligase I to establish a continuous strand also on the lagging strand (9, 19, 20, 51, 55).TAg functions in infected cells rely heavily on specific associations with host proteins; for example, TAg interacts with RPA, Pol-prim, and topoisomerase I to replicate viral DNA. Selective interactions with the host p180 and p48 subunits of Pol-prim were shown to be responsible for species-specific replication of simian virus 40 (SV40) and murine polyomavirus (mPyV) DNAs, respectively (8, 47, 50). The subunits of Pol-prim are highly conserved since 88, 80, 89, and 90% of the amino acids are identical between human and murine p180, p68, p58, and p48, respectively. Biochemical studies have shown that TAg interacts independently with all four subunits of Pol-prim (8, 12, 57). Moreover, the p180, p58, and p48 subunits of Pol-prim also physically bind to RPA (7, 11, 57). RPA and TAg binding sites in the Pol-prim complex are essential for SV40 DNA replication in vitro since the presence of an excess of these purified binding peptides diminishes viral DNA replication in vitro (52, 53). Interestingly, species specificity requires the viral origin of DNA replication, whereas physical protein-protein interactions of purified protein complexes are not host specific in the absence of viral origin DNA (29, 42).Consistent with other polyomaviruses, analyses of BKV TAg-dependent DNA replication recently revealed that BKV DNA cannot be replicated in murine cells and that cell extracts are able to mimic this behavior (28). Furthermore, a BKV DNA replication system with the purified human proteins Pol-prim, RPA, topoisomerase I, and BKV TAg was inhibited by murine extracts, whereas SV40 DNA replication was not. Further investigations revealed that the presence of inhibitory activities (IAs) in extracts from murine cells blocks BKV DNA replication at an early step of TAg-mediated unwinding of the BKV origin of replication. Detailed analyses using the BKV monopolymerase DNA replication system, which we report here, show that Pol-prim functions as a species-specific factor associated with core origin functions. In addition, we reveal that the inhibitory activities in murine extracts, which are associated with origin-flanking sequence-dependent factor(s), regulate BKV DNA replication in murine cell extracts in a Pol-prim-independent manner.     

Caveolar endocytosis is critical for BK virus infection of human renal proximal tubular epithelial cells

In recent years, BK virus (BKV) nephritis after renal transplantation has become a severe problem. The exact mechanisms of BKV cell entry and subsequent intracellular trafficking remain unknown. Since human renal proximal tubular epithelial cells (HRPTEC) represent a main natural target of BKV nephritis, analysis of BKV infection of HRPTEC is necessary to obtain additional insights into BKV biology and to develop novel strategies for the treatment of BKV nephritis. We coincubated HRPTEC with BKV and the cholesterol-depleting agents methyl beta cyclodextrin (MBCD) and nystatin (Nys), drugs inhibiting caveolar endocytosis. The percentage of infected cells (detected by immunofluorescence) and the cellular levels of BKV large T antigen expression (detected by Western blot analysis) were significantly decreased in both MBCD- and Nys-treated HPRTEC compared to the level in HRPTEC incubated with BKV alone. HRPTEC infection by BKV was also tested after small interfering RNA (siRNA)-dependent depletion of either the caveolar structural protein caveolin-1 (Cav-1) or clathrin, the major structural protein of clathrin-coated pits. BKV infection was inhibited in HRPTEC transfected with Cav-1 siRNA but not in HRPTEC transfected with clathrin siRNA. The colocalization of labeled BKV particles with either Cav-1 or clathrin was investigated by using fluorescent microscopy and image cross-correlation spectroscopy. The rate of colocalization of BKV with Cav-1 peaked at 4 h after incubation. Colocalization with clathrin was insignificant at all time points. These results suggest that BKV entered into HRPTEC via caveolae, not clathrin-coated pits, and that BKV is maximally associated with caveolae at 4 h after infection, prior to relocation to a different intracellular compartment.     

miR-582-5p Is Upregulated in Patients with Active Tuberculosis and Inhibits Apoptosis of Monocytes by Targeting FOXO1

Macrophage apoptosis is a host innate defense mechanism against tuberculosis (TB). In this study, we found that percentage of apoptotic cells in peripheral blood monocytes from patients with active TB was lower than that from healthy controls (p<0.001). To understand whether microRNAs can modulate apoptosis of monocytes, we investigated differentially expressed microRNAs in patients with active TB. miR-582-5p was mainly expressed in monocytes and was upregulated in patients with active TB. The apoptotic percentage of THP-1 cells transfected with miR-582-5p mimics was significantly lower than those transfected with negative control of microRNA mimics (p<0.001), suggesting that miR-582-5p could inhibit apoptosis of monocytes. To our knowledge, the role of miR-582-5p in regulating apoptosis of monocytes has not been reported so far. Systematic bioinformatics analysis indicated that FOXO1 might be a target gene for miR-582-5p and its 3′UTR contains potential binding sites for miR-582-5p. To determine whether miR-582-5p could influence FOXO1 expression, miR-582-5p mimics or negative control of microRNA mimics were transfected into THP-1 cells. RT-PCR and western blot analysis showed that the miR-582-5p could suppress both FOXO1 mRNA and protein expression. Co-transfection of miR-582-5p and FOXO1 3′UTR-luciferase reporter vector into cells demonstrated that significant decrease in luciferase activity was only found in reporter vector that contained a wild type sequence of FOXO1 3′UTR, suggesting that miR-582-5p could directly target FOXO1. In conclusion, miR-582-5p inhibited apoptosis of monocytes by down-regulating FOXO1 expression and might play an important role in regulating anti-M. tuberculosis directed immune responses.     

BK Virus as a Cofactor in the Etiology of Prostate Cancer in Its Early Stages

Prostate cancer has been projected to cause almost 10% of all male cancer deaths in the United States in 2007. The incidence of mutations in the tumor suppressor genes Rb1 and p53, especially in the early stages of the disease, is low compared to those for other cancers. This has led to the hypothesis that a human virus such as BK virus (BKV), which establishes a persistent subclinical infection in the urinary tract and encodes oncoproteins that interfere with these tumor suppressor pathways, is involved. Previously, we detected BKV DNA in the epithelial cells of benign and proliferative inflammatory atrophy ducts of cancerous prostate specimens. In the present report, we demonstrate that BKV is present at a much lower frequency in noncancerous prostates. Additionally, in normal prostates, T-antigen (TAg) expression is observed only in specimens harboring proliferative inflammatory atrophy and prostatic intraepithelial neoplasia. We further demonstrate that the p53 gene from atrophic cells expressing TAg is wild type, whereas tumor cells expressing detectable nuclear p53 contain a mix of wild-type and mutant p53 genes, suggesting that TAg may inactivate p53 in the atrophic cells. Our results point toward a role for BKV in early prostate cancer progression.     

BK virus-plasmid expression vector that persists episomally in human cells and shuttles into Escherichia coli.

We describe a novel expression vector, pBK TK-1, that persists episomally in human cells that can be shuttled into bacteria. This vector includes sequences from BK virus (BKV), the thymidine kinase (TK) gene of herpes simplex virus type 1, and plasmid pML-1. TK+-transformed HeLa and 143 B cells contained predominantly full-length episomes. There were typically 20 to 40 (HeLa) and 75 to 120 143 B vector copies per cell, although some 143 B transformants contained hundreds. Low-molecular-weight DNA from TK+-transformed cells introduced into Escherichia coli were recovered as plasmids that were indistinguishable from the input vector. Removal of selective pressure had no apparent effect upon the episomal status of pBK TK-1 molecules in TK+-transformed cells. BKV T antigen may play a role in episomal replication of pBK TK-1 since this viral protein was expressed in TK+ transformants and since a plasmid that contained only the BKV origin of replication was highly amplified in BKV-transformed human cells that synthesize BKV T antigen.     

Robust expression of p27Kip1 induced by viral infection is critical for antiviral innate immunity

Influenza A virus (IAV) infection regulates the expression of numerous host genes. However, the precise mechanism underlying implication of these genes in IAV pathogenesis remains largely unknown. Here, we employed isobaric tags for relative and absolute quantification (iTRAQ) to identify host proteins regulated by IAV infection. iTRAQ analysis of mouse lungs infected or uninfected with IAV showed a total of 167 differentially upregulated proteins in response to the viral infection. Interestingly, we observed that p27Kip1, a potent cyclin‐dependent kinase inhibitor, was markedly induced by IAV both at mRNA and protein levels through in vitro and in vivo studies. Furthermore, it was shown that innate immune signalling positively regulated p27Kip1 expression in response to IAV infection. Ectopic expression of p27Kip1 in A549 cells dramatically inhibited IAV replication, whereas, p27Kip1 knockdown significantly enhanced the virus replication. in vivo experiments demonstrated that p27Kip1 knockout (KO) mice were more susceptible to IAV than wild‐type (WT) mice: exhibiting higher viral load in lung tissue, faster body‐weight loss, reduced survival rate and more severe organ damage. Moreover, we found that p27Kip1 overexpression facilitated the degradation of viral NS1 protein, caused a dramatic STAT1 activation and promoted the expression of IFN‐β and several critical antiviral interferon‐stimulated genes (ISGs). Increased p27Kip1 expression also restricted infections of several other viruses. Conversely, IAV‐infected p27Kip1 KO mice exhibited a sharp increase in NS1 protein accumulation, reduced level of STAT1 activation and decreased expression of IFN‐β and the ISGs in the lung compared to WT animals. These findings reveal a key role of p27Kip1 in enhancing antiviral innate immunity.     

Mutations in the external loops of BK virus VP1 and urine viral load in renal transplant recipients

Polyomavirus‐associated nephropathy (PVAN) is a major complication that occurs after renal transplantation and is induced by reactivation of the human polyomavirus BK (BKV). The structure of the viral capsid protein 1 (VP1) is characterized by the presence of external loops, BC, DE, EF, GH, and HI, which are involved in receptor binding. The pathogenesis of PVAN is not well understood, but viral risk factors are thought to play a crucial role in the onset of this pathology. In an attempt to better understand PVAN pathogenesis, the BKV‐VP1 coding region was amplified, cloned, and sequenced from the urine of kidney transplant recipients who did, and did not, develop the pathology. Urine viral loads were determined by using real time quantitative PCR (Q‐PCR). Amino acid substitutions were detected in 6/8 patients, and 6/7 controls. The BC and EF loop regions were most frequently affected by mutations, while no mutations were found within the GH and HI loops of both patients and controls. Some mutations, that were exclusively detected in the urine of PVAN patients, overlapped with previously reported mutations, although a correlation between changes in amino acids and the development of PVAN was not found. Urine viral loads were higher than that of the proposed cut‐off loads for identification of patients that are at a high risk of developing PVAN (10⁷ copies/ml), both in the PVAN and control groups, thus confirming that urine viral load is not a useful predictive marker for the development of PVAN. J. Cell. Physiol. 222:195–199, 2010. © 2009 Wiley‐Liss, Inc.     

Novel pandemic influenza A (H1N1) virus infection modulates apoptotic pathways that impact its replication in A549 cells

It is not well-known whether apoptosis signaling affects influenza virus infection and reproduction in human lung epithelial cells. Using A549 cell line, we studied the relationship of some apoptosis-associated molecules with novel pandemic influenza A (H1N1) virus, A/California/04/2009. Infected cells displayed upregulated Fas ligand, activated FADD and caspase-8, and downregulated FLIP in the extrinsic apoptotic pathway. p53 expression increased and Bcl-X_L expression decreased in the intrinsic pathway. Expression of pre-apoptotic molecules (FasL, FADD, and p53) increased virus replication, while inhibition of activity of FADD, caspase-8 and caspase-3, and expression of anti-apoptotic proteins (FLIP and Bcl-X_L) decreased virus replication. p38, ERK and JNK from MAPK pathways were activated in infected cells, and inhibition with their inhibitors diminished virus replication. In the p38 superfamily, p38α expression increased viral RNA production, while expression of p38β and p38γ decreased. These data indicated that influenza virus induces apoptotic signaling pathways, which benefit virus replication.     

Human alpha-defensins inhibit BK virus infection by aggregating virions and blocking binding to host cells

BK virus (BKV) is a polyomavirus that establishes a lifelong persistence in most humans and is a major impediment to success of kidney grafts. The function of the innate immune system in BKV infection and pathology has not been investigated. Here we examine the role of antimicrobial defensins in BKV infection of Vero cells. Our data show that alpha-defensin human neutrophil protein 1 (HNP1) and human alpha-defensin 5 (HD5) inhibit BKV infection by targeting an early event in the viral lifecycle. HD5 treatment of BKV reduced viral attachment to cells, whereas cellular treatment with HD5 did not. Colocalization studies indicated that HD5 interacts directly with BKV. Ultrastructural analysis revealed HD5-induced aggregation of virions. HD5 also inhibited infection of cells by other related polyomaviruses. This is the first study to demonstrate polyomavirus sensitivity to defensins. We also show a novel mechanism whereby HD5 binds to BKV leading to aggregation of virion particles preventing normal virus binding to the cell surface and uptake into cells.     

Involvement of cytoskeletal components in BK virus infectious entry

Posttransplant reactivation of BK virus (BKV) in the renal allograft progresses to polyomavirus-associated nephropathy in 1% to 8% of kidney recipients. Graft dysfunction and loss in 30% to 45% of polyomavirus-associated nephropathy-affected patients are secondary to extensive tubular epithelial cell injury induced by the lytic replication of BKV. The early events in productive BKV infection are not thoroughly understood. We have previously shown that BKV enters cells by caveola-mediated endocytosis. In this report we examine the role of microfilaments and microtubules during early viral infection. Our results show that BKV infection of Vero cells is sensitive to nocodazole-induced disassembly of the microtubule network for the initial 8 hours following virus binding. In contrast, suppression of microtubule turnover with the stabilizing agent paclitaxel has no effect on BKV infectivity. Selective disassembly of the actin filaments with latrunculin A does not impede BKV infection, while inhibition of microfilament dynamics with jasplakinolide results in reduced numbers of viral antigen-positive cells. These data demonstrate that BKV, like other polyomaviruses, relies on an intact microtubule network during early infection. BKV, however, does not share the requirement with the closely related JC virus for an intact actin cytoskeleton during intracellular transport.     

Expression of viral early functions in rat 3Y1 cells infected with human papovavirus BK.

A plaque morphology mutant (pm-522) of BK virus (BKV) with a small deletion at map unit 0.72 can readily transform rat 3Y1 cells, but wild-type BKV (wt-501) cannot. We examined the expression of the viral early functions in BKV (wt-501 or pm-522)-infected 3Y1 cells within a 2-week period after infection, before foci of transformed cells became detectable, to know how the difference between the two BKVs occurs. After a high-multiplicity infection, comparable amounts of free viral DNA (forms I and II) were found by Southern blotting analyses to persist in the nuclei of the cells infected with wt and pm BKVs. Whereas the proportion of T antigen-positive cells, as revealed by the indirect immunofluorescence method with complement, remained at a level of 60% in pm BKV infection, the level of T antigen-positive cells in wt BKV infection decreased from the initial 45% to 1% on day 9. The results obtained by the immunoprecipitation analyses of radiolabeled proteins from the infected cells were consistent with the immunofluorescence data. Viral early mRNA was detectable on day 2 and increased on day 9 in pm BKV infection, but in wt BKV infection, the low level of early mRNA detected on day 2 disappeared on day 9. Cell DNA synthesis and cell growth were enhanced more in pm BKV infection than in wt BKV infection. The low level of viral DNA synthesis that occurred in the infected rat cells was more prominent in pm BKV infection than in wt BKV infection. These data indicate that the expression of viral early functions continued much longer in pm BKV-infected rat cells than in wt BKV-infected rat cells, where the expression was probably repressed soon after infection. Continued T antigen production directed by the unintegrated viral genomes appears to be required for efficient transformation of rat cells by BKV.     

Long noncoding RNA UCA1 regulates HCV replication and antiviral response via miR-145-5p/SOCS7/IFN pathway

Hepatitis C virus (HCV) infection involves a variety of viral and host factors, which leads to the dysregulation of number of relevant genes including long noncoding RNAs (LncRNAs). LncRNA urothelial carcinoma-associated 1 (UCA1) has been reported to be upregulated in HCV-infected individuals. In a bid to elucidate on the contribution of UCA1 on HCV replication, we infected Huh7.5 cells with cell culture-derived HCV and found that UCA1 expression was elevated in time- and dose-dependent manners. Functionally, UCA1 knockdown by siRNA upregulated interferon (IFN) responses, thereby increasing the expression of interferon-stimulating genes (ISGs), and subsequently suppressing HCV replication. Bioinformatics analysis and experimental results indicated that, functioning as competitive endogenous RNA, UCA1 could sponge microRNA (miR)-145-5p, which targeted suppressor of cytokine signaling 7 (SOCS7) mRNA and subsequently mediated SOCS7 silencing. Moreover, SOCS7 protein exerted an inhibitory effect on IFN responses, thereby facilitating HCV replication. Taken together, at first, our findings demonstrate that UCA1 can counteract the expression of miR-145-5p, thereby upregulating the level of SOCS7, and in turn leading to the suppression of antiviral response in Huh7.5 cells.     

Large T Antigen Promotes JC Virus Replication in G2-arrested Cells by Inducing ATM- and ATR-mediated G2 Checkpoint Signaling

Large T antigen (TAg) of the human polyomavirus JC virus (JCV) possesses DNA binding and helicase activities, which, together with various cellular proteins, are required for replication of the viral genome. We now show that JCV-infected cells expressing TAg accumulate in the G₂ phase of the cell cycle as a result of the activation of ATM- and ATR-mediated G₂ checkpoint pathways. Transient transfection of cells with a TAg expression vector also induced G₂ checkpoint signaling and G₂ arrest. Analysis of TAg mutants with different subnuclear localizations suggested that the association of TAg with cellular DNA contributes to the induction of G₂ arrest. Abrogation of G₂ arrest by inhibition of ATM and ATR, Chk1, and Wee1 suppressed JCV genome replication. In addition, abrogation of the G₂-M transition by Cdc2 depletion disabled Wee1 depletion-induced suppression of JCV genome replication, suggesting that JCV replication is facilitated by G₂ arrest resulting from G₂ checkpoint signaling. Moreover, inhibition of ATM and ATR by caffeine suppressed JCV production. The observation that oligodendrocytes productively infected with JCV in vivo also undergo G₂ arrest suggests that G₂ checkpoint inhibitors such as caffeine are potential therapeutic agents for JCV infection.     

Interaction between simian virus 40 large T antigen and insulin receptor substrate 1 is disrupted by the K1 mutation, resulting in the loss of large T antigen-mediated phosphorylation of Akt

The cellular kinase Akt is a key controller of cellular metabolism, growth, and proliferation. Many viruses activate Akt due to its beneficial effects on viral replication. We previously showed that wild-type (WT) simian virus 40 (SV40) large T antigen (TAg) inhibits apoptosis via the activation of PI3K/Akt signaling. Here we show that WT TAg expressed from recombinant adenoviruses in U2OS cells induced the phosphorylation of Akt at both T308 and S473. In contrast, Akt phosphorylation was eliminated by the K1 mutation (E107K) within the retinoblastoma protein (Rb) binding motif of TAg. This suggested that Akt phosphorylation may depend on TAg binding to Rb or one of its family members. However, in Rb-negative SAOS2 cells depleted of p107 and p130 by using small hairpin RNAs (shRNAs), WT TAg still mediated Akt phosphorylation. These results suggested that the K1 mutation affects another TAg function. WT-TAg-mediated phosphorylation of Akt was inhibited by a PI3K inhibitor, suggesting that the effects of TAg originated upstream of PI3K; thus, we examined the requirement for insulin receptor substrate 1 (IRS1), which binds and activates PI3K. Depletion of IRS1 by shRNAs abolished the WT-TAg-mediated phosphorylation of Akt. Immunoprecipitation studies showed that the known interaction between TAg and IRS1 is significantly weakened by the K1 mutation. These data indicate that the K1 mutation disrupts not only Rb binding but also IRS1 binding, contributing to the loss of activation of PI3K/Akt signaling.     

MicroRNA-221 modulates RSV replication in human bronchial epithelium by targeting NGF expression

Background

Early-life infection by respiratory syncytial virus (RSV) is associated with aberrant expression of the prototypical neurotrophin nerve growth factor (NGF) and its cognate receptors in human bronchial epithelium. However, the chain of events leading to this outcome, and its functional implications for the progression of the viral infection, has not been elucidated. This study sought to test the hypothesis that RSV infection modulates neurotrophic pathways in human airways by silencing the expression of specific microRNAs (miRNAs), and that this effect favors viral growth by interfering with programmed death of infected cells.

Methodology

Human bronchial epithelial cells infected with green fluorescent protein-expressing RSV (rgRSV) were screened with multiplex qPCR arrays, and miRNAs significantly affected by the virus were analyzed for homology with mRNAs encoding neurotrophic factors or receptors. Mimic sequences of selected miRNAs were transfected into non-infected bronchial cells to confirm the role of each of them in regulating neurotrophins expression at the gene and protein level, and to study their influence on cell cycle and viral replication.

Principal Findings

RSV caused downregulation of 24 miRNAs and upregulation of 2 (p<0.01). Homology analysis of microarray data revealed that 6 of those miRNAs exhibited a high degree of complementarity to NGF and/or one of its cognate receptors TrKA and p75^NTR. Among the selected miRNAs, miR-221 was significantly downregulated by RSV and its transfection in bronchial epithelial cells maximally inhibited gene and protein expression of NGF and TrKA, increased apoptotic cell death, and reduced viral replication and infectivity.

Conclusions/Significance

Our data suggest that RSV upregulates the NGF-TrKA axis in human airways by silencing miR-221 expression, and this favors viral replication by interfering with the apoptotic death of infected cells. Consequently, the targeted delivery of exogenous miRNAs to the airways may provide a new strategy for future antiviral therapies based on RNA interference.