Qimin Chen

IFP35 Is Involved in the Antiviral Function of Interferon by Association with the Viral Tas Transactivator of Bovine Foamy Virus

ABSTRACT Interferon-induced proteins (IFPs) exert multiple functions corresponding to diverse interferon signals. However, the intracellular functions of many IFPs are not fully characterized. Here, we report that IFP35, a member of the IFP family with a molecular mass of 35 kDa, can interact with the bovine Tas (BTas) regulatory protein of bovine foamy virus (BFV). The interaction involves NID2 (IFP35/Nmi homology domain) of IFP35 and the central domain of BTas. The overexpression of IFP35 disturbs the ability of BTas to activate viral-gene transcription and inhibits viral replication. The depletion of endogenous IFP35 by interfering RNA can promote the activation of BFV, suggesting an inhibitory function of IFP35 in viral-gene expression. In addition, IFP35 can interact with the homologous regulatory protein of prototype FV and arrest viral replication and repress viral transcription. Our study suggests that IFP35 may represent a novel pathway of interferon-mediated antiviral activity in host organisms that plays a role in the maintenance of FV latency.

Activation of c-Jun N-terminal kinase (JNK) pathway by HSV-1 immediate early protein ICP0.

Abstract The immediate early protein ICP0 encoded by herpes simplex virus 1 (HSV-1) is believed to activate transcription and consequently productive infection. The precise mechanisms of ICP0-mediated transactivation are under intensive study. Here, we demonstrate that ICP0 can strongly activate AP-1 responsive genes specifically. This activation is inhibited by c-Jun (S73A), c-Jun (S63/73A), TAK1 (K63W), but not by p38 (AF), ERK1 (K71R), ERK2 (K52R) and TRAF6 (C85A/H87A). We further investigate the relevancy of ERK, JNK and p38 MAPK pathways using their respective inhibitors PD98059, SP600125 and SB202190. Only SP600125 significantly attenuates the AP-1 responsive gene activation by ICP0. Consistent with these, the JNK is remarkably activated in response to ICP0, and this JNK activation is shown to be significantly attenuated by TAK1 (K63W). It turns out that ICP0 interacts specifically with TAK1 and stimulates its kinase activity. These findings reveal a new molecular mechanism ICP0 explores to regulate gene expression.

Microtubule-dependent retrograde transport of bovine immunodeficiency virus.

Microtubules are essential components of the cytoskeleton that participate in a variety of cellular processes such as cell division and migration. In addition, there is a growing body of evidence implicating a role for microtubules in intracellular viral transport. In this study, we found that pharmacological disruption of microtubules remarkably blocked bovine immunodeficiency virus (BIV) movement from the cell periphery to the perinuclear region, a process known as retrograde transport. A similar effect was observed by inhibiting function of the microtubule‐associated motor protein dynein. By yeast two‐hybrid assay, we found that the capsid protein (CA) of BIV interacted with the dynein light‐chain component LC8. Immunoprecipitation and GST‐pulldown assays further demonstrated an interaction between CA and LC8 in mammalian cells. In addition, our data revealed LC8 as a linker between BIV particles and microtubules. Retrograde transport of BIV was significantly inhibited by knockdown of LC8 expression. Our findings present the first evidence that incoming BIV particles employ host microtubule/dynein machinery for transport towards the perinuclear region. In addition, our data indicate that the LC8–CA interaction is a potential target for the design of antiviral strategies.

Regulation of Microtubule Assembly and Stability by the Transactivator of Transcription Protein of Jembrana Disease Virus

Microtubules are cytoskeletal polymers consisting of tubulin subunits that take part in diverse cell activities. Many viruses hijack cellular motor proteins to move on microtubules toward the cell interior during the entry process and toward the plasma membrane during the egress period. In addition, viruses often remodel microtubules to facilitate the generation of infectious progeny. In this study, we found that the transactivator of transcription protein of Jembrana disease virus (Jtat) bound tubulin and microtubules both in cells and in the purified system. Microtubule co-sedimentation and co-localization assays revealed a robust interaction of Jtat with microtubules. Tubulin turbidity assay further showed that Jtat promoted tubulin polymerization in vitro in a concentration-dependent manner. Moreover, Jtat promoted the partitioning of cellular tubulin toward the polymeric form, increased the level of tubulin acetylation, and significantly enhanced the cold stability of cellular microtubules. In addition, Jtat-mediated disruption of microtubule dynamics induced the release of Bim from microtubules, leading to profound apoptosis. These results not only identify Jtat as an important viral regulator of microtubule dynamics but also indicate that Jtat-induced apoptosis might contribute to Jembrana disease pathogenesis.

BTat, a trans-acting regulatory protein, contributes to bovine immunodeficiency virus-induced apoptosis

Bovine immunodeficiency virus (BIV) is a member of the lentivirus subfamily of retroviruses highly related to human immunodeficiency virus in morphologic, antigenic and genomic features. BIV is known to induce chronic pathological changes in infected hosts, which are often associated with the development of immune‐mediated lesions. However, the molecular events underlying the cytopathic effect of BIV remain poorly understood. In this study, BIV was found to induce apoptotic cell death, and a small trans‐acting regulatory protein encoded by BIV, BTat, was found to participate in the pro‐apoptotic action of BIV. Introduction of exogenous BTat to cells triggered apoptosis dramatically, as revealed by assays such as terminal deoxynucleotidyl transferase‐mediated dUTP nick‐end labelling, nuclear morphology analysis, flow cytometry, and cleavages of caspases and poly(ADP‐ribose)polymerase. Interestingly, the pro‐apoptotic effect of BTat was found to be mediated through its interaction with cellular microtubules and its interference with microtubule dynamics. These results provide the first evidence that induction of apoptosis may contribute to the cytopathic effect of BIV. In addition, these results uncover a novel role for BTat in regulating microtubule dynamics in addition to its conventional role in regulating gene transcription.

Dimerization of BTas is required for the transactivational activity of bovine foamy virus.

The BTas protein of bovine foamy virus (BFV) is a 249-amino-acid nuclear regulatory protein which transactivates viral gene expression directed by the long terminal repeat promoter (LTR) and the internal promoter (IP). Here, we demonstrate the BTas protein forms a dimeric complex in mammalian cells by using mammalian two hybrid systems and cross-linking assay. Functional analyses with deletion mutants reveal that the region of 46-62aa is essential for dimer formation. Furthermore, our results show that deleting the dimerization region of BTas did not affect the localization of BTas, but that it did result in the loss of its transactivational activity on the LTR and IP. Furthermore, BTas (Delta46-62aa) retained binding ability to the LTR and IP similar to that of the wild-type BTas. These data suggest the dimerization region is necessary for the transactivational function of BTas and is crucial to the replication of BFV.

A new indicator cell line established to monitor bovine foamy virus infection.

In order to improve the accuracy for quantitating the bovine foamy virus (BFV) in vitro, we developed a baby hamster kidney cell (BHK)-21-derived indicator cell line containing a plasmid that encodes the firefly luciferase driven by the BFV long terminal repeat promoter (LTR, from −7 to 1012). The BFV titer could be determined by detecting the luciferase expression since the viral trans-activator BTas protein activates the promoter activity of the LTR. One clone, designated BFVL, was selected from ten neomycin-resistant clones. BFVL showed a specific and inducible dose- and time-dependent luciferase activity in response to BFV infection. Although the changes in luciferase activity of BFVL peaked at 84 h post infection, it was possible to differentiate infected and uninfected cells at 48 h post infection. A linear relationship was established between the multiplicity of infection (MOI) of BFV and the activated ratio of luciferase expression in BFVL. Moreover, the sensitivity of the BFVL-based assay for detecting infectious BFV was 10,000 times higher than the conventional CPE-based assay at 48 h post infection. These findings suggest that the BFVL-based assay is rapid, easy, sensitive, quantitative and specific for detection of BFV infection.

Establishment of an indicator cell line to quantify Bovine Foamy Virus infection

A cell line derived from baby hamster kidney (BHK‐21) cells was transfected with the enhanced green fluorescent protein gene driven by the bovine foamy virus (BFV) long terminal repeat (LTR) to establish a BFV indicator cell line (BICL). Among 48 clones, one clone was chosen for its little constitutive enhanced green fluorescent protein (EGFP) expression and high level of EGFP expression after activation by BFV infection. By detecting the EGFP expression of the BFV indicator cell line, the titers of BFV were quantified by the end point method. As a result, the titer determined by the EGFP based assay 5–6 days post infection (d.p.i.) was 100 fold higher than traditional assays measuring cytopathic effects 8–9 d.p.i.. Moreover, the EGFP based assay was also used to determine the titer of those cells infected by BFV without inducing cytopathic effects. Using this simple and rapid assay, we examined the in vitro host range of BFV. It was found that BFV can productively infect various cell lines derived from bovine, human, rat and monkey. (© 2008 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)

The requirements and mechanism for capsid assembly and budding of bovine foamy virus

Summary.Little is known about assembly of non-primate foamy virus (FV) such as bovine foamy virus (BFV). To help determine the requirements for assembly of BFV, we constructed BFV-Gag expression plasmids containing all or part of the gag gene, with or without modification by addition of myristate (Myr). Each construct was transfected alone, and with pFenv, into Sf-9 insect cells. The results showed that only the entire Gag could transit through nucleus, which is required for BFV viral assembly in the cytoplasm. Unlike other retroviruses (but like primate foamy viruses), BFV requires the coexpression of the Env protein for viral particle budding. In the case of BFV, this occurs at the plasma membrane rather than the endoplasmic reticulum (ER), due to lack of a functional ER retrieval signal (ERRS). The results also showed that addition of a Myr-membrane targeting signal to the C-terminus of Gag could restore the budding from plasma membrane, implying that Myr-membrane targeting signal could substitute for Env protein in budding.

Guanine tetrad and palindromic sequence play critical roles in the RNA dimerization of bovine foamy virus

SummaryRetroviruses are unique in having a diploid genome. However, the RNA sequences and structures that link the two RNA molecules are different. To identify the dimer linkage site of bovine foamy virus (BFV), complementary DNAs were used to interfere with RNA dimerization of BFV. We found that two sites, designated SI and SII, within a 53-base RNA fragment, were essential for BFV dimerization in vitro. SI consists of a potential guanine tetrad (GGGGC), which overlaps the primer binding site, while SII contains 15 nucleotides including a palindromic sequence, UCCCUAGGGA. Masking either of the sites completely abolished RNA dimer formation. Furthermore, a deletion of SII was introduced into a BFV infectious DNA clone; we found that deletion of SII significantly increased expression of BFV transactivator Borf-1. Interestingly, we also found that this deletion abolished viral infectivity. These results suggest that dimerization might play a unique role in the BFV life cycle.