The Effect of Epstein-Barr Virus Latent Membrane Protein 2 Expression on the Kinetics of Early B Cell Infection and Immortalization

Epstein-Barr virus (EBV) has been associated with the development of several human malignancies. Infection of B cells with wild-type EBV in vitro leads to activation and proliferation that result in efficient production of lymphoblastoid cell lines (LCLs). The majority of latent genes are expressed during early infection, including latent membrane protein 2 (LMP2). Currently, the role of LMP2 in B cell proliferation is controversial; some studies have shown that LMP2 is dispensable, while others report it is important role for this process. However, each of these experimental systems were limited by either wild-type virus contamination or use of incomplete viral genomes (mini-EBV), which precluded clear assessments of the effects of LMP2 during early infection. In this study I investigated the effect of LMP2 on early B cell infection and subsequent immortalization via complete recombinant EBV with knockouts of either or both isoforms of the LMP2 gene, LMP2A and LMP2B (Δ2A, Δ2B and Δ2A/Δ2B). Infection of B cells with LMP2A knockout viruses resulted in marked decreases in activation and proliferation relative to wild-type, and higher percentages of apoptotic B cells. Δ2B virus infection exhibited activation levels comparable to wild-type, but with fewer numbers of proliferating B cells. The stability of viral latency was determined for early B cell infection by evaluating latent and lytic gene expression with or without lytic stimulation via the B cell receptor (BCR). Infection with wild-type, Δ2A and Δ2B viruses with or without BCR stimulation did not result in changes in viral latency, whereas stimulation of BCR signaling in Δ2A/Δ2B-infected cells resulted in decreased LMP1 expression, suggesting loss of stability in viral latency. The long-term effects of LMP2 deletion on B cell outgrowth were investigated using LCL establishment assays, which revealed that LMP2A, but not LMP2B, is critical for efficient immortalization of B cells in vitro. Loss of both isoforms promoted the least activation, proliferation and LCL formation. This study enhances our knowledge of events required for B cell transformation by EBV, and clearly shows the public health relevance of understanding genes involved in tumorigenesis for the future pursuit of more effective treatments for EBV-associated malignancies

The Effect of Epstein-Barr Virus Latent Membrane Protein 2 Expression on the Kinetics of Early B Cell Infection

Infection of human B cells with wild-type Epstein-Barr virus (EBV) in vitro leads to activation and proliferation that result in efficient production of lymphoblastoid cell lines (LCLs). Latent Membrane Protein 2 (LMP2) is expressed early after infection and previous research has suggested a possible role in this process. Therefore, we generated recombinant EBV with knockouts of either or both protein isoforms, LMP2A and LMP2B (Δ2A, Δ2B, Δ2A/Δ2B) to study the effect of LMP2 in early B cell infection. Infection of B cells with Δ2A and Δ2A/Δ2B viruses led to a marked decrease in activation and proliferation relative to wild-type (wt) viruses, and resulted in higher percentages of apoptotic B cells. Δ2B virus infection showed activation levels comparable to wt, but fewer numbers of proliferating B cells. Early B cell infection with wt, Δ2A and Δ2B viruses did not result in changes in latent gene expression, with the exception of elevated LMP2B transcript in Δ2A virus infection. Infection with Δ2A and Δ2B viruses did not affect viral latency, determined by changes in LMP1/Zebra expression following BCR stimulation. However, BCR stimulation of Δ2A/Δ2B cells resulted in decreased LMP1 expression, which suggests loss of stability in viral latency. Long-term outgrowth assays revealed that LMP2A, but not LMP2B, is critical for efficient long-term growth of B cells in vitro. The lowest levels of activation, proliferation, and LCL formation were observed when both isoforms were deleted. These results suggest that LMP2A appears to be critical for efficient activation, proliferation and survival of EBV-infected B cells at early times after infection, which impacts the efficient long-term growth of B cells in culture. In contrast, LMP2B did not appear to play a significant role in these processes, and long-term growth of infected B cells was not affected by the absence of this protein. © 2013 Wasil et al

Control of Phage Bxb1 Excision by a Novel Recombination Directionality Factor

Mycobacteriophage Bxb1 integrates its DNA at the attB site of the Mycobacterium smegmatis genome using the viral attP site and a phage-encoded integrase generating the recombinant junctions attL and attR. The Bxb1 integrase is a member of the serine recombinase family of site-specific recombination proteins and utilizes small (<50 base pair) substrates for recombination, promoting strand exchange without the necessity for complex higher order macromolecular architectures. To elucidate the regulatory mechanism for the integration and excision reactions, we have identified a Bxb1-encoded recombination directionality factor (RDF), the product of gene 47. Bxb1 gp47 is an unusual RDF in that it is relatively large (˜28 kDa), unrelated to all other RDFs, and presumably performs dual functions since it is well conserved in mycobacteriophages that utilize unrelated integration systems. Furthermore, unlike other RDFs, Bxb1 gp47 does not bind DNA and functions solely through direct interaction with integrase–DNA complexes. The nature and consequences of this interaction depend on the specific DNA substrate to which integrase is bound, generating electrophoretically stable tertiary complexes with either attB or attP that are unable to undergo integrative recombination, and weakly bound, electrophoretically unstable complexes with either attL or attR that gain full potential for excisive recombination

. DNA-Binding Properties of Bxb1 gp47

<p>(A and B) The ability of gp47 to bind <i>attP, attB, attL,</i> and <i>attR</i> was determined using native gel electrophoresis. Binding of gp47 or gpInt to <i>attP</i> and <i>attB</i> (A) or <i>attL</i> and <i>attR</i> (B) was performed with either gpInt (0.072 μM), or increasing concentrations of gp47 (0.45, 0.89, 1.78, and 3.56 μM). </p

Substrate-Dependent Interaction of Bxb1 gpInt and gp47

<div><p>(A and B) Binding of gpInt and gp47 to <i>attP</i>/ <i>attB</i>/ <i>attL</i>/ <i>attR</i> was monitored by native gel electrophoresis. Radiolabeled DNA fragments (̃300 bp) were incubated with either gpInt (0.072 μM) alone or gpInt with increasing concentrations of gp47 (0.45, 0.89, 1.78, and 3.56 μM). The positions of DNA–gpInt complexes (cmplx I) as well as tertiary complexes containing DNA, gpInt, and gp47 (cmplx II) are shown. </p> <p>(C and D) The presence of gp47 in the tertiary complexes shown in (A) was determined by the ability of α-His antibodies to supershift complexes observed by native gel electrophoresis. α-His antibodies were either added to reactions containing DNA, gpInt, and gp47 (indicated as lane 1), or first preincubated with gp47 for 30 min and then added to reactions containing DNA and gpInt (lane 2). The protein–DNA complexes were separated from free DNA on a 5% native PAGE. The positions of the tertiary complexes of gp47, gpInt, and DNA as well as the antibody supershifted complexes are indicated.</p> <p>(E and F) Bxb1 gp47 is required for trapping a synaptic complex in excision. A suicide substrate version of <i>attL</i> DNA (5′ radiolabeled at both ends) was used that has a nick on the top strand positioned four bases to the 5′ side of the scissile bond. Bxb1 gpInt (72 nM) binds normally to this substrate to form Complex I (cmplx I), but when <i>attR</i> partner DNA (200 bp) and gp47 (3.56 μM) is added, no recombinant products are released. Instead, a prominent slow-moving complex is observed that absolutely requires Bxb1 gp47 for its formation. We have identified this as a synaptic complex using 2D-PAGE (F). In brief, a vertical gel slice was removed from the last lane in panel E, incubated with proteinase K and SDS, and then electrophoresed through a second dimension. Approximately 50% of the radiolabeled DNAs in this complex correspond to <i>attP</i> recombinant product and 50% correspond to a cleaved half-site. The bottom of the gel slice containing unbound <i>attL</i> DNA was removed prior to the second dimension of electrophoresis. Further details on the characterization of these suicide substrates will be described in future publications. </p></div

Confirmation of Site-Specific Recombination and Bxb1 Integrase Dependence

<div><p>(A) DNA from five sucrose-resistant transformants of the excision tester strain transformed with either pPG1 (empty vector), pPGX1, or pPGX6b was examined by PCR for the presence of <i>attB, attL,</i> and <i>attR</i>. Transformation with pPGX1 and pPGX6b leads to the presence of a product amplified with <i>attB</i>-specific primers; no product is observed using primers that amplify <i>attL</i> and <i>attR.</i> DNA from a Bxb1 lysogen and from clones transformed with the empty vector that were used as controls show a product corresponding to <i>attL</i> and <i>attR,</i> whereas DNA from wild-type <i>mc²155 </i> shows the presence of a product corresponding to <i>attB</i>. </p> <p>(B) An <i>int⁻</i> excision tester strain was created (as in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0040186#pbio-0040186-g001" target="_blank">Figure 1</a>A) using transient expression of gpInt. The int⁻ tester strain was then transformed with pAIK5 (gpInt alone), pX6b (gp47 alone), or pAIK5+47 (gpInt + gp47), and the frequency of suc^R colonies (and therefore excision) determined. </p> <p>CFU, colony-forming units</p></div

In Vitro Excisive Recombination Using gp47

<div><p>(A) E. coli BL21(DE3)pLysS transformed with pET28a and pPGgp47 were grown to an A₆₀₀ of 0.6 at 30 °C and induced for an additional 4 h at 22 °C with 0.6 mM IPTG. The cells were lysed in lysis buffer (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0040186#s3" target="_blank">Materials and Methods</a>) and partially purified by passage through a Ni-NTA column followed by elution with 150 mM imidazole; I, S, Ft, W, and E represent the insoluble fraction, soluble fraction, flow-through from the Ni-NTA column, washes with the indicated concentration of imidazole, and 150 mM elution from the Ni-NTA column, respectively. The induced cells of pPGgp47 show the presence of an approximately 32-kDa protein (as indicated) that is absent from the pET28a expression lanes and is abundant in the insoluble fraction. Molecular weight markers are shown in lane M and their corresponding sizes indicated. </p> <p>(B) Integrative recombination was performed as described previously using a supercoiled <i>attP</i> substrate, a linear 50-bp <i>attB</i> DNA, and increasing concentrations of gpInt [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0040186#pbio-0040186-b034" target="_blank">34</a>]: panel a, in the absence of any additional protein; panel b, in the presence of partially purified gp47; panel c, with addition of a control extract. Lanes 1–5 contain 0.36, 0.18, 0.09, 0.045, and 0.0225 μM of gpInt respectively. Panels b and c contain 1.78 μM of gp47 and an equivalent amount of the control extract respectively, in addition to gpInt. The positions of the supercoiled substrate and the linear recombinant product are indicated. The small (50 bp) linear <i>attB</i> substrate migrates fast and is not shown. </p> <p>(C) Excisive recombination was carried out in recombination buffer (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0040186#s3" target="_blank">Materials and Methods</a>) using a 367-bp <i>attL</i> in a supercoiled plasmid and a 377-bp linear <i>attR</i> partner DNA. Lanes 1–5 of panel a contain increasing concentrations of gp47 (0.89, 1.78, 2.67, 3.56, and 5.34 μM), lanes 7–11 of panel b contain an equivalent amount of the control protein. Control reactions lacking either the partner <i>attR</i> DNA (lane12), gp47 (lane 13), or gpInt (lane14) are shown in panel c. The positions of the supercoiled <i>attL</i> substrate and the linear recombinant product are indicated. </p> <p>(D) Excisive recombination reactions with varying sizes of linear DNA substrates show that only small substrate sites are required. Panel a shows recombination between a supercoiled plasmid containing a 367-bp <i>attL</i> and varying sizes of linear <i>attR</i> partner DNA as indicated. Panel b shows recombination between a supercoiled plasmid containing a 377-bp <i>attR</i> and varying sizes of linear <i>attL</i> partner DNAs. The positions of supercoiled substrates and linear recombinant products are indicated. </p></div

Identification of the Bxb1 RDF

<div><p>(A) The hygromycin-resistance cassette, sucrose-sensitivity cassette <i>(sacB),</i> and a portion of the Bxb1 DNA containing <i>attP</i> and <i>int</i> were cloned into an integrative plasmid containing the plasmid ColE1 origin of E. coli (OriE) <i>,</i> and the resultant plasmid pPGA1 was transformed into M. smegmatis mc²155. Expression of <i>int</i> drives integration of the plasmid into the host <i>attB</i> site leading to the formation of <i>attL</i> and <i>attR</i> sites flanking the <i>hyg</i> and <i>sacB</i> cassettes. The resultant strain, designated as the excision tester strain, is resistant to hygromycin and sensitive to the presence of sucrose. An excisive recombination event between <i>attL</i> and <i>attR</i> results in the removal (and subsequent loss) of the intervening DNA containing <i>int, hyg,</i> and <i>sacB;</i> the strain consequently becomes <i>hyg^S</i> and <i>suc^R</i> and can thereby be monitored by the appearance of colonies in the presence of sucrose. </p> <p>(B) A segment of the Bxb1 genome containing genes involved in integration and DNA replication as well as the corresponding portion of the phage L5 genome is shown in the upper part; related genes are colored accordingly. The lower part of the figure shows the region of Bxb1 DNA present in plasmids exhibiting excision activity. Following isolation of plasmid pPGX1—which is active in promoting sucrose resistance in the excision tester strain shown in (A)—plasmid derivatives containing deletions from both ends of pPGX1 were constructed, introduced into the excision tester strain, and scored for the appearance of <i>suc^R</i> colonies. </p> <p>CFU, colony-forming units</p></div

Confirmation of Bxb1 gp47 as the Phage RDF

<div><p>(A) The solid bar shows the portion of Bxb1 DNA from 35,976–36,811 bp contained within pPGX6b with the regions flanking gene <i>47</i> in dark grey. Truncation derivatives of pPGX6b were constructed as shown and tested for excision activity as in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0040186#pbio-0040186-g001" target="_blank">Figure 1</a>. The number of sucR colonies obtained upon transformation of the excision tester strain with each of the derivatives is shown. </p> <p>(B) Plasmid pPGX6b was randomly mutagenized by passage through a mutator strain, and excision defective mutants were selected (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0040186#pbio-0040186-g001" target="_blank">Figure 1</a>). The positions of 20 excision-defective mutants are shown as solid vertical lines (red), all of which lie within gene <i>47</i>. Arrows indicate ORFs in all six reading frames of pPGX6b, with Bxb1 gene <i>47</i> shown in red. </p> <p>(C) The amino acid changes in Bxb1 gp47 corresponding to each of the 15 base changes in pPGX6b are shown; the five nonsense substitutions are marked with an asterisk (*).</p> <p>(D) The locations of five nonsense mutations inactive in excision are shown using solid vertical lines. An excision tester strain previously transformed with plasmids expressing gp47 (wild type) or either of the two nonsense mutations (Q154Am or W85Op) was transformed with plasmids expressing nonsense suppressors generated by modification of tRNAs encoded by mycobacteriophage L5 [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0040186#pbio-0040186-b041" target="_blank">41</a>] (as indicated) and scored for the appearance of sucR colonies; the numbers of sucR colonies are shown. </p> <p>CFU, colony-forming units</p></div