Heterogeneity and Breadth of Host Antibody Response to KSHV Infection Demonstrated by Systematic Analysis of the KSHV Proteome

<div><p>The Kaposi sarcoma associated herpesvirus (KSHV) genome encodes more than 85 open reading frames (ORFs). Serological evaluation of KSHV infection now generally relies on reactivity to just one latent and/or one lytic protein (commonly ORF73 and K8.1). Most of the other polypeptides encoded by the virus have unknown antigenic profiles. We have systematically expressed and purified products from 72 KSHV ORFs in recombinant systems and analyzed seroreactivity in US patients with KSHV-associated malignancies, and US blood donors (low KSHV seroprevalence population). We identified several KSHV proteins (ORF38, ORF61, ORF59 and K5) that elicited significant responses in individuals with KSHV-associated diseases. In these patients, patterns of reactivity were heterogeneous; however, HIV infection appeared to be associated with breadth and intensity of serological responses. Improved antigenic characterization of additional ORFs may increase the sensitivity of serologic assays, lead to more rapid progresses in understanding immune responses to KSHV, and allow for better comprehension of the natural history of KSHV infection. To this end, we have developed a bead-based multiplex assay detecting antibodies to six KSHV antigens.</p></div

Heatmap showing seroreactivity of HAMB and RDP subjects to each antigen.

<p>A. Subjects with documented KSHV-associated diseases. B. Healthy blood donors. Subjects are shown in rows, antigens in columns. Color intensity is proportional to background-subtracted optical density (OD). HIV seropositivity is indicated in red; latent KSHV proteins are identified in green.</p

Bead-based assay.

<p><b>A</b>. Comparison of reactivity to individual antigens in RDP and HAMB subjects (Mann-Whitney test) <b>B</b>. Receiver operating characteristics. <b>C</b>. Assessment of signal specificity by antigen pre-adsorption. MFI, Median Fluorescence Intensity.</p

Characteristics of study participants.

#<p>For all subjects, the United States is the country of current domicile, but not necessarily the country of birth; African regions described using United Nations designations.</p><p>*Patients had Kaposi sarcoma and/or primary effusion lymphoma and/or multicentric Castleman's disease in remission (not symptomatic); some had multiple diagnoses.</p>†<p>CD4 counts at time of serologic evaluation available on 33 of 36 HIV-infected subjects.</p

Identification of a Cryptic Bacterial Promoter in Mouse (<i>mdr1a</i>) P-Glycoprotein cDNA

<div><p>The efflux transporter P-glycoprotein (P-gp) is an important mediator of various pharmacokinetic parameters, being expressed at numerous physiological barriers and also in multidrug-resistant cancer cells. Molecular cloning of homologous cDNAs is an important tool for the characterization of functional differences in P-gp between species. However, plasmids containing mouse <i>mdr1a</i> cDNA display significant genetic instability during cloning in bacteria, indicating that <i>mdr1a</i> cDNA may be somehow toxic to bacteria, allowing only clones containing mutations that abrogate this toxicity to survive transformation. We demonstrate here the presence of a cryptic promoter in mouse <i>mdr1a</i> cDNA that causes mouse P-gp expression in bacteria. This expression may account for the observed toxicity of <i>mdr1a</i> DNA to bacteria. Sigma 70 binding site analysis and GFP reporter plasmids were used to identify sequences in the first 321 bps of <i>mdr1a</i> cDNA capable of initiating bacterial protein expression. An <i>mdr1a</i> M107L cDNA containing a single residue mutation at the proposed translational start site was shown to allow sub-cloning of <i>mdr1a</i> in <i>E</i>. <i>coli</i> while retaining transport properties similar to wild-type P-gp. This mutant <i>mdr1a</i> cDNA may prove useful for efficient cloning of <i>mdr1a</i> in <i>E</i>. <i>coli</i>.</p></div

Mutations and their position in <i>mdr1a</i> cDNA after transformation into <i>E</i>. <i>coli</i>.

<p>The pCR-Blunt-II-TOPO vector containing <i>mdr1a</i> cDNA was used to transform <i>E</i>.<i>coli</i> and 20 colonies were selected for small-scale bacterial growth, plasmid purification, and analysis of plasmid DNA by agarose gel electrophoresis. Sixteen of the colonies selected for analysis contained large insertion or deletion mutations (not shown), and the remaining four colonies containing potentially correct <i>mdr1a</i> cDNA were sequenced. Each of the four sequenced plasmids contained mutated <i>mdr1a</i> cDNA, indicating a 100% mutational rate. Mutations observed were classified as point, insertion, or deletion mutations. Wild-type <i>mdr1a</i> cDNA (top) is compared to sequenced, “mutant” <i>mdr1a</i> cDNA (bottom). Insertion and point mutations are indicated in red in the mutant cDNA. Deleted base pairs are highlighted in red in wild-type cDNA, and the resulting mutated cDNA sequence is shown below. A) A point mutation (C→T) at location 1027 bp resulting in an introduced stop codon into <i>mdr1a</i> cDNA. B) A single adenine base pair insertion at location 1033 bp. C) Two examples of deletion mutations. All mutations resulted either directly (point mutations) or indirectly (insertion or deletion mutations) in the introduction of a stop codon into <i>mdr1a</i> cDNA. Locations of the introduced stop codons are indicated.</p

Identification of putative transcriptional and translational start sites in <i>mdr1a</i> cDNA.

<p>An <i>in silico</i> sigma 70 binding site analysis was used to screen for a putative cryptic bacterial promoter in <i>mdr1a</i> cDNA. (A) Sequence of <i>mdr1a</i> cDNA shown with predicted -35 and extended -10 elements (red). The location in the cDNA for each element is noted above the sequence. (B) Predicted Shine-Dalgarno and ATG of a methionine that is in-frame with regards to the <i>mdr1a</i> ORF.</p

Schematic of GFP fusion constructs.

<p>GFP reporter constructs were generated to assess the ability of sequences of <i>mdr1a</i> cDNA to drive protein expression. Black lines represent DNA sequences fused upstream of GFP. GFP cDNA is represented in green and the presence of introduced RBSs are shown in blue where applicable. The p300-GFP plasmid contains the first 300 bps of <i>mdr1a</i> fused to GFP immediately preceded by a strong RBS. The p321-GFP plasmid contains the first 321 bps of <i>mdr1a</i> fused to GFP without the presence of an introduced RBS. pGFP and λpR-GFP were used as positive and negative control for GFP expression, respectively. p321-GFP and p-321-M107L-GFP are equivalent with the exception of the methionine to leucine mutation at position 107 in the amino acid sequence.</p

Fluorescence of <i>E</i>. <i>coli</i> transformed with GFP fusion plasmids.

<p>(A) Confocal images of <i>E</i>. <i>coli</i> transformed with GFP fusion plasmids. From left to right, columns represent fluorescence, bright field, and a merged image. Yellow bars indicate 20 μm. (B) Fluorescence of <i>E</i>. <i>coli</i> transformed with GFP fusion plasmids. Fluorescence spectroscopy was used to determine levels of GFP fluorescence in bacteria transformed with GFP plasmids. Confocal images and fluorescence spectroscopy values for bacteria transformed with λpR-GFP plasmids were omitted due to the signal saturation resulting from high GFP fluorescence. Data represent the mean and standard deviation of five individual colonies from one transformation event. Multiple groups were analyzed using one-way ANOVA where significance was defined as p < 0.05 in GraphPad Prism.</p

Mouse P-gp model in apo (open) conformation with methionine residue at position 107 highlighted.

<p>(A) Mouse P-gp is shown with the methionine to leucine mutation highlighted in TM1-ECL1 by the presence of a space filled leucine residue. The region of the drug-binding pocket (DBP) is indicated. (B) Enlarged view of M107L mutation. Figures were generated using PyMOL from PDB deposit 4M1M [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0136396#pone.0136396.ref016" target="_blank">16</a>].</p