Activation of CCR2+ human proinflammatory monocytes by human herpesvirus-6B chemokine N-terminal peptide

Abstract

Human monocytes expressing CCR2 with CD14 and CD16 can mediate antigen presentation, and promote inflammation, brain infiltration and immunosenescence. Recently identified roles are in human immunodeficiency virus infection, tuberculosis and parasitic disease. Human herpesvirus 6B (HHV-6B) encodes a chemokine, U83B, which is monospecific for CCR2, and is distinct from the related HHV-6A U83A, which activates CCR1, CCR4, CCR5, CCR6 and CCR8 on immune effector cells and dendritic cells. These differences could alter leukocyte-subset recruitment for latent/lytic replication and associated neuroinflammatory pathology. Therefore, cellular interactions between U83A and U83B could help dictate potential tropism differences between these viruses. U83A specificity is maintained in the 38-residue N-terminal spliced-truncated form. Here, we sought to determine the basis for the chemokine receptor specificity differences and identify possible applications. To do this we first analysed variation in a natural host population in sub-Saharan Africa where both viruses are equally prevalent and compared these to global strains. Analyses of U83 N-terminal variation in 112 HHV-6A and HHV-6B infections identified 6/ 38 U83A or U83B-specific residues. We also identified a unique single U83A-specific substitution in one U83B sequence, 'U83BA'. Next, the variation effects were tested by deriving N-terminal (NT) 17-mer peptides and assaying activation of ex vivo human leukocytes, the natural host and cellular target. Chemotaxis of CCR2+ leukocytes was potently induced by U83B-NT, but not U83BA-NT or U83A-NT. Analyses of the U83B-NT activated population identified migrated CCR2+, but not CCR5+, leukocytes. The U83BA-NT asparagine-lysine14 substitution disrupted activity, thus defining CCR2 specificity and acting as a main determinant for HHV-6A/B differences in cellular interactions. A flow-cytometry-based shape-change assay was designed, and used to provide further evidence that U83B-NT could activate CCR2+CD14+CD16+ monocytes. This defines a potential antiviral target for HHV-6A/B disease and novel peptide immunomodulator for proinflammatory monocytes.

Analyses of germline, chromosomally integrated human herpesvirus 6A and B genomes indicate emergent infection and new inflammatory mediators

“…The U83 chemokine gene is distinct between HHV-6A and HHV-6B strains, encoding up to 13 % amino acid differences (French et al, 1999). The HHV-6A (U83A) and HHV-6B (U83B) chemokines have distinct specificities which determine chemoattraction or diversion of different leukocyte subsets for infection or immune evasion, thus an early component of cellular tropism as well as mediator of innate immunity (Catusse et al, 2007(Catusse et al, , 2009Clark et al, 2013;Dewin et al, 2006;Lüttichau et al, 2003). U83 also has a varied gene structure, with N-terminal length variation determining production of the encoded mature secreted chemokine, coupled with control by cell-directed splicing which truncates the chemokine gene early in replication to encode an antagonist (Dewin et al, 2006).…”

“…Alternatively, pending an appropriate transcript secondary to the first initiating codon or leaky scanning of the ribosome from an adequate but less than perfect initiating codon (23G, no +1G) to a favoured one (23G, +1G) (GAAATGT to GTTATGG) (Kozak, 1989(Kozak, , 1995, this could result in utilization of the second internal in-frame initiating methionine codon. Even under these conditions, this would encode a product that is not secreted, hence inactive, or only very inefficiently secreted, since the encoded signal sequence is poorly predicted to be utilized, termed here the 'short' form of U83A or U83B (Clark et al, 2013;Dewin et al, 2006;Sjahril et al, 2009) (see virus reference sequences in Figs 1, 2 and S1, available in the online Supplementary Material).…”

“…The gene structure of the U83 chemokine gene was further examined in the HHV-6A/B virus infections from Zambia, since to date this is the only region where natural HHV-6A infant infections are equally prevalent with HHV-6B infections (Bates et al, 2009;Clark et al, 2013;Kasolo et al, 1997) and, therefore, the structure of virus U83A in particular can be examined here. Fig.…”

“…DNA extracted from blood, except sample 21846 from cerebrospinal fluid (CSF). monocytic and dendritic cells expressing the CCL2 receptor CCR2 (Bates et al, 2009;Clark et al, 2013;Niu & Kolattukudy, 2009). These effects on innate immunity may underlie other intracellular cardiotropic pathogens such as parvovirus B19 (Tzoulaki et al, 2013).…”

“…3 U83RP1 59-TGCCATATCACACATCGAG-39; and U51 with: IU51SF1 59-GTCAATACGGATGGGGTTTTG-39) and IU51SR2 59-CAGCGC-CGAAGATCTATTCT-39 (all nucleotide primers synthesized by SigmaAldrich). To amplify the U83 locus (primers BHU83F, U83FP1, U83RP1), a semi-nested PCR procedure was used for some samples using primers BHU83F1 and U83RP1, then a second step using primers U83FP1 and U83RP1; some additional primers were used to confirm sequence differences as described by Bates et al (2009) and Clark et al (2013). To amplify the chromosome 17p telomere integration site, primers were 17p (59-AACATCGAATCCACGGATTGCTTTGTGTAC-39) and HHV-6 DRR (59-CATAGATCGGGACTGCTTGAAAGCGC-39) in a PCR to generate a 1.5 kb amplicon confirmed by sequencing, as described by Arbuckle et al (2010) and Britt-Compton et al (2006).…”