Identification and Characterization of U83A Viral Chemokine, a Broad and Potent β-Chemokine Agonist for Human CCRs with Unique Selectivity and Inhibition by Spliced Isoform

Abstract

Leukotropic human herpesvirus 6 (HHV-6) establishes a persistent infection associated with inflammatory diseases and encodes chemokines that could chemoattract leukocytes for infection or inflammation. HHV-6 variant A encodes a distant chemokine homolog, U83A, and a polymorphism promoting a secreted form was identified. U83A and three N-terminal modifications were expressed and purified, and activities were compared with a spliced truncated isoform, U83A-Npep. U83A efficiently and potently induced calcium mobilization in cells expressing single human CCR1, CCR4, CCR6, or CCR8, with EC50 values <10 nM. U83A also induced chemotaxis of Th2-like leukemic cells expressing CCR4 and CCR8. High-affinity binding, 0.4 nM, was demonstrated to CCR1 and CCR5 on monocytic/macrophage cells, and pretreatment with U83A or modified forms could block responses for endogenous ligands. U83A-Npep acted only as antagonist, efficiently blocking binding of CCL3 to CCR1 or CCR5 on differentiated monocytic/macrophage leukemic cells. Furthermore, CCL3 induction of calcium signaling via CCR1 and CCL1 induced chemotaxis via CCR8 in primary human leukocytes was inhibited. Thus, this blocking by the early expressed U83A-Npep could mediate immune evasion before finishing the replicative cycle. However, late in infection, when full-length U83A is made, chemoattraction of CCR1-, CCR4-, CCR5-, CCR6-, and CCR8-bearing monocytic/macrophage, dendritic, and T lymphocyte cells can facilitate dissemination via lytic and latent infection of these cells. This has further implications for neuroinflammatory diseases such as multiple sclerosis, where both cells bearing CCR1/CCR5 plus their ligands, as well as HHV-6A, have been linked. Applications also discussed include novel vaccines/immunotherapeutics for cancer and HIV as well as anti-inflammatories.

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

“…This included 38 sequences described here from clinical strains in Zambia which were compared to 74 available on GenBank, from Japan, Germany, USA, DR Congo and Uganda (total 112). This comparison showed U83 variation between HHV-6A and B species, as previously identified (Bates et al, 2009;Dewin et al, 2006;French et al, 1999), typified by alignments from laboratory reference strains, HHV-6A strain U1102 and HHV-6B strain Z29 (Fig. 1).…”

“…This specificity is in contrast to properties of the homologous chemokine U83A, encoded by HHV-6A. The U83A chemokine has broad chemokine receptor specificity: CCR1, CCR4, CCR5, CCR6 and CCR8, yet does not include CCR2 (Catusse et al, 2007;Dewin et al, 2006). The properties of CCL2 have been shown to be essential in a number of systems, including HIV, where it is critical for mediating monocyte movement across the blood-brain barrier and for subsequent correlates to neuroinflammatory disease (Buckner et al, 2011;Lentz et al, 2011;Williams et al, 2012).…”

“…In contrast, U83A is specific for chemokine receptors, CCR5 or CCR6, which are present on dendritic cells and may affect antigen presentation by different pathways. Experiments with a spliced version of U83A, which encodes a truncated version of U83A, U83A-N, show the chemokine binding specificity is retained in the N-terminal half of the molecule (Catusse et al, 2007;Dewin et al, 2006). Here, this is further explored by analysing strain variants in order to define U83B specificity for CCR2.…”

Microarray-based determination of the lytic cascade of human herpesvirus 6B

“…Similarly, these virion-associated transcripts are characterized by their detection at early times during infection and are transcribed maximally only at the time of viral assembly (Bechtel et al, 2005;Bresnahan & Shenk, 2000). The functions of two HHV-6B biphasic genes have been reported: U21 encodes a protein that interferes with major histocompatibility complex class I surface expression (Glosson & Hudson, 2007), whilst U83 encodes two isoforms of viral chemokines capable of chemoattracting leukocytes and may therefore aid virus spread by recruiting uninfected cells during primary infection (Dewin et al, 2006).…”

In Vitro and In Vivo Transactivation of HIV-1 by Human Herpesvirus 6

“…The full-length form acts as an agonist while the spliced form acts as an antagonist that interacts with other chemokine receptors, i.e. CCR1, CCR4, CCR5, CCR6 and CCR8, and is expressed on T cells, monocytes/macrophages, and dendritic cells (Dewin et al, 2006). Gene U22 also codes for yet another chemokine (French et al, 1999).…”