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. 1999 Sep 14;96(19):10881-6.
doi: 10.1073/pnas.96.19.10881.

Cytomegalovirus-encoded beta chemokine promotes monocyte-associated viremia in the host

N Saederup  1 , Y C Lin, D J Dairaghi, T J Schall, E S Mocarski
Affiliations

Cytomegalovirus-encoded beta chemokine promotes monocyte-associated viremia in the host

N Saederup et al. Proc Natl Acad Sci U S A. .

Abstract

Chemokine homologs are encoded by many large DNA viruses, suggesting that they contribute to control of host leukocyte transmigration and trafficking during viral infection. Murine cytomegalovirus carries a CC (beta) chemokine homolog gene giving rise to two related proteins, murine cytomegalovirus chemokine 1 and 2 (MCK-1 and MCK-2). MCK-1 peptide was found to induce calcium signaling and adherence in murine peritoneal macrophages. Cells bearing human chemokine receptor CCR3 and the human macrophage THP1 cell line were responsive to MCK-1. This pattern suggested that MCK-1 might act as an agonist, promoting leukocyte trafficking during viral infection. Consistent with this prediction, MCK-1/MCK-2 mutant viruses exhibit dramatically reduced peak levels of monocyte-associated viremia in experimentally infected mice. Thus, MCK-1/MCK-2 appears to promote host leukocyte migration to initial sites of infection and may be responsible for attracting monocytes or macrophages that efficiently disseminate virus in the host.

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Figures

Figure 1
Figure 1
MCK-1 induces Ca2+ signaling in murine PECs. (A). Predicted MCK-1 amino acid sequence (3) showing the similarity of this protein to three murine β chemokines, RANTES, JE (the homolog of human MCP-1), and MIP1α. The predicted signal sequence cleavage sites of the displayed chemokines are indicated in blue, including MCK-1 threonine 19, at which the synthetic MCK-1 peptide commences. (B–G). Digital fluorescence video microscopy frames of synthetic MCK-1 induced Ca2+ flux in glass-adherent PECs. Cells harvested by lavage from uninfected (B–D) or RM427+-infected (E–G) 10-week-old BALB/c mice were labeled with 1 μM Fura-2-AM, and levels of free intracellular Ca2+ in individual cells, indicated on the digitally calculated color scale inserts on each panel, were measured by spectrofluorometric video microscopy before (B and E), 12 sec after addition of 250 nM MCK-1 (C and F), and 1 (D) and 2 (G) min later. The full series E-G can be viewed at http://cmgm.stanford.edu/∼saederup/
Figure 2
Figure 2
MCK-1-induced Ca2+ flux leads to tight adherence. (A) Percentage of PECs from mock-infected mice responding to MCK-1, with three independent experiments shown. (B) Percentage of PECs from RM427+-infected mice (48 h post-i.p. inoculation) responding to MCK-1 (gray bars), with results from five independent experiments shown. Percentage of PECs that were virus-infected based on β-galactosidase expression also is shown (black bars). The digital images shown in Fig. 1 of PECs from mock-infected mice are from experiment 2 (Exp. #2), and those from RM427+-infected mice are from experiment 4. (C). Induction of tight adherence by MCK-1. The percentage of total adherent PECs that were loosely attached and that fluxed in response to MCK-1 (gray bars) and the percentage that adhered tightly to glass after responding to MCK-1 (black bars) are shown. Data are from the five experiments shown in B.
Figure 3
Figure 3
Murine CMV MCK-1/MCK-2 mutant construction and evaluation of viremia during infection of mice. (A) Map of the HindIII K, L, and J fragments of wild-type murine CMV (WT MCMV; K181+ strain) showing the position of the m129 and m131 ORFs (17), as open boxes, and the predicted MCK-1 ORF within m131 as a filled box. The murine CMV ie1/ie2/ie3 transcriptional enhancer and the arrangement of ie1, ie2, ie3, sgg1, and γ0.85 transcripts also are shown with splicing patterns indicated on the arrows depicting individual transcripts. The lacZ insertion mutations in the three recombinant viruses (RM461, RM4485, RM427+) as well as the deletion mutation disrupting γ0.85 transcript expression in RM4485 are depicted below the map. The genome structure and growth properties of RM461, which carries a lacZ insert disrupting the γ0.85 transcript expression, has been described (12, 29). Expression of the lacZ gene in all recombinant viruses was regulated by a 199-bp human CMV ie1/ie2 promoter (αHCMV) fragment (−219 to −19 relative to the transcription start site) (22). This promoter fragment exhibits immediate early expression kinetics when adjacent to the murine CMV transcriptional enhancer, as in RM427+ and RM4485, and delayed early expression kinetics in RM461 (12). (B) Levels of viremia after infection with K181+ (□) or RM4485 (⋄). BALB/c mice 3 or 4 weeks of age were inoculated by the i.p. route with 1 × 106 plaque-forming units of virus, and PBLs were harvested by heart puncture at 3, 5, and 7 days postinfection. Infection rates were expressed as the percentage of total PBLs that scored positive in an infectious centers assay on monolayers of NIH 3T3 cells. Error bars represent standard deviation of the means of titers in five mice. (C) Levels of viremia after co-infection with K181+ and RM4485. Mice were co-inoculated by the i.p. route with a 1:1 mixture of K181+ and RM4485 at 1 × 106 plaque-forming units, and PBLs were harvested as described in B. Infectious centers were stained to reveal β-galactosidase expression and to differentiate RM4485 from K181+ plaques. (D) Levels of viremia after s.c. inoculation of RM427+ (■) or RM4485 (⋄) inoculated into one or two hind footpads were analyzed as above, except the results of individual mice were plotted as separate points. The dashed line in each panel represents the limit of detection.
Figure 4
Figure 4
Impact of MCK-1/MCK-2 on viral infection. After inoculation of murine CMV, virus gene expression and DNA replication ensues within the first 24 h and is accompanied by an innate inflammatory response. As virus-infected cells begin producing progeny virus, the MCK-1/MCK-2 gene product is made and either sustains or increases the recruitment of a permissive monocyte/macrophage (Mono/Mac) population that can be directed efficiently to blood by an as yet unknown (but possibly MCK-1/MCK-2-determined) process that results in more efficient dissemination.
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