Marek's disease virus-encoded vIL-8 gene is involved in early cytolytic infection but dispensable for establishment of latency

Abstract

Marek's disease, a lymphoproliferative disease of chickens, is caused by an alphaherpesvirus, Marek's disease virus (MDV). This virus encodes a virokine, vIL-8, with general homology to cellular CXC chemokines such as interleukin-8 (IL-8) and Gro-alpha. To study the function of vIL-8 gene, we deleted both copies of vIL-8 residing in the terminal repeat long and internal repeat long region of the viral genome and generated a mutant virus with vIL-8 deleted, rMd5/DeltavIL-8. Growth kinetics study showed that vIL-8 gene is dispensable for virus replication in cell culture. In vivo, the vIL-8 gene is involved in early cytolytic infections in lymphoid organs, as evidenced by limited viral antigen expression of rMd5/DeltavIL-8. However, the rMd5/DeltavIL-8 virus is unimpaired in virus replication in the feather follicle epithelium. vIL-8 does not appear to be important for establishment of latency, since rMd5/DeltavIL-8 and the wild-type virus have similar viremia titers at 14 days postinfection, a period when the virus titer comes primarily from reactivated latent genomes. Nevertheless, because of the impaired cytolytic infections, the overall transformation efficiency of the virus with vIL-8 deleted is much lower, as reflected by the reduced number of transformed cells at 5 weeks postinoculation and the presence of fewer gross tumors. Importantly, the revertant virus that restored the expression of vIL-8 gene also restored the wild-type phenotype, indicating the deficient phenotypes are results of vIL-8 deletion. One of the interesting differences between the MDV vIL-8 gene and its cellular counterpart is the presence of a DKR (Asp-Lys-Arg) motif instead of ELR (Glu-Leu-Arg) preceding the invariable CXC motif. To study the significance of this variation, we generated recombinant MDV, rMd5/vIL-8-ELR, carrying the ELR motif. Both in vitro and in vivo studies revealed that the DKR motif is as competent as ELR in pathogenesis of MDV.

FIG. 1.

Construction of recombinant virus with deletion of the vIL-8 gene. (A) The MDV genome consists of terminal repeat long (TR_L) and short (TR_S), internal repeat long (IR_L) and short (IR_S), and unique long (U_L) and unique short (U_S) DNA segments. (B) Schematic representation of overlapping clones generated to reconstitute an infectious virus from a very virulent strain of MDV (Md5). The restriction enzymes used to generate each cosmid clone and their positions are indicated. (C) Cosmids SN5/ΔvIL8 and A6/ΔvIL8 have the vIL8 coding sequences deleted by ClaI and NcoI digestions. The locations of the restriction enzymes used to introduce the deletions are indicated.

FIG. 2.

Western blot analysis of supernatant from rMd5/ΔvIL-8-RV (lane 1), rMd5/ΔvIL-8 (clones 1 and 2 in lanes 2 and 3), rMd5 (lane 4), rMd5/vIL-8-ELR (clones 1 and 2 in lanes 5 and 6), and mock infected DEF (lane 7) using anti-vIL-8 rabbit polyclonal sera. The vIL-8 protein is about 18 kDa.

FIG. 3.

Southern blot analysis of DNA isolated from recombinant MDVs. (A) Viral DNA was digested with EcoRI and probed with all five radiolabeled cosmids. (B) Viral DNA was digested with BamHI (lanes 1 to 6) or double digested with BamHI/SalI (lanes 1′ to 6′) and hybridized with the 3.1-kb MDV BamHI fragment containing the vIL8 gene. Lanes: 1 and 1′, uninfected DEF; 2 and 2′, rMd5; 3, 4, 3′, and 4′, rMd5/vIL-8-ELR (clones 1 and 2); 5, 6, 5′, and 6′, rMd5/ΔvIL-8 (clones 1 and 2). BamHI single digestion produces a 3.1-kb band in both rMd5 and rMd5/vIL-8-ELR viruses and a 2.3-kb fragment in rMd5/ΔvIL-8 virus. BamHI/SalI double digestion results in two bands (1.8 and 1.3 kb) in rMd5 and a single band (3.1 kb) in rMd5/vIL-8-ELR, due to loss of the SalI site.

FIG. 4.

In vitro growth kinetics of rMd5, rMd5/ΔvIL-8 (clones 1 and 2), and rMd5/vIL-8-ELR (clones 1 and 2) viruses. DEF were infected with approximately 100 PFU of the indicated viruses, and at 1, 2, 3, 4, and 5 days postinfection, the cells were harvested and their titers were determined on fresh DEF. The experiment was performed in duplicate, and the titer is indicated as PFU for each 60-mm dish. Error bars in the figure show the standard deviation of the mean.

FIG. 5.

Immunohistochemistry of lymphoid organs (the tissues in each column from top to bottom represent bursa, thymus, and spleen) of 15 × 7 MDV maternal antibody-negative chickens 6 days after inoculation with control (A, B, and C), rMd5 (D, E, and F), rMd5/ΔvIL-8 (G, H, and I), rMd5/vIL-8-ELR (J, K, and L), or rMd5/ΔvIL-8-RV (M, N, and O). MAb against pp38 (H19) was used for the staining. Antigen expression in lymphoid organs is severely impaired only in rMd5/ΔvIL-8, showing that vIL-8 is involved in early cytolytic infection in lymphocytes.

FIG. 6.

Immunohistochemical analysis of FFE cells from inoculated chickens. FFE cells were sampled at 2 weeks postinoculation. All of the recombinant viruses rMd5 (A), rMd5/ΔvIL-8 (B), and rMd5/vIL-8-ELR (C) expressed viral antigen in FFEs, indicating that the second lytic infection is not impaired in either the vIL-8 gene deletion or vIL-8 gene mutations. No viral antigen was detected in the control chickens (D).

FIG. 7.

Viral titers at 6, 8, 14, and 35 days postinoculations in peripheral blood lymphocytes of 15 × 7 chickens inoculated with rMd5, rMd5/ΔvIL-8, and rMd5/vIL-8-ELR. Five chickens from each experimental group were tested, and titrations were performed in duplicate. The titer is indicated as PFU/10⁶ peripheral blood lymphocytes. Error bars in the figure show the standard deviation of the mean.