Figure 1.
Transgenic Expression of v-Bcl-2 in Thymocytes
(A) Schematic illustration of the γHV68 genome, v-Bcl-2 genomic region, and transgene construct.
(B) Real-time RT-PCR quantitation of v-Bcl-2 expression in transgenic thymocytes (left) and splenocytes (right).
(C) Total number of thymocytes in v-Bcl-2 transgenic and nontransgenic mice.
(D) Percentage of CD4 or CD8 single-positive, double-positive (DP) and double-negative (DN) thymocytes in v-Bcl-2 transgenic and nontransgenic mice.
Figure 2.
v-Bcl-2 Inhibits Cell Death in Thymocytes
(A) Survival of DP thymocytes from v-Bcl-2A (left) and v-Bcl-2B (right) mice 48 h following intraperitoneal injection with various doses of dexamethasone.
(B and C) Survival of DP thymocytes from v-Bcl-2A and v-Bcl-2B mice 48 h following (B) 250 rads of γ-irradiation and (C) intraperitoneal injection with 30 μg of anti-CD3ɛ antibody.
Figure 3.
Solution Structure of γHV68 v-Bcl-2
(A and B) Ribbon representation of (A) γHV68 v-Bcl-2 and (B) Bcl-xL with BH1, BH2, BH3, and BH4 regions in magenta, red, green, and yellow, respectively. Helices are numbered with respect to Bcl-xL.
(C) Connolly surface for γHV68 v-Bcl-2 calculated using a probe radius of 1.4 Å. Residues are colored as follows: Leu, Val, Ile, Phe, Tyr, Trp, Met, and Ala in yellow; Arg, Lys, and His in blue; Asp and Glu in red; all others in gray. The hydrophobic groove is indicated by an arrow.
(D) Structural and sequence alignment of KSHV and γHV68 v-Bcl-2 proteins with Bcl-2 and Bcl-xL.
Figure 4.
Mutagenesis of γHV68 v-Bcl-2 BH3 Binding Groove
(A) Ribbon representation of γHV68 v-Bcl-2 showing residues that contact Bak peptide (in green and R87) and the SGR residues that were mutated (in magenta).
(B) Growth of yeast following transformation with the indicated constructs (above, bar graph) and Western blot of SGR/AAA and wild-type v-Bcl-2 from transformed yeast (below, blot).
(C) Overlay of the 15N-HSQC spectra of SGR/AAA (red contours) and wild-type (black contours) v-Bcl-2 showing conservation of structure between mutant and wild-type proteins.
Figure 5.
SGR/AAA Mutant Viruses Replicate Normally In Vitro and In Vivo
(A) Shown are schematic illustrations of the genomes of γHV68, v-cyclin.LacZ, and SGR/AAA mutant viruses with the engineered PstI site underlined, and v-Bcl-2 containing the SGR/AAA mutation.
(B) Southern blot of γHV68, v-cyclin.LacZ, and SGR/AAA mutant viruses. Expected bands (kb) for PstI/BamHI digest: γHV68, 1.5, 1.3, and 1.2; v-cyclin.LacZ, 1.3, 1.2, 1.1, and 0.05; and SGR/AAA, 1.5, 1.2, 0.8, and 0.6. Expected bands (kb) for PstI/HindIII digest: γHV68, 7.5, 1.2, 0.9, and 0.4; v-cyclin.LacZ, 7.1, 4.3, 1.2, 0.9, 0.4, and 0.07; and SGR/AAA, 7.5, 1.2, 0.6, 0.39, and 0.36.
(C) Multistep growth curves of SGR/AAA mutants and γHV68.
(D) Acute splenic titers at 4 or 9 dpi from mice infected with SGR/AAA mutants.
Figure 6.
SGR/AAA Mutant Viruses Exhibit Defects in Chronic Infection In Vivo
Ex vivo reactivation (A) and persistent replication (B) of γHV68, Δv-Bcl-2, or SGR/AAA mutant viruses at 16 or 42 dpi of IFNγ−/− mice. No significant differences were observed between the two independent isolates of SGR/AAA mutants, and data from these two groups were pooled.
Table 1.
Summary of Chronic Infection Results