Virus reconstituted from infectious bacterial artificial chromosome (BAC)-cloned murine gammaherpesvirus 68 acquires wild-type properties in vivo only after excision of BAC vector sequences

Abstract

We studied the in vivo biological properties of viruses reconstituted from the genome of murine gammaherpesvirus 68 (MHV-68) cloned as an infectious bacterial artificial chromosome (BAC). Recombinant virus RgammaHV68A98.01, containing BAC vector sequences, is attenuated in vivo as determined by (i) viral titers in the lungs during the acute phase of infection, (ii) the extent of splenomegaly, and (iii) the number of latently infected spleen cells reactivating virus in an ex vivo reactivation assay. Since the BAC vector sequences were flanked by loxP sites, passaging the virus in fibroblasts expressing Cre recombinase resulted in the generation of recombinant virus RgammaHV68A98.02, with biological properties comparable to those of wild-type MHV-68. On the basis of these data we conclude (i) that excision of BAC vector sequences from cloned MHV-68 genomes is critical for reconstitution of the wild-type phenotypic properties of this virus and (ii) that the BAC-cloned MHV-68 genome is suitable for the construction of mutants and mutant libraries whose phenotypes can be reliably assessed in vivo.

FIG. 1

Genomic structures of wild-type MHV-68, RγHV68A98.01, and RγHV68A98.02. Top line, left end of the unique sequence of the MHV-68 wild-type genome and the adjacent terminal repeats (TR). The genome of the recombinant virus RγHV68A98.01 contains in addition to the BAC vector (BAC) the genes for the guanosine phosphoribosyltransferase (gpt) and the green fluorescent protein (gfp), flanked by loxP sites (middle line). After excision of the loxP-flanked sequences, only one loxP site is left in the genome of recombinant virus RγHV68A98.02 (bottom line).

FIG. 2

BAC-derived virus RγHV68A98.01 is attenuated in vivo, whereas BAC-derived virus RγHV68A98.02 shows in vivo properties very similar to those of wild-type (wt) MHV-68. C57BL/6 mice were intranasally infected with 5 × 10⁴ PFU of either wild-type MHV-68, RγHV68A98.01, or RγHV68A98.02. Viral titers in the lungs were determined 3 and 6 days after infection (A and B). The extent of splenomegaly was determined by counting spleen cell numbers (C and D) and measuring the weight of the spleen 17 days after infection (E and F). The level of viral reactivation was determined by a limiting-dilution reactivation assay 17 days after infection (G and H). In panels A and B, titers of individual mice (n = 5) and median values are shown. Data shown in panels C to F are means ± standard deviations of three individual mice per group. Data in panels G and H are from pools of three mice per group. Both at day 3 and day 6 after infection, viral titers of RγHV68A98.01 are significantly lower than those of wild-type MHV-68 (P = 0.046 and 0.001, respectively; unpaired Student's t test) (A). The means of the cell numbers and the means of the spleen weights of the three groups of mice are significantly different (P = 0.0156 for cell number, and P = 0.0045 for spleen weight; one-way analysis of variance) (C and E). The number of spleen cells which reactivated MHV-68 was significantly lower in mice infected with RγHV68A98.01 than in mice infected with wild-type MHV-68 (P = 0.048; paired Student's t test) (G). cpe, cytopathic effect.

FIG. 3

BAC-derived virus RγHV68A98.01 is also attenuated in SCID mice. C57BL/6 and SCID mice were intranasally infected with 1,000 PFU of RγHV68A98.01 or RγHV68A98.02. Viral titers in the lungs were determined 6 days after infection. Titers of individual mice (n = 5) and median values are shown. Both in C57BL/6 mice and in SCID mice, viral titers of RγHV68A98.01 were significantly lower than those of RγHV68A98.02 (P = 0.003 for both groups; unpaired Student's t test).