Olfactory Entry Promotes Herpesvirus Recombination

Abstract

Herpesvirus genomes show abundant evidence of past recombination. Its functional importance is unknown. A key question is whether recombinant viruses can outpace the immunity induced by their parents to reach higher loads. We tested this by coinfecting mice with attenuated mutants of murid herpesvirus 4 (MuHV-4). Infection by the natural olfactory route routinely allowed mutant viruses to reconstitute wild-type genotypes and reach normal viral loads. Lung coinfections rescued much less well. Attenuated murine cytomegalovirus mutants similarly showed recombinational rescue via the nose but not the lungs. These infections spread similarly, so route-specific rescue implied that recombination occurred close to the olfactory entry site. Rescue of replication-deficient MuHV-4 confirmed this, showing that coinfection occurred in the first encountered olfactory cells. This worked even with asynchronous inoculation, implying that a defective virus can wait here for later rescue. Virions entering the nose get caught on respiratory mucus, which the respiratory epithelial cilia push back toward the olfactory surface. Early infection was correspondingly focused on the anterior olfactory edge. Thus, by concentrating incoming infection into a small area, olfactory entry seems to promote functionally significant recombination. IMPORTANCE All organisms depend on genetic diversity to cope with environmental change. Small viruses rely on frequent point mutations. This is harder for herpesviruses because they have larger genomes. Recombination provides another means of genetic optimization. Human herpesviruses often coinfect, and they show evidence of past recombination, but whether this is rare and incidental or functionally important is unknown. We showed that herpesviruses entering mice via the natural olfactory route meet reliably enough for recombination routinely to repair crippling mutations and restore normal viral loads. It appeared to occur in the first encountered olfactory cells and reflected a concentration of infection at the anterior olfactory edge. Thus, natural host entry incorporates a significant capacity for herpesvirus recombination.

Keywords: herpesviruses; host entry; olfactory; recombination.

FIG 1

Nasal infection by MuHV-4 mutants lacking normal latency. (a) A schematic sketch of MuHV-4 mutations shows the linear genome flanked by terminal repeats (TR), with expanded views below. Replication-deficient ORF50DEL MuHV-4 has most of ORF50 exon 2 replaced by luciferase plus a polyadenylation site. ORF73FS MuHV-4 has a frameshift in ORF73, which encodes the viral episome maintenance protein. The ΔL virus mutation additionally deletes 11 kb from the genome left end, removing ORFs M1-M4. M50 MuHV-4 has an MCMV IE1 promoter inserted in the 5′ untranslated region of ORF50. p1 to p6 show the locations of primers used to identify the ORF50DEL, M50, and ORF73FS mutations. (b) C57BL/6 mice were infected nasally (10⁵ PFU in 5 μl without anesthesia) with wild-type, M50, or ORF73FS virus. Titers in noses were determined by plaque assay. Titers in superficial cervical lymph nodes (SCLN) and spleens were determined by infectious-center assay. Symbols show individual mice; bars show means. The dashed line shows the detection limit. Significant differences in virus recovery relative to the wild type are shown.

FIG 2

Recovery of latency after nasal coinfection with latency-deficient MuHV-4 mutants. (a) C57BL/6 mice were infected nasally (10⁵ PFU) with wild-type, M50, or ORF73FS virus or a 1:1 M50/ORF73FS mix. Weights of spleens 25 days later are shown, with significant splenomegaly for the wild type and mixed infections. Symbols show individual mice; bars show means. (b) Titers in the spleens in panel a were determined for latent virus by infectious-center assay. Symbols show individuals; bars show means. The dashed line shows the lower limit of assay detection. Mixed M50/ORF73FS infection yielded significantly more virus than either single infection. No preformed infectious virus was recovered by parallel titer of freeze-thawed spleen samples. (c) Viruses cloned (c1 to c4) from spleens of mixed-infection mice were genotyped by PCR across the M50 insertion site (primers p1 and p2 in Fig. 1a). PCR products were resolved by agarose gel electrophoresis and stained with ethidium bromide. WT and M50, wild-type and M50 input viruses; Mw, molecular weight markers. Sequencing of the main (268 bp) product of cloned virus DNA confirmed identity with the wild type. (d) DNA from the cloned viruses in panel c was genotyped by PCR across the ORF73 frameshift (primers p5 and p6 in Fig. 1a). PCR products were digested or not with BstEII, resolved by agarose gel electrophoresis, and stained with ethidium bromide. 73, ORF73FS input viruses. As the viruses were cloned prior to analysis, we interpret the minor residual 0.43-kb product for BstEII-digested c3 DNA as incomplete digestion. DNA sequencing of the main (428 bp) undigested product of the cloned viruses confirmed identity with the wild type. (e) C57BL/6 mice were infected as for panel a. Three months later, latent virus was detected by infectious-center assay. M50/ORF73FS coinfection yielded significantly more virus than either single infection and was indistinguishable from the wild type. Symbols show individual mice; bars show means. The dashed line shows the detection limit. (f) The spleens in panel e were assayed for viral genomes by quantitative PCR of extracted DNA. Viral copies are expressed relative to cellular β-actin copies amplified in parallel. M50/ORF73FS coinfection yielded significantly more viral genomes than either single infection and was indistinguishable from the wild type.

FIG 3

Recovery of latency after coinfection by M50 and ORF73FSΔL mutants. (a) C57BL/6 mice were infected nasally (10⁵ PFU) with wild-type, M50, or ORF73FSΔL virus or a 1:1 M50/ORF73FSΔL mix. We also infected mice with a control ΔL single mutant. Spleens were infectious-center assayed for latent infection 25 days later. Symbols show individual mice; bars show means. The dashed line shows the detection limit. M50/ORF73FSΔL coinfection yielded significantly more latency than either virus alone. No preformed infectious virus was recovered by parallel titer of freeze-thawed spleen samples. (b) To test the viruses recovered from spleens of individual coinfected mice for full rescue, they were inoculated into the lungs of naive C57BL/6 mice (10³ PFU). Splenic infectious-center assays 12 days later showed titers equivalent to those of the wild type.

FIG 4

Little evidence of MuHV-4 recombination after lung coinfection. (a) Wild-type, M50, or ORF73FS virus or a 1:1 M50/ORF73FS mix was inoculated into the lungs of C57BL/6 mice (10⁴ PFU in 30 μl under anesthesia). Spleens were infectious-center assayed for latent virus 17 days later. Symbols show individual mice; bars show means. The dashed line is the detection limit. (b) Mice were infected as for panel a. One hundred days later, DNA from spleens was assayed for viral DNA by quantitative PCR. Values are expressed relative to the cellular DNA load assayed in parallel for each sample. Symbols show individual mice; bars show means. (c) Mice were infected as for panel a. Twelve days later, SCLN and MLN were infectious-center assayed for latent virus.

FIG 5

Rescue of MCMV mutants after coinfection in the nose but not the lungs. (a) Wild-type, M33, or M78 MCMV or a 1:1 M33/M78 mix was inoculated into the lungs of BALB/c mice (10⁴ PFU in 30 μl under anesthesia). Eighteen days later, salivary glands were plaque assayed for infectious virus. Symbols show individual mice; bars show means. The dashed line is the detection limit. Only wild-type infection yielded recoverable virus. (b) BALB/c mice were infected nasally (10⁵ PFU) with M33 or M78 MCMV or a 1:1 M33/M78 mix. Eighteen days later, salivary glands were plaque assayed for infectious virus. Symbols show individual mice; bars show means. Only the mixed infection yielded recoverable virus. (c) DNA of virus cloned from mixed infection salivary glands in panel b was checked for M33 mutation by PCR. 33 and 78, M33 and M78 input viruses. The predicted wild-type band is 572 bp. The upper 33 sample band corresponds to the expected size for the lacZ cassette insertion of the mutant (4.4 kb). The source of the lower (2.2 kb) 33 sample band is unclear, but the 33 sample is clearly free of wild-type DNA, and no recovered clone shows a mutant M33 locus. (d) The same DNA samples were checked for M78 mutation. Clone 2 appeared to be a parental M78 virus. The other 3 clones had a wild-type M78 locus, indicating recombination.

FIG 6

Early recombinational rescue of MuHV-4 after nasal coinfection. (a) C57BL/6 mice were infected nasally (10⁵ PFU) with wild-type, M50, or ORF73FS MuHV-4 or a 1:1 M50/ORF73FS mix. Eight days later, latent virus in SCLN was infectious-center assayed. Symbols show individual mice; bars show means. The dashed line is the detection limit. Mixed infections gave higher titers than either component single infection, although the wide spread meant that as a group they were not significantly higher than M50 alone. (b) Viruses cloned from SCLN for each mouse in panel a were given i.n. to naive mice under anesthesia to inoculate the lungs (10³ PFU in 30 μl). One clone from each positive mouse was given to one naive mouse. Fourteen days later, spleens were infectious-center assayed for latent infection. Symbols show individual mice; bars show means. (c) C57BL/6 mice were infected nasally (10⁵ PFU in 5 μl) with wild-type, ORF50DEL, or ORF73FS MuHV-4 or a 1:1 ORF50DEL/ORF73FS mix. Eighteen days later, SCLN and spleens were infectious-center assayed for latent virus. Symbols show individual mice; bars show means. Mixed infections gave significantly higher titers than either component single infection. (d) Mice infected as for panel c were infectious-center assayed for splenic virus after 25 days. Mixed infections gave significantly higher titers than either component single infection. (e) DNA of viruses cloned (c1 to c4) from mixed-infection spleens for panel d was PCR amplified across the ORF50 deletion site (primers p3 and p4 in Fig. 1a). The PCR products were resolved by agarose gel electrophoresis and stained with ethidium bromide. 50, ORF50DEL input viruses. DNA sequencing of cloned virus PCR products confirmed identity with the wild type. (f) DNA from the mixed-infection clones in panel e was PCR amplified across the ORF73 frameshift site (primers p5 and p6 in Fig. 1a). The PCR products were digested or not with BstEII, resolved by agarose gel electrophoresis, and stained with ethidium bromide. As in Fig. 2d, as c1 to c4 were cloned prior to analysis; the minor residual 0.43-kb band after incubation with BstEII digestion presumably reflected incomplete digestion rather than mutant DNA. Sequencing of the undigested PCR products confirmed identity with the wild type.

FIG 7

Recombinational rescue after serial mixed infection. C57BL/6 mice were infected nasally (10⁵ PFU) with ORF50DEL MuHV-4. Five days later, they were infected nasally (10⁵ PFU) with ORF73FS MuHV-4. Controls were given either just ORF50DEL or just ORF73FS. Eighteen days after ORF73FS infection, spleens were infectious-center assayed for latent virus. Symbols show individual mice; bars show means. The dashed line shows the detection limit. Serial mixed infection yielded significantly more recoverable virus than either single infection.

FIG 8

Infection at the olfactory/respiratory border. C57BL/6 mice were infected nasally with wild-type MuHV-4 (10⁵ PFU). One to 3 days later, nose sections were stained for viral lytic antigens with a polyclonal rabbit serum (brown) and counterstained with hemalum (blue). OE, olfactory epithelium; RE, respiratory epithelium. Representative sections show foci of infection on the olfactory side of the olfactory/respiratory border.