Specific phenotypic restoration of an attenuated virus by knockout of a host resistance gene - PubMed (original) (raw)

Specific phenotypic restoration of an attenuated virus by knockout of a host resistance gene

D A Leib et al. Proc Natl Acad Sci U S A. 2000.

Abstract

To produce disease, viruses must enter the host, multiply locally in host tissues, spread from the site of entry, and overcome or evade host immune responses. At each stage in this infectious process, specific microbial and host genes determine the ultimate virulence of the virus. Genetic approaches have identified many viral genes that play critical roles in virulence and are presumed to target specific components of the host innate and acquired immune response. However, formal proof that a virulence gene targets a specific protein in a host pathway in vivo has not been obtained. Based on cell culture studies, it has been proposed that the herpes simplex virus type 1 gene ICP34.5 (ICP, infected cell protein) enhances neurovirulence by negating antiviral functions of the IFN-inducible double-stranded RNA-dependent protein kinase R or PKR [Chou, J., Chen, J.J., Gross, M. & Roizman, B. (1995) Proc. Natl. Acad. Sci. USA 92, 10516-10520]. Herein, we show that a virus that has been attenuated by deletion of ICP34.5 exhibits wild-type replication and virulence in a host from which the PKR gene has been deleted. We show that restoration of virulence is specific to ICP34.5 and PKR by using additional host and viral mutants. The use of recombinant viruses to infect animals with null mutations in host defense genes provides a formal genetic test for identifying in vivo mechanisms and targets of microbial virulence genes.

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Figures

Figure 1

Figure 1

IFN-mediated induction of the antiviral state through independent pathways by RNase L and PKR. For RNase L, IFNs interacting with their receptors lead to expression of 2-5A synthetase, which, after double-stranded RNA binding, produces 2′,5′-oligoadenylates that lead to RNase L activation and blockage of viral replication. IFN also induces expression of PKR, which is also activated through double-stranded RNA binding. One of the activities of PKR is to phosphorylate eIF-2α leading to cessation of protein synthesis and blockage of viral replication. To evade this antiviral mechanism, HSV-1 ICP34.5 binds to protein phosphatase 1α and dephosphorylates eIF-2α, lifting the block on viral replication. Mice with null mutations in the IFN receptors, RNase L, and PKR (gray boxes) were used in this study to dissect the interaction of ICP34.5 with these pathways.

Figure 2

Figure 2

Replication of control virus (closed squares) and Δ34.5 (open triangles) in corneas. (A) Growth in wild-type 129/B6 mice (one experiment with four to six samples per time point; P < 0.01 by two-tailed _t_ test). (_B_) PKR-deficient mice (three experiments with four samples per time point in each; _P_ > 0.8). (C) Wild-type 129 mice (two experiments with four samples per time point in each; P < 0.01). (_D_) IFN-αβγ receptor-deficient mice (two experiments with four samples per time point in each; _P_ > 0.27). (E) Wild-type C57BL/6 mice (one experiment with four to six samples per time point; P < 0.01). (F) RNase L-deficient mice (three experiments with four samples per time point in each; P < 0.02). All data were analyzed by two-tailed t tests, and values shown are logarithmic means (± SEM). Control virus and Δ34.5 are both in the background of strain 17. Limit of detection is 10 pfu.

Figure 3

Figure 3

Replication of control virus (solid bars) and Δ34.5 (shaded bars) in trigeminal ganglia 3 days after infection of wild-type 129/B6 mice (one experiment; four samples per time point; P < 0.001), PKR-deficient mice (three experiments; four samples per time point in each; _P_ > 0.9), wild-type 129 mice (two experiments with four samples per time point in each; P < 0.001), IFN-αβγ receptor-deficient mice (two experiments with four samples per time point in each; _P_ > 0.3), wild-type C57BL/6 mice (one experiment with six samples per time point; P < 0.001), and RNase L-deficient mice (three experiments with four samples per time point in each; P < 0.01). Values shown are logarithmic means (± SEM). Control virus and Δ34.5 are both in the background of strain 17.

Figure 4

Figure 4

Replication of KOS, Δtk, Δvhs, 17Δtk, and 17ΔtkR in corneas and trigeminal ganglia. Replication in corneas is shown for KOS (closed squares), Δtk (open triangles), and Δvhs (open circles) in wild-type 129 mice (A), IFN-αβγR−/− mice (B), and PKR-deficient mice (C). (D) Growth in trigeminal ganglia of wild-type 129, PKR-deficient, and IFN-αβγ receptor-deficient mice of KOS (solid bars), Δtk (shaded bars), and Δvhs (open bars). Replication in corneas is also shown for 17Δtk (open triangles) and 17ΔtkR (closed squares) in wild-type 129/B6 mice (E) and PKR−/− mice (F). (G) Growth in trigeminal ganglia of wild-type 129/B6 and PKR-deficient mice of 17ΔtkR (shaded bars) and 17Δtk (solid bars). Values shown are logarithmic means from four to six samples (± SEM). Δtk and Δvhs are in the background of strain KOS. 17Δtk and 17ΔtkR are in the background of strain 17.

Figure 5

Figure 5

Survival of wild-type, IFN receptor-deficient, and PKR-deficient mice after intracerebral infection. After intracerebral infection, survival of wild-type 129 (closed squares), IFN-αβ receptor-deficient (open diamonds), and IFN-αβγ receptor-deficient (closed diamonds) mice is shown for control virus (A) and Δ34.5 (B). Survival of wild-type 129/B6 (closed squares) and PKR-deficient (open triangles) mice is shown for control virus (C) and Δ34.5 virus (D). At least 10 mice were used over two experiments to generate the data shown. Control virus and Δ34.5 are both in the background of strain 17.

Figure 6

Figure 6

Survival of wild-type, RNase L-deficient, and PKR-deficient mice after intracerebral infection. After intracerebral infection, survival of wild-type C57BL/6 (open triangles) and RNase L-deficient (closed squared) mice is shown for control virus (A) and Δ34.5 (B). Survival of wild-type 129/B6 (closed squares) and PKR-deficient (open triangles) mice is shown for 17Δtk (C) and 17ΔtkR virus (D). At least 10 mice were used over two experiments to generate each curve. Control virus, Δ34.5, 17Δtk, and 17ΔtkR are all in the background of strain 17.

Figure 7

Figure 7

Survival of wild-type 129 and PKR-deficient mice after corneal infection. Survival of wild-type 129 (closed squares) and PKR-deficient (open triangles) mice is shown for control virus (A) and Δ34.5 (B). At least 10 mice were used over two experiments to generate each curve. Control virus and Δ34.5 are both in the background of strain 17.

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