1. Volume 1, Issue 1, Pages 23-35 (March 2007)
HSV-1 ICP34.5 Confers Neurovirulence by Targeting the Beclin 1 Autophagy Protein 
Anthony Orvedahl, Diane Alexander, Zsolt Tallóczy, Qihua Sun, Yongjie Wei, Wei Zhang, Dennis Burns, David A. Leib, Beth Levine 
Cell Host & Microbe 
Volume 1, Issue 1, Pages (March 2007)
DOI: /j.chom
Copyright © 2007 Elsevier Inc. Terms and Conditions

2. Figure 1 ICP34.5 Inhibits Beclin 1-Dependent Autophagy in Yeast
(A) Yeast two-hybrid interactions of ICP34.5 and Beclin 1. +, positive reaction within 8 hr; −, lack of positive reaction at 24 hr. Nucleotide position of genes fused to plasmid is indicated next to human beclin 1 constructs.
(B) Quantitation of starvation-induced autophagy in atg6Δ yeast transformed with plasmids indicated below the x axis. Results represent mean ± SEM for triplicate samples. For each sample, a minimum of 100 cell profiles were analyzed, and cell profiles with one or more autophagosome within the vacuole were scored as positive. Similar results were obtained in five independent experiments.
(C) Representative DIC photomicrographs of atg6Δ yeast transformed with indicated plasmids following starvation for 4 hr. Arrows denote representative autophagosomes.
Cell Host & Microbe 2007 1, 23-35DOI: ( /j.chom )
Copyright © 2007 Elsevier Inc. Terms and Conditions

3. Figure 2
ICP34.5 Binds to Beclin 1 in Mammalian Cells and Inhibits Beclin 1-Mediated Autophagy
(A) Coimmunoprecipitation of Flag-tagged Beclin 1 with ICP34.5 in HEK293 cells transfected with the indicated plasmids.
(B) Coimmunoprecipitation of endogenous Beclin 1 with ICP34.5 in beclin 1+/+ or beclin 1−/− mouse ES cells infected with the indicated virus.
(C) Quantitation of autophagy, as measured by GFP-LC3 punctations, in MCF7 cells transfected with plasmids expressing Beclin 1 and/or ICP34.5 during growth in normal media (open bars) or starvation media (filled bars). Results represent mean ± SEM for triplicate samples. For each sample, a minimum of 100 cells was analyzed. Similar results were obtained in five independent experiments.
(D) Representative photomicrographs of images used for quantitative analyses in (C). Arrow denotes representative punctate GFP-LC3 dot corresponding to an autophagosome.
Cell Host & Microbe 2007 1, 23-35DOI: ( /j.chom )
Copyright © 2007 Elsevier Inc. Terms and Conditions

4. Figure 3
A Twenty-Amino-Acid Region of ICP34.5 Is Required for Beclin 1 Binding and Inhibition of Autophagy
(A) Schematic representation of ICP34.5 showing position of Beclin 1-binding region (amino acids 68–87) and GADD34 homology region.
(B) Coimmunoprecipitation of Myc-tagged wild-type or indicated mutant ICP34.5 and Flag-tagged Beclin 1 in MCF7.beclin 1 cells.
(C) Quantitation of autophagy, as measured by GFP-LC3 punctate regions in MCF7.beclin 1 cells transfected with the indicated HSV-1 ICP34.5 plasmids during growth in normal media (open bars) or starvation media (filled bars). Results represent mean ± SEM for triplicate samples. For each sample, a minimum of 100 cells was analyzed. Similar results were obtained in three independent experiments.
Cell Host & Microbe 2007 1, 23-35DOI: ( /j.chom )
Copyright © 2007 Elsevier Inc. Terms and Conditions

5. Figure 4
The Beclin 1-Binding Domain of ICP34.5 Is Dispensable for ICP34.5-Mediated Blockade of Host-Cell Shutoff
(A) Coimmunoprecipitation of endogenous Beclin 1 with ICP34.5 in SK-N-SH cells infected with HSV-1 ΔICP34.5 (17termA) or its marker rescue virus (17termAR), or HSV Δ68–87 or its marker rescue (HSV Δ68–87R).
(B) Viral replication of HSV Δ68–87 and HSV Δ68–87R in SK-N-SH neuroblastoma cells. Data shown represent mean ± SEM geometric titer.
(C) Western blot detection of the serine 51 phosphorylated form of eIF2α in SK-N-SH neuroblastoma cells 16 hr after infection with the indicated virus.
(D) 35S-labeled cellular proteins in SK-N-SH cells 16 hr after infection with the indicated virus.
Cell Host & Microbe 2007 1, 23-35DOI: ( /j.chom )
Copyright © 2007 Elsevier Inc. Terms and Conditions

6. Figure 5
The Beclin 1-Binding Domain of ICP34.5 Is Required for Inhibition of Autophagy in Neurons
(A) Representative electron micrographs of primary sympathetic neurons infected with the indicated virus. Arrows denote representative autophagosomes that would be scored as positive in (B). Scale bars, 1 μm.
(B) Quantitation of the number of autophagosomes per cell profile in primary sympathetic neurons infected with the indicated virus. Results shown represent mean value ± SEM for 50 cells per experimental condition.
Cell Host & Microbe 2007 1, 23-35DOI: ( /j.chom )
Copyright © 2007 Elsevier Inc. Terms and Conditions

7. Figure 6
An HSV-1 Recombinant Virus Containing a Mutation in ICP34.5 that Abrogates Binding to Beclin 1 Is Neuroattenuated In Vivo
(A) Survival of C57BL/6J mice infected intracerebrally with 5 × 105 pfu of either HSV Δ68–87 or its marker rescue (HSV Δ68–87R). Results shown represent survival data combined from four independent infections. Similar results were observed in each experiment.
(B) Viral replication of HSV Δ68–87 and HSV Δ68–87R in brain tissue of infected mice at indicated time after infection. Lower limit of detection = 1.7. Data shown represent mean ± SEM geometric titer for seven to ten mice per experimental group per time point.
(C) Representative images of H&E staining (left panels) and HSV-1 antigen staining (right panels) in basal ganglia from mice infected with HSV Δ68–87R (upper panels) or HSV Δ68–87 (bottom panels) on day 5 postinfection. Arrows in upper left panel indicate eosinophilic, necrotic neurons with pyknotic nuclei. Scale bars, 50 μm in left panels and 200 μm in right panels.
Cell Host & Microbe 2007 1, 23-35DOI: ( /j.chom )
Copyright © 2007 Elsevier Inc. Terms and Conditions

8. Figure 7
Restoration of Neurovirulence of a Beclin 1-Binding-Deficient Virus in pkr−/− Mice
(A) Survival of wild-type 129 Ev/Sv mice infected intracerebrally with 1 × 105 pfu of either HSV Δ68–87 or its marker rescue (HSV Δ68–87R). (B) Survival of 129 Ev/Sv pkr−/− mice infected intracerebrally with 1 × 105 pfu of either HSV Δ68–87 or its marker rescue (HSV Δ68–87R). (A and B) Results shown represent survival data combined from two to three independent infections. Similar results were observed in each experiment. (C) Viral replication of HSV Δ68–87 and HSV Δ68–87R in brain tissue of infected 129 Ev/Sv mice at indicated time after infection. (D) Viral replication of HSV Δ68–87 and HSV Δ68–87R in brain tissue of infected 129 Ev/Sv pkr−/− mice at indicated time after infection. For (C) and (D), data shown represent mean ± SEM geometric titer for three to five mice per experimental group per time point.
Cell Host & Microbe 2007 1, 23-35DOI: ( /j.chom )
Copyright © 2007 Elsevier Inc. Terms and Conditions