1993, 67(8):4484. J. Virol. Arnheiter W J Mitchell, R J De Santo, S D Zhang, W F Odenwald and H protein Hox 1.3. homeodomain transgenic mice is altered by the Herpes simplex virus pathogenesis in http://jvi.asm.org/content/67/8/4484 Updated information and services can be found at: These include: CONTENT ALERTS more�� cite this article), Receive: RSS Feeds, eTOCs, free email alerts (when new articles http://jvi.asm.org/site/misc/reprints.xhtml Information about commercial reprint orders: http://journals.asm.org/site/subscriptions/ To subscribe to to another ASM Journal go to: on February 6, 2012 by University of Groningen http://jvi.asm.org/ Downloaded from

JOURNAL OF VIROLOGY, Aug. 1993, p. 4484-4491 0022-538X/93/084484-08$02.00/0 Copyright ��) 1993, American Society for Microbiology Herpes Simplex Virus Pathogenesis in Transgenic Mice Is Altered by the Homeodomain Protein Hox 1.3 WILLIAM J. MITCHELL,`* RONALD J. DE SANTO,2 SHANG-DING ZHANG,3 WARD F. ODENWALD,3 AND HEINZ ARNHEITER2 Laboratory of Experimental Neuropathology, 1 Laboratory of Viral and Molecular Pathogenesis, 2 and Neurogenetics Unit, Laboratory of Neurochemistry,3 National Institute of Neurological Disorders and Stroke, Bethesda, Maryland 20892 Received 19 June 1992/Accepted 26 April 1993 The DNA sequence TAAT is the core binding motif for the mouse homeodomain protein Hox 1.3 (proposed new name, Hoxa-5). These sequences are present within the multiple TAATGARAT regulatory motifs in the promoters of the immediate-early genes which control herpes simplex virus type 1 replication. To investigate the role of this homeodomain protein in the regulation of herpes simplex virus gene expression and pathogenesis, transgenic mice containing a mouse Hox 1.3 cDNA under the control of the virus- and interferon-inducible Mx 1 promoter were generated. After infection of transgenic mice with herpes simplex virus, Hox 1.3 RNA and protein were expressed at the sites ofvirus replication. In these transgenic mice, herpes simplex virus replication, spread of virus through the host, and virus-induced mortality were markedly enhanced. Increased spread and replication of herpes simplex virus were also observed in cultured fibroblasts from transgenic mice. This finding suggests that in vivo, Hox 1.3 may increase viral spread by increasing viral replication at the level of the individual infected cells. These results demonstrate that expression of a transgene encoding a single host protein, Hox 1.3, alters the pathogenesis of experimental herpes simplex virus infection. We conclude that a protein that belongs to a class of DNA-binding proteins which are best known for their role in regulating embryonic development may also regulate herpesvirus pathogenesis. Infections with herpes simplex virus (HSV), though often asymptomatic, may cause a variety of human diseases, including recurrent genital and orofacial lesions, keratitis, conjunctivitis, encephalitis, and disseminated infections of newborns (28). Host factors are partly responsible for deter- mining the outcome of infection. The relative contributions of these factors are difficult to assess in humans but can be studied more readily in animal models. HSV infections in mice are influenced by the host genetic background, and multiple dominantly inherited genes are involved (10). Even though these results have been known for a long period of time, no particular host gene that can control the outcome of experimental HSV-1 infection in animals has so far been identified. We have examined the question of whether host genes which encode transcriptional regulators can control HSV-1 pathogenesis in transgenic mice. Regulation of viral immediate-early (IE) genes controls HSV-1 gene expression (24) and may determine the outcome of infections such as the level of acute replication or the commitment to a latent infection in which HSV expression is restricted within neurons. Viral genes are divided into three classes (IE, early, and late) which are coordinately ex- pressed (24). In vitro, HSV-1 IE genes are regulated in part by the interaction of the TAATGARAT motifs with protein complexes composed of viral and host proteins (13, 21, 22, 26). A complex composed of the viral protein VP16 (also termed a-TIF or VMW65), the host octamer-binding protein Oct-1, and other host proteins serves as an activator of IE gene transcription. The octamer-binding protein Oct-2 may act as a repressor of IE gene transcription in neuronal cell types (9). Since binding sites are present for a number of host * Corresponding author. transcription factors in the regulatory sequences of the HSV-1 IE genes (8, 13, 18, 21, 22, 26), it is reasonable to assume that these proteins may play a role in controlling viral replication in animals. Binding sites containing the TAAT core binding motif for the mouse homeodomain protein Hox 1.3 (proposed new name, Hoxa-5) (25) are present within the TAATGARAT regulatory sequences of the promoters of HSV-1 IE genes (18, 19). To test whether a host protein which is a sequence-specific DNA-binding protein could affect the regulation of HSV-1 and thus the pathogenesis of HSV-1-induced disease, transgenic mice in which the Hox 1.3 protein is expressed under the control of a virus-inducible regulatory element were generated. We found that in these transgenic mice, HSV-1 infection leads to induction of the transgene at the sites of viral replication and that Hox 1.3 expression leads to an alteration of viral pathogenesis. This model shows that a protein belonging to a class of proteins which are best known for their role in development has a profound effect on viral pathogenesis. MATERIALS AND METHODS Transgenic mice. The cDNA insert of cAmb-1 (20) was restricted with SnaBI and ScaT, and the fragment containing the Hox 1.3 cDNA (nucleotides -120 through +872) was isolated. Plasmid pSS-2-2 (unpublished data) containing the Mx promoter (5) and the mouse 3-globin sequence (7) cloned into pSP65 was linearized at the single BamHI site at the junction between the promoter and the ,-globin sequence. The linearized plasmid and the Hox 1.3 cDNA were made blunt ended with Klenow enzyme and ligated. The 4.3-kb XbaI fragment (Fig. 1A) from the final construct was isolated and purified, and approximately 200 copies were injected (1, 3) into each (C57BL/6 x C3H)F1 x (C57BL/6 x C3H)F1 one-cell embryo. Transgenic founders were identified by 4484 Vol. 67, No. 8 on February 6, 2012 by University of Groningen http://jvi.asm.org/ Downloaded from