Thomas Michael Kristie, Ph.D.

Research in the Molecular Genetics Section focuses on the mechanisms involved in gene expression of the alpha-herpesviruses: herpes simplex (HSV) and varicella zoster (VZV). As with all classes of herpesviruses, infection with HSV or VZV results in a primary infection followed by the establishment of a lifelong latent infection. Periodically, these latent viruses can reactivate or re-enter the lytic replication state, resulting in recurrent disease.

For herpes simplex virus, infection results in establishment of latency specifically in neurons of sensory ganglia, and reactivation produces disease that can range from mild oral or genital lesions to keratoconjunctivitis, blindness, neurological issues, and encephalitis.

Credit: NIAIDFigure 1

Figure 1. The cycles of viral lytic and latency-reactivation. Upon primary infection with herpes simplex virus, the viral genome is injected into the nucleus, and viral genes are expressed in a regulated, sequential manner beginning with IE. IE products are required for the expression of later E and L genes. Progeny virus may then infect local sensory neurons where the virus becomes quiescent and lytic genes are not expressed. Various stress stimuli result in re-entry into the lytic replication cycle and production of virus that is released from the neurons and re-infects cells to produce recurrent disease.

As with its cellular hosts, viral gene expression is tightly regulated, and expression is dictated by a complex series of cellular and viral regulatory proteins that promote the expression of several classes of genes in a sequential manner (Immediate Early – Early – DNA replication – Late). The expression of Immediate Early (IE) genes is induced rapidly upon initial lytic infection. Importantly, coordinated expression of these genes is also critical for the initiation of reactivation from latency.

IE genes are exceptionally complex and are regulated by multiple families of transcription factors and coactivators.

Credit: NIAIDFigure 2

Figure 2. Complex regulation of viral IE gene transcription. Multiple cellular and viral DNA binding transcription factors of distinct families bind regulatory sites in the viral IE promoter-enhancer domains. The primary means of regulation is via the assembly of enhancer complexes that include the cellular coactivator HCF-1. This protein also interacts and mediates the transcriptional potential of many factors that contribute to IE transcription. (Cellular transcription factors and coactivators, GAPB, Sp1, OCT-1 CREB3, FHL2; Viral activators, VP16, ORF10, IE62).

A critical component of this reguatory network is the cellular transcriptional activator HCF-1. While multiple DNA binding transcription factors regulate IE genes, this coactivator HCF-1 interacts with and modulates the transcriptional potential.

In addition to these direct factors, the virus is also regulated by an overlay of regulation that is determined by the host cell’s chromatin modulation machinery. Recent data have demonstrated that HCF-1 interacts with or is a component of numerous cellular chromatin modulation complexes, and it is in this capacity that it plays a significant role in modulating viral IE gene expression. Studies in the MGS are focused on the role of HCF-1-mediated chromatin control of HSV and other herpesvirus infection.

Credit: NIAIDFigure 3

Figure 3. HCF-1 chromatin modulation complexes participate in regulation of viral IE genes. HCF-1 interacts with or is a component of multiple chromatin modulation complexes including histone acetyltransferases (ATAC, MOF), deacetylases (Sin3A), methyltransferases (Set1/MLLs), demethylases (LSD1, JMJD2s, PHF8), and histone chaperones (Asf1). Upon infection, the virus accumulates nucleosomes bearing repressive chromatin marks on viral IE promoter regions (e.g., histone H3K9-methylation). An HCF-1 complex recruited to the viral IE promoters contains required demethylases and methyltransferases to convert these marks to transcriptionally active states (e.g., histone H3K4-methylation).

In addition to its role in regulation of the viral IE gene expression upon initial infection, HCF-1 also plays a significant role in replication of the viral genome, presumably via required chromatin modulation of DNA replication. The protein interacts with histone chaperones and localizes to foci of viral DNA synthesis at later times in infection. Continuing projects investigate the HCF-1 role and complexes in both viral and cellular DNA replication.

Credit: NIAIDIncorporation of HCF-1 in viral replication factories

Figure 4. Incorporation of HCF-1 in viral replication factories. Later in infection, viral replication foci coalesce into replication factories (characterized by staining with the viral-encoded, single-stranded DNA binding protein UL29).

While HCF-1 is a critical component of the viral lytic replication cycle, it is also hypothesized to be a determinant of the latency-reactivation cycles. The MGS has demonstrated that HCF-1 is uniquely sequestered in the cytoplasm of sensory neurons where the virus establishes latency and is rapidly transported to the nucleus upon stimulation that results in viral reactivation. The MGS continues to investigate the role of HCF-1, its associated factors, and chromatin modulation components in the control of latency-reactivation.

Credit: NIAIDFigure 5

Figure 5. HCF-1 is sequestered in the cytoplasm of sensory neurons and is rapidly relocalized to the nucleus upon stimuli that results in viral reactivation from latency.Immunohistochemical staining of unstimulated sensory ganglia (trigeminal ganglia) shows punctate cytoplasmic localization of HCF-1. Stimulation results in the accumulation of HCF-1 in the nuclei.

These studies have also led to the development of novel chromatin modulation inhibitors that block viral gene expression and provide proof of principle for the development of epigenetic inhibitors as novel antivirals.

Credit: NIAIDFigure 6

Figure 6. Inhibition of HCF-1-associated chromatin modulation enzymes blocks viral reactivation from latency. TCP (tranylcypromine), a Mono-Amine Oxidase Inhibitor (MAOI) inhibits the activity of the histone H3K9 demethylase LSD1. Reactivation of HSV-1 from latently infected sensory ganglia is blocked by MAOIs and other LSD1 inhibitors, resulting in suppression of viral progeny production. Immunofluorescent staining of stimulated ganglia shows neurons undergoing viral reactivation (Control) as determined by expression of the viral single strand binding protein UL29. Acyclovir (ACV), a DNA replication inhibitor, does not block the expression of viral proteins and the initiation of viral reactivation. In contrast, MAOIs prevent expression of viral IE genes and suppress reactivation at early stages.

Dr. Kristie received his Ph.D. from the Committee on Virology at the University of Chicago for his work with Dr. Bernard Roizman on the regulation of herpes simplex virus gene expression. As a postdoctoral fellow with Dr. Philip Sharp at the Center for Cancer Research, Massachusetts Institute of Technology, Dr. Kristie focused on the interaction of components involved in the formation of transcriptional enhancer complexes. Dr. Kristie joined the NIAID Laboratory of Viral Diseases in 1993, became a senior investigator in 2000, and became chief of the Molecular Genetics Section in 2001.

Memberships

• American Association for the Advancement of Science
• American Society for Biochemistry and Molecular Biology
• American Society for Microbiology

Advisory Boards

• Journal of Biomedical Science
• JOVE

Awards

• 2009 Norman P. Salzman Memorial Mentor Award in Virology
• 2010 NIAID Merit Award

  For insights into the molecular mechanisms regulating the transcriptional program of herpesviruses that have led to the identification of new therapeutic targets

• 2012 Elected Fellow, American Academy of Microbiology