Biography of Patricia G. Spear

Herpes infections are caused by two genetically related viruses or serotypes: herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2). HSV-1 infection usually manifests as oral fever blisters, whereas HSV-2 causes genital lesions. Both serotypes can establish lifelong latent infections, and HSV infections can cause significant morbidity in immunocompromised individuals, such as those who receive organ transplants or are infected with HIV. In rare cases, HSV infections can be fatal to newborns and can cause encephalitis or meningitis in adults. Much of our current understanding of the virus is a result of the pioneering work of Patricia G. Spear. Through years of dedication, she has identified and characterized the proteins present on the surfaces of viruses and cells that allow HSV to enter its host.

Figure 1.

Patricia G. Spear

In recognition of her seminal research on HSV, Spear was elected to the American Academy of Microbiology in 1999 and to the American Academy of Arts and Sciences in 2003. For 16 years, she was chair of the department of microbiology and immunology at Northwestern University (Evanston, IL), and she recently completed a term as president of the American Society for Virology. In 2002, Spear was elected to the National Academy of Sciences. In her Inaugural Article, published in this issue of PNAS (1), she demonstrates that amino acid substitutions in HSV glycoprotein D (gD) reduce gD binding to one of its cellular receptors, nectin-1. These substitutions selectively abrogate viral entry and cell fusion via nectins but not via other receptors (1). Nectins are likely to be the principal receptors that HSV uses to enter neurons, the cell type in which HSV establishes latent infection. Thus, HSV mutant strains that cannot enter neurons via nectin binding could be candidates for a safe, live-virus vaccine.

The Beginning of a Prolific Career

In 1960, Spear enrolled in Florida State University in Tallahassee on a nursing scholarship, but, early on, she suspected that nursing was not for her. Spear distinctly remembers that her honors adviser, Laurence Chalmers, sensed that she was uncertain about a life in medicine. “He said after our first meeting, `You should come back and talk to me when you want to change majors,”' she recalls. With Chalmers' support, she switched her major to bacteriology, a course that she was enjoying at the time. In 1964, Spear received a B.A. in bacteriology with a minor in chemistry. She remained at Florida State for one year in a master's program in bacteriology. In search of more rigorous scientific training, Spear then enrolled in the doctoral program in virology at the University of Chicago.

At Chicago, Spear conducted her thesis research on HSV under the guidance of Bernard Roizman. She remembers, “I was just fascinated with what the virus did to cells. I thought it was mind-blowing how the virus could change the shape and behavior of the cell before killing it off.” This fascination marked the beginning of a career devoted almost exclusively to HSV research. During her graduate school days, Spear was among the first to identify the many protein and glycoprotein constituents of HSV. With some difficulty, she purified the virion from infected cells and used electrophoresis to determine the number of viral proteins (2). “You have to remember that in those days, the late'60s, we didn't have the tools available today,” she recalls fondly. “The biggest advances at that time included the Laemmli gels that allowed you to separate proteins of different sizes by electrophoresis! It became clear that the virion had at least thirty proteins, if not more, so it was a much more complicated structure than anyone had realized.” After receiving her Ph.D. in virology in 1969, Spear remained in Roizman's lab for 2 years as a postdoctoral fellow, continuing to characterize the vast array of HSV virion proteins. She credits her time with Roizman as having the greatest impact on her career: “It was from Bernard and my fellow graduate students that I first really experienced the joys and frustrations of research.”

In 1971, Spear went to The Rockefeller University (New York) as a postdoctoral fellow in the laboratory of Gerald Edelman, the 1972 Nobel Laureate in Physiology or Medicine. She joined Edelman's group with the aim of learning more about protein chemistry but received intensive training in immunology instead. “His lab had recently completed the first sequence of an immunoglobulin the hard way—true protein sequencing—and that was part of the work that won him the Nobel Prize,” she says. As a natural corollary to his Nobel work, Edelman wanted to delve into cellular immunology and cell–cell interactions. Thus, Spear studied the developing immune system in mice, determining when the spleen and thymus became populated with functional T cells and B cells (3, 4).

Two years later, now armed with training in bacteriology, virology, and immunology, Spear became an assistant professor in the department of microbiology at her alma mater, the University of Chicago. Returning to her graduate training, she chose to make HSV her research focus: “I was able to use my graduate school credentials to get funding for my initial work as a faculty member. I had my own lab, and I had grants even before I started. Back then, you could get a grant with a good idea.” Like most new faculty, Spear was slightly apprehensive about distinguishing her work from her former adviser's work; however, Spear quickly established an independent line of research. She recalls, “I was able to make my mark at the University of Chicago by developing a new area of research studying HSV glycoproteins,” and she was promoted to associate professor in 1978 and to full professor in 1982. During her tenure at Chicago, she described various glycoproteins of the HSV viral envelop: “This was slow, slogging work of developing antibodies, trying to characterize which glycoproteins were in the virion, and analyzing viral mutations.” Well worth the toil, Spear's research established the modern framework for understanding HSV infection.

HSV Glycoproteins and Viral Binding

In addition to describing the surface glycoproteins, during her early years at Chicago, Spear characterized HSV glycoprotein functions in cell fusion and immune responses. By examining temperature-sensitive viral mutants, she determined that one of the viral glycoproteins, gB, promotes cell fusion whereas another, gC, could modulate or suppress fusion activity (5). Cell fusion occurs in HSV lesions in vivo and may be one mechanism by which the virus spreads from cell to cell. In addition, Spear and colleagues determined that viral glycoprotein gE bound the Fc region of immunoglobulin G (6) and that gC could protect virus from neutralization by complement (7).

Later, Spear turned her focus to the cellular side of the HSV infection equation by searching for cell-surface receptors that were important for viral entry. She determined that heparan sulfate, a carbohydrate component of certain proteoglycans, serves as the initial cell-surface receptor for both HSV serotypes (8–11). “We had a hard time convincing the world that [heparan sulfate] was important and that it wasn't just a non-specific binding interaction,” she remembers. Nevertheless, by showing that virus binding to heparan sulfate-deficient cells was severely impaired, she and her colleagues demonstrated that heparan sulfate was necessary for the virus to efficiently infect the cell (8, 10). Furthermore, analysis of viral mutants revealed that two of the viral glycoproteins, gB and gC, could mediate binding of HSV to cell-surface heparan sulfate (9, 11). According to Spears, this work “opened up a cottage industry, and, since then, it has been shown that many viruses, including almost all of the herpesviruses, interact with heparan sulfate.”

Cellular Entry Receptors

In 1987, Spear was recruited to Northwestern University Medical School as chair of the department of microbiology and immunology. “Moving to Northwestern was the best career move I ever made,” she said of the highly collegial environment. At Northwestern, Spear continued to research viral binding to cell-surface heparan sulfate but knew that this interaction was not sufficient for viral penetration into the cell: “We realized that heparan sulfate wasn't the whole story and that, although binding to heparan sulfate might be essential for HSV to grab on to a cell, there had to be other receptors involved.”

Figure 2.

Current members of the Spear lab group. Back row from the left: Nanette Susmarski, Sharmila Manoj, Miri Yoon, Joann Taylor, Erick Lin, Craig Steffel, Melissa McNeely, Frank Struyf, and Ann Zago. Seated from the left: Cheryl Jogger, Dr. Spear, and Nancy Ruel.

Working with several students and postdoctoral fellows, Spear began a labor-intensive project of expression cloning to identify the cell-surface proteins that mediate HSV entry. As an experimental tool, they used Chinese hamster ovary (CHO) cells, which expressed heparan sulfate on the surface but were resistant to viral entry. By transfecting the CHO cells with cDNA libraries from HSV-susceptible human cells, they identified plasmids that could render CHO cells vulnerable to viral penetration. Ultimately, Spear and colleagues identified three different classes of entry receptors, discoveries that she considers her greatest contribution to HSV research.

“Moving to Northwestern was the best career move I ever made.”

The first mediator of HSV entry to be identified was a cell-surface protein called herpes virus entry mediator (HVEM), cloned by Rebecca Montgomery (12). Next, Morgyn Warner and Robert Geraghty characterized two members of the immunoglobulin superfamily, nectin-1 (originally HveC) and nectin-2 (originally HveB), as the second class of HSV entry receptors (13, 14). Nectin-1 is expressed in human cells of epithelial and neuronal origin and is the prime candidate receptor that allows HSV to spread on mucosal surfaces and infect cells of the nervous system.

The third class of HSV entry receptors emerged when Spear's postdoctoral fellow, Deepak Shukla, was cloning the mouse homologs of HVEM, nectin-1, and nectin-2. “He discovered something unusual—that the enzyme 3-O-sulfotransferase could actually make the CHO cells susceptible to HSV entry,” said Spear. Through an extensive collaboration with Robert Rosenberg at the Massachusetts Institute of Technology in Cambridge, MA, Jeffrey Esko at the University of California at San Diego, and Gary Cohen and Roselyn Eisenberg at the University of Pennsylvania in Philadelphia, Spear and Shukla determined that 3-O-sulfotransferase generated viral entry receptors by modifying cell-surface heparan sulfate (15). Spear recalls this serendipitous finding with amusement, “So now we had heparan sulfate playing two roles: plain vanilla heparan sulfate mediating virus binding and interactions with the two viral glycoproteins gB and gC, and the specially modified 3-O-sulfated heparan sulfate acting as an entry receptor.” Through collaborations with Gary Cohen and Roselyn Eisenberg, Spear established that HSV gD is the ligand for all three known classes of HSV entry receptors (15–17).

A New Paradigm for Viral Entry

Currently, Spear has two major research foci, which she addresses in her Inaugural Article (1). First, she and her colleagues are working to understand how the virus physically fuses with the cell membrane. They already have determined that fusion depends on the expression of an entry receptor for gD as well as four viral glycoproteins: gB, gD, gH, and gL (18). Given this, Spear says the obvious question is, “Why does this virus require four different glycoproteins and a gD receptor to initiate fusion when many viruses can do it with one glycoprotein and receptor?” The second major focus of her research is to understand the relative roles of all of the cellular entry receptors in HSV disease, such as how the differential expression of entry receptors on various cell populations influences infection and pathogenesis.

In her Inaugural Article (1), Spear and colleagues used mutational analysis to determine which domains of viral gD interact with the various entry receptors, HVEM, nectin-1, nectin-2, or 3-O-sulfated heparan sulfate, to mediate fusion (1, 19). They generated a panel of gD mutants, all of which exhibit restricted receptor usage, but each of which retains activity with a different receptor or set of receptors. “Now, when we build these mutations back into the virus,” she says, “we can challenge mice with these viruses and see what kind of cells become infected and how this restriction of receptor specificity alters pathogenesis.” Spear speculates “that the virus probably uses different entry receptors to get into different cell types.” For example, Spear postulates that the entry receptor most important for infecting cells of epithelial and neuronal origin is probably nectin-1, whereas HVEM is probably most important for infection of lymphocytes and other leukocytes (1, 12).

“Why does this virus require four different glycoproteins and a gD receptor?”

The larger implications for the work described in Spear's Inaugural Article (1) are on the potential impact on vaccine development. HSV can establish latent infections in neurons, persisting in the body for life. Live-virus vaccines are more effective than vaccines made of killed virus or viral protein subunits, yet Spear cautions that live HSV has the potential to establish permanent infection: “This has been a hang up for HSV vaccine development. However, if the viral mutants cannot use nectins as an entry receptor, they might not be able to infect neurons, but yet retain ability to infect other cells.” To circumvent this, Spear says a transient infection with live mutant viruses would allow the body to develop immunity without risking latent infection: “If you can use a live virus vaccine that doesn't get into neurons, you might have an effective, safe vaccine.”

Spear recently has found some more time to focus on her work on virus–cell fusion and differential cell infection. After serving as chair of the department of microbiology and immunology at Northwestern for 16 years, Spear stepped down in 2003, and she is currently on research sabbatical in her own laboratory. Spear concedes that, despite the enormity of her previous research findings, there are still many questions to solve: “Before our work with HSV and others' work with HIV, people tended to think of viral entry as: a virus binds a single receptor, presto chango, it's in the cell. I think we have broadened people's perspectives that viral entry is a much more complicated process than any of us imagined.”