Discovery of antivirals against smallpox Stephen C. Harrisona,b, Bruce Albertsc, Ellie Ehrenfeldd, Lynn Enquiste, Harvey Finebergf, Steven L. McKnightg, Bernard Mossh, Michael O’Donnelli, Hidde Ploeghj, Sandra L. Schmidk, K. Peter Walterl, and Julie Theriotm aHarvard Medical School, Howard Hughes Medical Institute, Seeley Mudd Building, Room 130, 250 Longwood Avenue, Boston, MA 02115; cNational Academy of Sciences, 2101 Constitution Avenue, NW, Washington, DC 20418; dLaboratory of Infectious Disease, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Building 50, Room 6120, 50 South Drive, Bethesda, MD 20892; ePrinceton University, 314 Schultz Laboratory, Washington Road, Princeton, NJ 08544; fInstitute of Medicine, 2101 Constitution Avenue, NW, Washington, DC 20418; gDepartment of Biochemistry, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75390; hLaboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Building 4, Room 229, 4 Center Drive, Bethesda, MD 20892; iLaboratory of DNA Replication, The Rockefeller University, Howard Hughes Medical Institute, 1230 York Avenue, New York, NY 10021; jDepartment of Pathology, Harvard Medical School, NRB, 77 Avenue Louis Pasteur, Boston, MA 02115; kDepartment of Cell Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037; lDepartment of Biochemistry and Biophysics, University of California School of Medicine, Howard Hughes Medical Institute, Box 0448, HSE 1001, San Francisco, CA 94143; and mDepartment of Biochemistry, Stanford University School of Medicine, Stanford, CA 94305 Contributed by Stephen C. Harrison, May 21, 2004 mallpox, a devastating infectious Whatever the likelihood of covertly dopoxviruses has a restricted and spe- disease dreaded throughout much held variola virus stocks, an intentional cific host array (Table 2). Humans are of recorded history, is caused by release of the virus would pose a serious the sole hosts of two poxviruses, variola the variola virus, a member of health threat and would probably pro- virus (smallpox virus) and molluscum Sthe poxviridae family. In the 20th cen- voke a global health crisis. The lethality contagiosum virus, although many mem- tury alone, smallpox deaths worldwide of the disease (up to 40%) and its ease bers of Orthopoxvirus, Parapoxvirus, and numbered in the millions. In 1980, after of transmissibility place variola virus at Yatapoxvirus can infect both animals and an intensive program of immunization the top of CDC’s list of high-threat humans (Table 2, zoonotic viruses). with vaccinia virus, a related but rela- (Category A) agents. For that reason, Vaccinia virus is the virus used in the tively nonpathogenic virus, the World the federal government rapidly in- variola virus vaccine, and it is widely Health Organization (WHO) declared creased its support of research related used as a model poxvirus in the labora- the disease eradicated. By 1983, all to the discovery of antiviral drugs tory. Its origins are obscure. known stocks of variola virus were in against smallpox. In addition to provid- The genomes of 30 poxviruses have two WHO collaborating centers: the ing potential therapy for infected peo- been completely sequenced, including U.S. Centers for Disease Control and ple, the availability of antiviral drugs several strains of variola and vaccinia Prevention (CDC) in Atlanta and (after could decrease the risks associated with viruses. At least two variants of variola a transfer in 1994) the Russian State the smallpox vaccine by providing a virus are known, and they cause two Research Center of Virology and Bio- treatment for vaccine-associated compli- forms of smallpox: variola major, with technology (the Vektor Institute) in cations. Ultimately, the development of a case fatality rate of 30–40%, and Novosibirsk. The WHO Committee on antiviral drugs against smallpox could variola minor, with a much reduced deter rogue states and terrorists from Orthopoxvirus Infections voted on sev- fatality rate of Ϸ1%. At the genome releasing variola virus because its impact eral occasions to recommend destruc- level, the two variants are very similar. would be diminished. Moreover, the tion of the stocks, but each time the They differ in Ϸ2% of the roughly same new therapies are likely to be use- decision was deferred to permit more 185,000 unique DNA nucleotides; es- ful in other poxvirus diseases, such as research on live variola virus. A 1999 sentially all of the encoded proteins monkeypox. National Academies report summarized are nearly identical. These comparisons suggest that the high fatality rate asso- and assessed scientific needs for live Scientific Opportunities variola virus (1). ciated with variola major may be at- Introduction to Poxviruses.n The poxvirus tributable to a small number of genetic The concern that undeclared stocks of family. Poxviruses are the largest known determinants, and they point to our variola virus might exist and that they animal viruses, with Ϸ200 distinct genes ignorance of the causes of poxvirus might be used as a bioterrorist weapon (5). They are DNA viruses that replicate pathogenesis in general and of variola (2) was heightened in late 2001 by the entirely in the cytoplasm. Thus, a subset virus pathogenesis in particular. Eradi- deliberate release of Bacillus anthracis, of their gene products carries out the the agent of anthrax, in the weeks after functions that are essential for the vi- the September 11, 2001, attacks. That ruses to be independent of the host-cell Abbreviations: WHO, World Health Organization; CDC, U.S. concern prompted a voluntary national- nucleus. The other viral gene products Centers for Disease Control and Prevention; IMV, intracel- preparedness effort to vaccinate health- lular mature vaccinia virion; EEV, extracellular enveloped use or modulate an astonishingly wide virion; ITR, inverted terminal repetition; IEV, intracellular care workers, first responders, and array of host-cell and immune-system enveloped virion; CEV, cell-associated virion; UDG, uracil members of the military against small- processes. DNA glycosylase; IV, immature virion; NIH, National Insti- pox. However, given the substantial side Poxviruses infect most vertebrates and tutes of Health; SARS, severe acute respiratory syndrome; effects, the risks associated with the FDA, Food and Drug Administration; DHHS, Department of invertebrates, causing a variety of dis- Health and Human Services; NIAID, National Institute of smallpox vaccine, and the absence of eases of veterinary and medical impor- Allergy and Infectious Diseases; BSL, biosafety level. information about an imminent bioter- tance. The one large family (Poxviridae) bTo whom correspondence should be addressed. E-mail: rorist attack, vaccination was not ac- has two main subfamilies, the chor- [email protected]. cepted by all members of those groups, dopoxvirinae, which infect vertebrates, nAdapted with permission from ref. 4 (Copyright 2003, ASM nor was it recommended for the general and the entomopoxvirinae, which infect Press). public by the government (3). insects (Table 1). Each of the chor- © 2004 by The National Academy of Sciences of the USA 11178–11192 ͉ PNAS ͉ August 3, 2004 ͉ vol. 101 ͉ no. 31 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0403600101 FROM THE ACADEMY: PERSPECTIVE Table 1. The poxvirus family: Poxviridae Subfamily Genus Type species Chordopoxvirinae Orthopoxvirus Vaccinia virus (vertebrate hosts) Parapoxvirus Orf virus Avipoxvirus Fowlpox virus Capripoxvirus Sheeppox virus Leporipoxvirus Myxoma virus Suipoxvirus Swinepox virus Molluscipoxvirus Molluscum contagiosum virus Yatapoxvirus Yaba monkey tumor virus Entomopoxvirinae Entomopoxvirus A Melolontha melolontha entomopoxvirus (MMEV) (insect hosts) Entomopoxvirus B Amsacta moorei entomopoxvirus (AMEV) Entomopoxvirus C Chironomus luridus entomopoxvirus (CLEV) cation of smallpox occurred at a time rus biology is derived largely from stud- biology are in place to dissect the virus– of limited knowledge of the molecular ies of this virus. The tools available for cell interactions. and cellular nature of the relevant host its study now include large collections of Poxvirus replication cycle. Poxviruses have a defense. Thus, during the times when it temperature-sensitive mutant strains as complex structure. Fig. 1A shows an was possible to study human infections, well as recombinant strains with specific electron micrograph of a cross section the molecular tools for dissecting the genes under inducible regulation. Sev- of the infectious intracellular mature host response were lacking. eral of the known gene products, such vaccinia virion (IMV) and a schematic Vaccinia virus has become the model as its enzymes, can be expressed and summary of the virion. The nature of the of choice, and our knowledge of poxvi- studied in vitro, and new tools of cell membrane envelope surrounding the IMV Table 2. Hosts of and infections caused by chordopoxvirinae Animal Virus reservoir Geographic distribution Transmission to other hosts Genus Molluscipoxvirus Molluscum contagiosum Humans Worldwide No transmission to other hosts Genus Orthopoxvirus Zoonotic viruses Monkeypox virus* Squirrels West and Central Africa Monkeys, humans Vaccinia virus Unknown Worldwide Humans, buffaloes, rabbits, cows Buffalopox virus† Unknown Asia Buffaloes, humans Camelpox virus Camels Africa, Asia Camels Cowpox virus‡ Rodents Europe, Asia Cats,