Uniqueness Reveals Weakness for Virus

The unique characteristics of a dangerous group of viruses may become their downfall. Researchers at HMS report the elucidation of the unusual machinery used by nonsegmented negative-sense (nsNS) RNA viruses to prepare mRNA for translation within host cells. The findings, in the May 30 Proceedings of the National Academy of Sciences, may contribute to new strategies for antiviral intervention.

Anatomy of a methylase. The viral polymerase (red, on left) found within vesicular stomatitis virus contains six regions (I–VI on right) that are conserved among all nonsegmented negative-sense RNA viruses. In this viral group, Region VI on the polymerase contains the unique methylase activity involved in mRNA capping, the molecular process that completes the nascent mRNA transcript. Experiments led by Sean Whelan modified amino acids in this region, revealing that multiple methylation reactions use a single binding site for the methyl donor, SAM.

Jianrong Li, a postdoctoral fellow in the lab of Sean Whelan, HMS assistant professor of microbiology and molecular genetics, along with research assistant Jennifer Wang, studied vesicular stomatitis virus (VSV), a relatively innocuous nsNS RNA virus. This viral group also contains several unsavory characters from measles and mumps (paramyxoviruses) to Ebola and Marburg (filoviruses).

As a virus prepares its RNA for translation, it appropriates the host cell’s ribosomes for protein production. The pathogen, however, must first use its own set of mRNA capping enzymes—a phosphatase, guanylyltransferase, and methyltransferase—to make the viral mRNA molecule stable and translatable by the host.

In nsNS viruses, the steps catalyzed by each of these enzymes occur in unique ways. Previously, other researchers had found that the nsNS viruses’ guanylyltransferase adds GDP to the cap, though in all other known capping reactions, the enzyme adds GMP. Whelan’s current study reveals the genetic and biochemical uniqueness of the methyltransferase, which carries out two methylations.

The methylase activities take place on a section of the much larger viral polymerase protein. Whelan and colleagues found that a single binding site for the methyl donor S-adenosyl-L-methionine (SAM) participates in both methylations. “It’s an economy from an evolutionary perspective,” he said. To uncover this, the researchers generated eight versions of VSV, each having amino acid substitutions within the binding region for the methyl donor. In these disabled versions of the virus, methylation of the cap decreased in vitro, and viral replication decreased in cell culture.

In three of the eight recombinant viruses, however, methylation still occurred on one of the two intended sites—either on a guanine or a ribose component of the cap. Whelan hypothesizes that the amino acids changed in these versions caused weakening but not complete inhibition of methyl-donor binding.

The discovery of this unique double-acting methylation site helps solidify the theory that VSV evolved an entirely separate, and efficient, capping mechanism from that of its hosts. These findings likely extend to other, more dangerous human pathogens in the nsNS RNA viral group such as rabies and Ebola.

This novelty in nsNS RNA viruses “might be an effective target to think about in developing therapeutics,” said Whelan. The virus has potential for use as a vaccine vector and as an oncolytic therapy. One challenge to exploiting VSV in this manner has been that the wild-type virus is not sufficiently attenuated and can cause disease in experimental animals. By crippling the methylase, it might be possible to generate stably attenuated versions of VSV.

—Kathleen Fink

No Deaf Child Left Behind: Expanding Screens for Hearing Loss

In the first days after birth, babies are subjected to a multitude of tests. Among them, hospitals screen each newborn for a variety of genetic conditions that with early detection and treatment can have dramatically improved outcomes. The genetic screens differ among states, but according to the National Newborn Screening and Genetics Resource Center, most test for phenylketonuria, congenital hypothyroidism, galactosemia, and sickle cell disease.

Cynthia Morton, the William Lambert Richardson professor of obstetrics, gynecology and reproductive biology and professor of pathology at HMS and Brigham and Women’s Hospital, and geneticist Walter Nance (HMS ’58) of the Medical College of Virginia want to add etiologically focused hearing loss screening to that list. Morton and Nance make their case in the May 18 New England Journal of Medicine.

Morton bases her recommendation on evidence that early identification of hearing loss can make a profound difference in a child’s language development. A study in the same issue of NEJM, for example, shows that children diagnosed with hearing loss before nine months of age have better language skills in mid-childhood compared with those diagnosed later. Research in 1998 found similar results for hearing loss identification and subsequent intervention in children before six months of age.

Almost all newborns in the United States are already screened for hearing loss using functional tests. These have value, but they also have limitations. Functional tests can miss genetic or viral conditions that cause hearing loss to set in weeks or months after the test. Functional tests also cannot always determine the root causes of hearing loss. “Etiology is so important,” said Morton. “We need to know if a child has hearing loss, and we need to know why. Something different might be done if we know the cause.” Additional tests may provide the etiology of a child’s hearing loss and assist doctors in determining whether certain interventions, such as a cochlear implant, would be beneficial.

Morton suggests, as a first step, screening those who fail functional tests for three known genetic defects and for cytomegalovirus, a common cause of lost hearing. As technology improves, molecular diagnostic DNA chips, such as the Deafness GeneChip being developed by Heidi Rehm, associate director of the HMS Center for Hereditary Deafness, and collaborators may allow efficient screening for many different hearing-associated genetic mutations. “This is our future,” said Morton. “When the genome sequence for each one of us costs a thousand dollars, that will be our newborn screen.”

—Elizabeth Dougherty

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