Genetic interactions between an essential 3' cis-acting RNA pseudoknot, replicase gene products, and the extreme 3' end of the mouse coronavirus genome.

The upstream end of the 3' untranslated region (UTR) of the mouse hepatitis virus genome contains two essential and overlapping RNA secondary structures, a bulged stem-loop and a pseudoknot, which have been proposed to be elements of a molecular switch that is critical for viral RNA synthesis. It has previously been shown that a particular six-base insertion in loop 1 of the pseudoknot is extremely deleterious to the virus. We have now isolated multiple independent second-site revertants of the loop 1 insertion mutant, and we used reverse-genetics methods to confirm the identities of suppressor mutations that could compensate for the original insertion. The suppressors were localized to two separate regions of the genome. Members of one class of suppressor were mapped to the portions of gene 1 that encode nsp8 and nsp9, thereby providing the first evidence for specific interactions between coronavirus replicase gene products and a cis-acting genomic RNA element. The second class of suppressor was mapped to the extreme 3' end of the genome, a result which pointed to the existence of a direct base-pairing interaction between loop 1 of the pseudoknot and the genomic terminus. The latter finding was strongly supported by phylogenetic evidence and by the construction of a deletion mutant that reduced the 3' UTR to its minimal essential elements. Taken together, the interactions revealed by the two classes of suppressors suggest a model for the initiation of coronavirus negative-strand RNA synthesis.

A hypervariable region within the 3' cis-acting element of the murine coronavirus genome is nonessential for RNA synthesis but affects pathogenesis.

The 3' cis-acting element for mouse hepatitis virus (MHV) RNA synthesis resides entirely within the 301-nucleotide 3' untranslated region (3' UTR) of the viral genome and consists of three regions. Encompassing the upstream end of the 3' UTR are a bulged stem-loop and an overlapping RNA pseudoknot, both of which are essential to MHV and common to all group 2 coronaviruses. At the downstream end of the genome is the minimal signal for initiation of negative-strand RNA synthesis. Between these two ends is a hypervariable region (HVR) that is only poorly conserved between MHV and other group 2 coronaviruses. Paradoxically, buried within the HVR is an octanucleotide motif (oct), 5'-GGAAGAGC-3', which is almost universally conserved in coronaviruses and is therefore assumed to have a critical biological function. We conducted an extensive mutational analysis of the HVR. Surprisingly, this region tolerated numerous deletions, rearrangements, and point mutations. Most striking, a mutant deleted of the entire HVR was only minimally impaired in tissue culture relative to the wild type. By contrast, the HVR deletion mutant was highly attenuated in mice, causing no signs of clinical disease and minimal weight loss compared to wild-type virus. Correspondingly, replication of the HVR deletion mutant in the brains of mice was greatly reduced compared to that of the wild type. Our results show that neither the HVR nor oct is essential for the basic mechanism of MHV RNA synthesis in tissue culture. However, the HVR appears to play a significant role in viral pathogenesis.

Magnetic resonance elastography of the plantar fat pads: Preliminary study in diabetic patients and asymptomatic volunteers.

OBJECTIVE: To test the feasibility of applying the magnetic resonance elastography (MRE) technique to map the elastic modulus of the plantar fat pads in diabetic and nondiabetic subjects. METHODS: A prototype MRE imaging apparatus was used to produce quantitative maps of the heel fat pad in a pilot study of 12 volunteers and 4 patients with diabetes with neuropathy. Anatomic images corresponding to MRE maps allowed precise selection of regions of interest in the fat. RESULTS: Magnetic resonance elastograms of the heel fat pads were successfully created; mean measurements in the volunteers and the diabetic patients were 4.85 and 5.26 kPa, respectively. CONCLUSION: It is feasible to perform MRE on the plantar fat pads and to produce elasticity maps. The trend toward stiffer fat pads, as demonstrated in patients with diabetes, suggests that the fat pads were qualitatively different. Magnetic resonance elastography offers great potential to investigate the mechanical properties of soft tissues in vivo noninvasively.

Cutting edge: virus-specific CD4+ memory T cells in nonlymphoid tissues express a highly activated phenotype.

Recent studies have shown that CD4(+) memory T cells persist in nonlymphoid organs following infections. However, the development and phenotype of these peripheral memory cells are poorly defined. In this study, multimerized MHC-Ig fusion proteins, with a covalently attached peptide sequence from the Sendai virus hemagglutinin/neuraminidase gene, have been used to identify virus-specific CD4(+) T cells during Sendai virus infection and the establishment of peripheral CD4(+) memory populations in the lungs. We show declining frequencies of virus-specific CD4(+) T cells in the lungs over the course of approximately 3 mo after infection. Like peripheral CD8(+) T cells, the CD4(+) have an acutely activated phenotype, suggesting that a high level of differentiation is required to reach the airways and persist as memory cells. Differences in CD25 and CD11a expression indicate that the CD4(+) cells from the lung airways and parenchyma are distinct memory populations.

Reliable generation and use of MHC class II:gamma2aFc multimers for the identification of antigen-specific CD4(+) T cells.

MHC tetramers have proven to be powerful reagents for the analysis of MHC class I-restricted T cells. However, generating similarly reliable reagents for MHC class II-restricted T cells has been elusive. Here we evaluated the utility of MHC class II:gamma2aFc multimers, which contain the MHC class II extracellular domains, with or without recombinantly attached peptides, dimerized via a fos-jun leucine zipper and attached to the hinge of murine IgG2a. We have successfully generated 24 multimers in either myeloma or Drosophila melanogaster S2 cells, with an average yield of 7 mg/L. 'Empty' MHC class II:gamma2aFc multimers were effectively used in peptide binding assays. Similar versions that contained recombinantly attached peptides stimulated T cells in an antigen-specific, MHC-restricted manner, and identified antigen-specific nai;ve and effector T cells by flow cytometry. Furthermore, we have successfully used these reagents to stain T cells generated following viral infection. Thus, MHC class II:gamma2aFc multimers are robust and reliable reagents for the analysis of MHC class II-restricted T cells.