The Luis Enjuanes team has made major contributions to our understanding of coronavirus replication, transcription, virus-host cell interactions, and pathogenesis and has been a leader in the development of coronavirus vectors. Enjuanes group developed the first infectious coronavirus cDNA clone using bacterial artificial chromosomes, which facilitated studies of virus replication and pathogenesis. They identified key viral and cellular proteins that interact with coronavirus RNA and that are critical for virus replication. His group also made major contributions to understanding how short distance and long distance RNA interactions were required for optimal coronavirus RNA transcription and replication. These very careful studies provided insight into how coronaviruses, which express the largest single stranded RNA molecule of any virus, are able to efficiently replicate.
The Luis Enjuanes team developed transgenic mice engineered to secrete virus-neutralizing antibodies in the milk during lactation. This strategy is generally useful for preventing severe disease in neonatal animals. Subsequent work from Enjuanes group has led to the development of candidate vaccines for the coronavirus that caused the Severe Acute Respiratory Syndrome. A consequence of these efforts to develop a live attenuated vaccine for SARS was the identification of novel SARS-CoV virulence factors. His team was the first to show how a single small membrane protein, known to be critical for virus assembly, also regulated cells stress and the unfolded protein response, even in the context of stress induced by exogenous viral infections or chemicals.
1. Sola, I., Castilla, J., Pintado, B., Sanchez-Morgado, J. M., Whitelaw, B., Clark, J., and, Enjuanes, L. 1998. Transgenic mice secreting coronavirus neutralizing antibodies into the milk. J. Virol. 72, 3762-7372
This manuscript shows as a proof of principle that the progeny of animals engineered to express pathogen-specific neutralizing recombinant monoclonal antibodies in breast milk are protected against those pathogens early after birth.
2. Castilla, J., Pintado, B., Sola, I., Sanchez-Morgado, J. M., and Enjuanes, L. 1998. Engineering passive immunity in transgenic mice secreting virus-neutralizing antibodies in milk. Nature Biotech. 16, 349-354
This manuscript describes in detail the generation of transgenic animals engineered to express virus neutralizing antibodies in breast milk, as a novel approach to protecting offspring against neonatal infections of the enteric tract.
3. Izeta, A., Smerdou, C., Alonso, S., Penzes, Z., Mendez, A., Plana-Duran, J., Enjuanes, L. 1998. Replication and packaging of transmissible gastroenteritis coronavirus derived synthetic minigenomes. J. Virol. 73, 1535-1545.
This manuscript describes the engineering of coronavirus minigenomes transcribed from cDNA. Analysis of the replication in trans of a collection of deletion mutants of this minigenome led to the identification of the smallest synthetically generated minigenome with the ability to replicate and encapsidate.
4. Almazan, F., Gonzalez, J. M., Penzes, Z., Izeta, A., Calvo, E., Plana-Duran, J., Enjuanes, L. 2000. Engineering the largest RNA virus genome as an infectious bacterial artificial chromosome. Proc. Nat. Acad. Sci. USA. 97, 5516-5521.
Here, Enjuanes and co-workers describe the development, for the first time, of a cDNA clone encoding an infectious coronavirus RNA genome using a bacterial artificial chromosome and nuclear expression of RNAs that are typically produced within the cytoplasm. This achievement facilitated reverse genetic studies of coronaviruses and opened up the possibility of employing CoV infectious cDNAs as vectors for vaccine development and gene delivery.
5. Gonzalez, J.M., Penzes, Z., Almazan, F., Calvo, E., Enjuanes, L. 2002. Stabilization of a full-length infectious cDNA clone of transmissible gastroenteritis coronavirus by insertion of an intron. J. Virol. 76, 4655-4661.
This manuscript shows that the stable propagation of long cDNAs, such as full-length coronavirus cDNA, in Escherichia coli was considerably improved by the insertion of an intron that disrupted toxic regions identified in the viral genome.
6. Sola, I., Alonso, S., Zuñiga, S., Balach, M., Plana-Duran, J. y Enjuanes, L. 2003.
Engineering transmissible gastroenteritis virus genome as an expression vector inducing lactogenic immunity. J. Virol. 77, 4357-4369.
This manuscript describes the use of a transmissible gastroenteritis coronavirus (TGEV) infectious cDNA to express heterologous genes and thereby induce lactogenic immunity that was transferred to progeny during lactation.
7. Zuñiga, S., Sola, I., Alonso, S., Enjuanes, L. 2004. Sequence motifs involved in the regulation of discontinuous coronavirus subgenomic RNA synthesis. J. Virol. 78, 980-994.
This manuscript describes a novel mechanism of base pair scanning critical for coronavirus transcription.
8. DeDiego, M. L., Alvarez, E., Almazan, F., Rejas, M. T., Lamirande, E., Roberts, A., Shieh, W. J., Zaki. S. R., Subbarao, K., and Enjuanes, L. 2007. A severe acute respiratory syndrome coronavirus that lacks the E gene is attenuated in vitro and in vivo. J. Virol. 81, 1701-1713.
This manuscript describes the engineering of SARS-CoV mutants deleted in E gene and showed that the E protein was a virulence factor. SARS-CoV lacking E gene were shown to be safe and effective vectors against challenge with virulent virus, suggesting that these viruses would be potentially useful as SARS-CoV vaccines.
9. Zuñiga, S., Sola, I., Moreno, J. L., Sabella, P., Plana-Duran, J., and Enjuanes, L. 2007. Coronavirus nucleocapsid protein is an RNA chaperone. Virology, 357, 215-227.
This manuscript demonstrated, for the first time, that a coronavirus protein has RNA chaperone activity.
10. Moreno J. L., Zuñiga S, Enjuanes L, and Sola I. 2008. Identification of a coronavirus transcription enhancer. J. Virol. 82, 3882-3893.
This manuscript describes, for the first time, the presence of a transcriptional enhancer in the coronavirus genome. The enhancer facilitated augmented expression of heterologous genes from recombinant coronavirus vectors.
11. Cruz, J. L. G., I. Sola, M. Becares, B. Alberca, J. Plana, L. Enjuanes, and S. Zuñiga. 2011. Coronavirus gene 7 counteracts host defenses and modulates virulence. PLoS Pathog. 7, e1002090.
This manuscript describes a novel coronavirus protein that antagonizes the cellular dsRNA-induced host antiviral response by inhibiting the function of the protein phosphatase 1 catalytic subunit (PP1c).
12. DeDiego, M. L., Nieto-Torres., J. L., Jimenez-Guardeño, J. M., Regla-Nava, J. A., Alvarez, E., Oliveros, J. C., Zhao, J., Fett, C., Perlman, S., and Enjuanes, L. 2011. Severe acute respiratory syndrome coronavirus envelope protein regulates stress response and inflammation. PLoS Pathog., doi: 10.1371/Journal.ppat.1002315.
This manuscript shows that the envelope protein of SARS-CoV is a virulence protein, inhibiting activation of the stress protein and reducing inflammation.