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ABSTRACT: The coronavirus nucleocapsid (N) protein plays an essential role in virion assembly via interactions with the large, positive-strand RNA viral genome and the carboxy-terminal endodomain of the membrane protein (M). To learn about the functions of N protein domains in the coronavirus mouse hepatitis virus (MHV), we replaced the MHV N gene with its counterpart from the closely related bovine coronavirus (BCoV). The resulting viral mutant was severely defective, even though individual domains of the N protein responsible for N-RNA, N-M, or N-N interactions were completely interchangeable between BCoV and MHV. The lesion in the BCoV N substitution mutant could be compensated for by reverting mutations in the central, serine- and arginine-rich (SR) domain of the N protein. Surprisingly, a second class of reverting mutations were mapped to the amino terminus of a replicase subunit, nonstructural protein 3 (nsp3). A similarly defective MHV N mutant bearing an insertion of the SR region from the severe acute respiratory syndrome coronavirus N protein was rescued by the same two classes of reverting mutations. Our genetic results were corroborated by the demonstration that the expressed amino-terminal segment of nsp3 bound selectively to N protein from infected cells, and this interaction was RNA independent. Moreover, we found a direct correlation between the N-nsp3 interaction and the ability of N protein to stimulate the infectivity of transfected MHV genomic RNA (gRNA). Our results suggest a role for this previously unknown N-nsp3 interaction in the localization of genomic RNA to the replicase complex at an early stage of infection.
Journal of Virology 10/2010; 84(19):10276-88. · 5.40 Impact Factor
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ABSTRACT: The coronavirus nucleocapsid protein (N), together with the large, positive-strand RNA viral genome, forms a helically symmetric nucleocapsid. This ribonucleoprotein structure becomes packaged into virions through association with the carboxy-terminal endodomain of the membrane protein (M), which is the principal constituent of the virion envelope. Previous work with the prototype coronavirus mouse hepatitis virus (MHV) has shown that a major determinant of the N-M interaction maps to the carboxy-terminal domain 3 of the N protein. To explore other domain interactions of the MHV N protein, we expressed a series of segments of the MHV N protein as fusions with green fluorescent protein (GFP) during the course of viral infection. We found that two of these GFP-N-domain fusion proteins were selectively packaged into virions as the result of tight binding to the N protein in the viral nucleocapsid, in a manner that did not involve association with either M protein or RNA. The nature of each type of binding was further explored through genetic analysis. Our results defined two strongly interacting regions of the N protein. One is the same domain 3 that is critical for M protein recognition during assembly. The other is domain N1b, which corresponds to the N-terminal domain that has been structurally characterized in detail for two other coronaviruses, infectious bronchitis virus and the severe acute respiratory syndrome coronavirus.
Journal of Virology 06/2009; 83(14):7221-34. · 5.40 Impact Factor
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Scott J Goebel,
Jill Taylor,
Bradd C Barr,
Timothy E Kiehn,
Hugo R Castro-Malaspina,
Cyrus V Hedvat,
Kim A Rush-Wilson,
Cassandra D Kelly,
Stephen W Davis,
William A Samsonoff, Kelley R Hurst,
Melissa J Behr,
Paul S Masters
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ABSTRACT: An unknown virus was isolated from a lung biopsy sample and multiple other samples from a patient who developed a lethal case of pneumonia following a peripheral blood stem cell transplant. A random PCR-based molecular screening method was used to identify the infectious agent as avian paramyxovirus 1 (APMV-1; a group encompassing Newcastle disease virus), which is a highly contagious poultry pathogen that has only rarely been found in human infections. Immunohistochemical analysis confirmed the presence of APMV-1 antigen in sloughed alveolar cells in lung tissue from autopsy. Sequence from the human isolate showed that it was most closely related to virulent pigeon strains of APMV-1. This is the most completely documented case of a systemic human infection caused by APMV-1 and is the first report of an association between this virus and a fatal disease in a human.
Journal of Virology 12/2007; 81(22):12709-14. · 5.40 Impact Factor
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ABSTRACT: The small envelope protein (E) plays a role of central importance in the assembly of coronaviruses. This was initially established by studies demonstrating that cellular expression of only E protein and the membrane protein (M) was necessary and sufficient for the generation and release of virus-like particles. To investigate the role of E protein in the whole virus, we previously generated E gene mutants of mouse hepatitis virus (MHV) that were defective in viral growth and produced aberrantly assembled virions. Surprisingly, however, we were also able to isolate a viable MHV mutant (DeltaE) in which the entire E gene, as well as the nonessential upstream genes 4 and 5a, were deleted. We have now constructed an E knockout mutant that confirms that the highly defective phenotype of the DeltaE mutant is due to loss of the E gene. Additionally, we have created substitution mutants in which the MHV E gene was replaced by heterologous E genes from viruses spanning all three groups of the coronavirus family. Group 2 and 3 E proteins were readily exchangeable for that of MHV. However, the E protein of a group 1 coronavirus, transmissible gastroenteritis virus, became functional in MHV only after acquisition of particular mutations. Our results show that proteins encompassing a remarkably diverse range of primary amino acid sequences can provide E protein function in MHV. These findings suggest that E protein facilitates viral assembly in a manner that does not require E protein to make sequence-specific contacts with M protein.
Journal of Virology 04/2007; 81(5):2249-62. · 5.40 Impact Factor
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Advances in experimental medicine and biology 02/2006; 581:163-73. · 1.09 Impact Factor
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ABSTRACT: The two major constituents of coronavirus virions are the membrane (M) and nucleocapsid (N) proteins. The M protein is anchored in the viral envelope by three transmembrane segments flanked by a short amino-terminal ectodomain and a large carboxy-terminal endodomain. The M endodomain interacts with the viral nucleocapsid, which consists of the positive-strand RNA genome helically encapsidated by N protein monomers. In previous work with the coronavirus mouse hepatitis virus (MHV), a highly defective M protein mutant, MDelta2, was constructed. This mutant contained a 2-amino-acid carboxy-terminal truncation of the M protein. Analysis of second-site revertants of MDelta2 revealed mutations in the carboxy-terminal region of the N protein that compensated for the defect in the M protein. To seek further genetic evidence corroborating this interaction, we generated a comprehensive set of clustered charged-to-alanine mutants in the carboxy-terminal domain 3 of N protein. One of these mutants, CCA4, had a highly defective phenotype similar to that of MDelta2. Transfer of the CCA4 mutation into a partially diploid MHV genome showed that CCA4 was a loss-of-function mutation rather than a dominant-negative mutation. Analysis of multiple second-site revertants of CCA4 revealed mutations in both the M protein and the N protein that could compensate for the original lesion in N. These data more precisely define the region of the N protein that interacts with the M protein. Further, we found that fusion of domain 3 of the N protein to the carboxy terminus of a heterologous protein caused it to be incorporated into MHV virions.
Journal of Virology 12/2005; 79(21):13285-97. · 5.40 Impact Factor
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ABSTRACT: How chemokines shape the immune response to viral infection of the central nervous system (CNS) has largely been considered within the context of recruitment and activation of antigen-specific lymphocytes. However, chemokines are expressed early following viral infection, suggesting an important role in coordinating innate immune responses. Herein, we evaluated the contributions of CXC chemokine ligand 10 (CXCL10) in promoting innate defense mechanisms following coronavirus infection of the CNS. Intracerebral infection of RAG1(-/-) mice with a recombinant CXCL10-expressing murine coronavirus (mouse hepatitis virus) resulted in protection from disease and increased survival that correlated with a significant increase in recruitment and activation of natural killer (NK) cells within the CNS. Accumulation of NK cells resulted in a reduction in viral titers that was dependent on gamma interferon secretion. These results indicate that CXCL10 expression plays a pivotal role in defense following coronavirus infection of the CNS by enhancing innate immune responses.
Journal of Virology 02/2004; 78(2):585-94. · 5.40 Impact Factor
