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Lili Kuo

Publications and source records attributed to Lili Kuo.

6 recordsLinked to original sources
PubMed2006

Exceptional flexibility in the sequence requirements for coronavirus small envelope protein function.

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.

Amino Acid Sequence↗
PubMed2005

A major determinant for membrane protein interaction localizes to the carboxy-terminal domain of the mouse coronavirus nucleocapsid protein.

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.

Animals↗
PubMed2003

The glycosylation status of the murine hepatitis coronavirus M protein affects the interferogenic capacity of the virus in vitro and its ability to replicate in the liver but not the brain.

The coronavirus M protein, the most abundant coronaviral envelope component, is invariably glycosylated, which provides the virion with a diffuse, hydrophilic cover on its outer surface. Remarkably, while the group 1 and group 3 coronaviruses all have M proteins with N-linked sugars, the M proteins of the group 2 coronaviruses [e.g., mouse hepatitis virus (MHV)] are O-glycosylated. The conservation of the N- and O-glycosylation motifs suggests that each of these types of carbohydrate modifications is beneficial to their respective virus. Since glycosylation of the M protein is not required for virus assembly, the oligosaccharides are likely to be involved in the virus-host interaction. In order to investigate the role of the M protein glycosylation in the host, two genetically modified MHVs were generated by using targeted RNA recombination. The recombinant viruses carried M proteins that were either N-glycosylated or not glycosylated at all, and these were compared with the parental, O-glycosylated, virus. The M protein glycosylation state did not influence the tissue culture growth characteristics of the recombinant viruses. However, it affected their interferogenic capacity as measured using fixed, virus-infected cells. Viruses containing M proteins with N-linked sugars induced type I interferons to higher levels than viruses carrying M proteins with O-linked sugars. MHV with unglycosylated M proteins appeared to be a poor interferon inducer. In mice, the recombinant viruses differed in their ability to replicate in the liver, but not in the brain, whereas their in vivo interferogenic capacity did not appear to be affected by their glycosylation status. Strikingly, their abilities to replicate in the liver correlated with their in vitro interferogenic capacity. This apparent correlation might be explained by the functioning of lectins, such as the mannose receptor, which are abundantly expressed in the liver but also play a role in the induction of interferon-alpha by dendritic cells.

Amino Acid Sequence↗
PubMed2003

The small envelope protein E is not essential for murine coronavirus replication.

The importance of the small envelope (E) protein in the assembly of coronaviruses has been demonstrated in several studies. While its precise function is not clearly defined, E is a pivotal player in the morphogenesis of the virion envelope. Expression of the E protein alone results in its incorporation into vesicles that are released from cells, and the coexpression of the E protein with the membrane protein M leads to the assembly of coronavirus-like particles. We have previously generated E gene mutants of mouse hepatitis virus (MHV) that had marked defects in viral growth and produced virions that were aberrantly assembled in comparison to wild-type virions. We have now been able to obtain a viable MHV mutant in which the entire E gene, as well as the nonessential upstream genes 4 and 5a, has been deleted. This mutant (Delta E) was obtained by a targeted RNA recombination method that makes use of a powerful host range-based selection system. The Delta E mutant produces tiny plaques with an unusual morphology compared to plaques formed by wild-type MHV. Despite its low growth rate and low infectious titer, the Delta E mutant is genetically stable, showing no detectable phenotypic changes after several passages. The properties of this mutant provide further support for the importance of E protein in MHV replication, but surprisingly, they also show that E protein is not essential.

Amino Acid Sequence↗
PubMed2002

Genetic evidence for a structural interaction between the carboxy termini of the membrane and nucleocapsid proteins of mouse hepatitis virus.

The coronavirus membrane (M) protein is the most abundant virion protein and the key component in viral assembly and morphogenesis. The M protein of mouse hepatitis virus (MHV) is an integral membrane protein with a short ectodomain, three transmembrane segments, and a large carboxy-terminal endodomain facing the interior of the viral envelope. The carboxy terminus of MHV M has previously been shown to be extremely sensitive to mutation, both in a virus-like particle expression system and in the intact virion. We have constructed a mutant, M(Delta)2, containing a two-amino-acid truncation of the M protein that was previously thought to be lethal. This mutant was isolated by means of targeted RNA recombination with a powerful host range-based selection allowed by the interspecies chimeric virus fMHV (MHV containing the ectodomain of the feline infectious peritonitis virus S protein). Analysis of multiple second-site revertants of the M(Delta)2 mutant has revealed changes in regions of both the M protein and the nucleocapsid (N) protein that can compensate for the loss of the last two residues of the M protein. Our data thus provide the first genetic evidence for a structural interaction between the carboxy termini of the M and N proteins of MHV. In addition, this work demonstrates the efficacy of targeted recombination with fMHV for the systematic genetic analysis of coronavirus structural protein interactions.

Amino Acid Sequence↗