Mauricio Comas-Garcia

University of California, Los Angeles, Los Angeles, California, United States

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Publications (5)14.06 Total impact

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    ABSTRACT: We have recently discovered (Cadena-Nava, et al. J. Virol. 2012: , 86, 3318) that the in vitro packaging of RNA by the capsid protein (CP) of Cowpea Chlorotic Mottle Virus is optimal when there is a significant excess of CP, specifically that complete packaging of all the RNA in solution requires sufficient CP to provide charge matching of the N-terminal positively-charged arginine-rich motifs (ARMS) of the CPs with the negatively-charged phosphate backbone of the RNA. We show here that packaging results from the initial formation of a "charge-matched" protocapsid consisting of RNA decorated by a disordered arrangement of CPs. This protocapsid reorganizes into the final, icosahedrally-symmetric, nucleocapsid by displacing the excess CPs from the RNA to the exterior surface of the emerging capsid, through electrostatic attraction between the ARMs of the excess CP and the negative charge density of the capsid exterior. As a test of this scenario we prepare CP mutants with "extra" and "missing" (relative to wildtype) cationic residues and show that a correspondingly smaller and larger excess of CP is needed, respectively, for complete packaging of RNA.
    Journal of virology. 06/2014;
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    ABSTRACT: For many viruses, the packaging of a single-stranded RNA (ss-RNA) genome is spontaneous, driven by capsid protein-capsid protein (CP) and CP-RNA interactions. Furthermore, for some multipartite ss-RNA viruses, copackaging of two or more RNA molecules is a common strategy. Here we focus on RNA copackaging in vitro by using cowpea chlorotic mottle virus CP and an RNA molecule that is short (500 nucleotides [nt]) compared to the lengths (≈3000nt) packaged in wild-type virions. We show that the degree of cooperativity of virus assembly depends not only on the relative strength of the CP-CP and CP-RNA interactions but also on the RNA being short: a 500nt RNA molecule cannot form a capsid by itself, so its packaging requires the aggregation of multiple CP-RNA complexes. By using fluorescence correlation spectroscopy we show that at neutral pH and sufficiently low concentrations RNA and CP form complexes that are smaller than the wild-type capsid and that four 500-nt RNAs are packaged into virus-like-particles (VLPs) only upon lowering the pH. Further, a variety of bulk-solution techniques confirm that fully ordered VLPs are formed only upon acidification. On the basis of these results we argue that the observed cooperativity involves equilibrium between multiple CP/RNA complexes.
    The journal of physical chemistry. B. 06/2014;
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    ABSTRACT: The strength of attraction between capsid proteins (CPs) of Cowpea Chlorotic Mottle Virus (CCMV) is controlled by the solution pH. Additionally, the strength of attraction between CP and the single-stranded (ss) RNA viral genome is controlled by ionic strength. By exploiting these properties, we are able to control and monitor the in vitro co-assembly of CCMV CP and ssRNA as a function of the strength of CP-CP and CP-RNA attractions. Using the techniques of velocity sedimentation and electron microscopy, we find that the successful assembly of nuclease-resistant virus-like particles (VLPs) depends delicately on the strength of CP-CP attraction relative to CP-RNA attraction. If the attractions are too weak, the capsid cannot form; if they are too strong, the assembly suffers from kinetic traps. Separating the process into two steps - by first turning on CP-RNA attraction, and then turning on CP-CP attraction - allows for the assembly of well-formed VLPs under a wide range of attraction strengths. These observations establish a protocol for the efficient in vitro assembly of CCMV VLPs, and suggest potential strategies that the virus may employ in vivo.
    Journal of Molecular Biology 10/2013; · 3.91 Impact Factor
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    ABSTRACT: While most T=3 single-stranded RNA (ssRNA) viruses package in vivo about 3,000 nucleotides (nt), in vitro experiments have demonstrated that a broad range of RNA lengths can be packaged. Under the right solution conditions, for example, cowpea chlorotic mottle virus (CCMV) capsid protein (CP) has been shown to package RNA molecules whose lengths range from 100 to 10,000 nt. Furthermore, in each case it can package the RNA completely, as long as the mass ratio of CP to nucleic acid in the assembly mixture is 6:1 or higher. Yet the packaging efficiencies of the RNAs can differ widely, as we demonstrate by measurements in which two RNAs compete head-to-head for a limited amount of CP. We show that the relative efficiency depends nonmonotonically on the RNA length, with 3,200 nt being optimum for packaging by the T=3 capsids preferred by CCMV CP. When two RNAs of the same length-and hence the same charge-compete for CP, differences in packaging efficiency are necessarily due to differences in their secondary structures and/or three-dimensional (3D) sizes. For example, the heterologous RNA1 of brome mosaic virus (BMV) is packaged three times more efficiently by CCMV CP than is RNA1 of CCMV, even though the two RNAs have virtually identical lengths. Finally, we show that in an assembly mixture at neutral pH, CP binds reversibly to the RNA and there is a reversible equilibrium between all the various RNA/CP complexes. At acidic pH, excess protein unbinds from RNA/CP complexes and nucleocapsids form irreversibly.
    Journal of Virology 09/2012; 86(22):12271-82. · 5.08 Impact Factor
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    ABSTRACT: Virus-like particles can be formed by self-assembly of capsid protein (CP) with RNA molecules of increasing length. If the protein "insisted" on a single radius of curvature, the capsids would be identical in size, independent of RNA length. However, there would be a limit to length of the RNA, and one would not expect RNA much shorter than native viral RNA to be packaged unless multiple copies were packaged. On the other hand, if the protein did not favor predetermined capsid size, one would expect the capsid diameter to increase with increase in RNA length. Here we examine the self-assembly of CP from cowpea chlorotic mottle virus with RNA molecules ranging in length from 140 to 12,000 nucleotides (nt). Each of these RNAs is completely packaged if and only if the protein/RNA mass ratio is sufficiently high; this critical value is the same for all of the RNAs and corresponds to equal RNA and N-terminal-protein charges in the assembly mix. For RNAs much shorter in length than the 3,000 nt of the viral RNA, two or more molecules are assembled into 24- and 26-nm-diameter capsids, whereas for much longer RNAs (>4,500 nt), a single RNA molecule is shared/packaged by two or more capsids with diameters as large as 30 nm. For intermediate lengths, a single RNA is assembled into 26-nm-diameter capsids, the size associated with T=3 wild-type virus. The significance of these assembly results is discussed in relation to likely factors that maintain T=3 symmetry in vivo.
    Journal of Virology 12/2011; 86(6):3318-26. · 5.08 Impact Factor

Publication Stats

31 Citations
14.06 Total Impact Points

Institutions

  • 2011–2013
    • University of California, Los Angeles
      • Department of Chemistry and Biochemistry
      Los Angeles, California, United States
  • 2012
    • University of California, Riverside
      • Department of Plant Pathology and Microbiology
      Riverside, California, United States