Murine Leukemia Virus Assembly Research Articles

Murine leukemia virus particle assembly quantitated by fluorescence microscopy: role of Gag-Gag interactions and membrane association.

In order to track the assembly of murine leukemia virus (MLV), we used fluorescence microscopy to visualize particles containing Gag molecules fused to fluorescent proteins (FPs). Gag-FP chimeras budded from cells to produce fluorescent spots, which passed through the same pore-size filters and sedimented at the same velocity as authentic MLV. N-terminal myristylation of Gag-FPs was necessary for particle formation unless wild-type Gag was coexpressed. By labeling nonmyristylated Gag with yellow FP and wild-type Gag with cyan FP, we could quantitate the coincorporation of two proteins into single particles. This experiment showed that nonmyristylated Gag was incorporated into mixed particles at approximately 50% the efficiency of wild-type Gag. Mutations that inhibit Gag-Gag interactions (K. Alin and S. P. Goff, Virology 216:418-424, 1996; K. Alin and S. P. Goff, Virology 222:339-351, 1996) were then introduced into the capsid (CA) region of Gag-FPs. The mutations P150L and R119C/P133L inhibited fluorescent particle formation by these Gag-FPs, but Gag-FPs containing these mutations could be efficiently incorporated into particles when coexpressed with wild-type Gag. When these mutations were introduced into nonmyristylated Gag-FPs, no incorporation into particles in the presence of wild-type Gag was detected. These data suggest that two independent mechanisms, CA interactions and membrane association following myristylation, cooperate in MLV Gag assembly and budding.

Effect of polymerase mutations on packaging of primer tRNAPro during murine leukemia virus assembly

The role of reverse transcriptase in selective encapsidation of the murine leukemia virus (MuLV) tRNA primer, tRNAPro, was investigated by examining the tRNA composition of several nonconditional pol mutants. One mutant, clone 23, which contains an altered polymerase about 40% smaller than the wild-type enzyme (B. I. Gerwin et al., J. Virol. 31:741-751, 1979) had a typical viral tRNA pattern, including normal levels of tRNAPro in free and 70S-associated 4S RNA. Another class of mutants, produced by Moloney murine leukemia virus-infected cell clone M13 and subclone M13/1, does not contain any detectable polymerase protein (A. Shields et al., Cell 14:601-609, 1978) and was found to have reduced amounts of tRNAPro in free 4S RNA. However, the level of tRNAPro associated with the genome was normal in the mutant virions. These results suggest that the reverse transcriptase protein is involved in the initial selection of tRNA primer during virus assembly, but not in the subsequent association of this tRNA with genomic RNA.

A morphological study on the ultrastructure and assembly of murine leukemia virus using a temperature-sensitive mutant restricted in assembly

T, a temperature-sensitive mutant of Moloney murine leukemia virus, was observed to produce large numbers of both normal particles and multiploids, (i.e., virions with more than one ribonucleoprotein component [RNP]) in all stages of assembly at the nonpermissive temperature. In thin sections, budding particles and extracellular immature particles show the general structural components characteristic of other RNA tumor viruses. Examination of the core shell confirms that it consists of polygonal subunits. The double striated tracks seen in negatively stained virions appear to be the edges of these subunits and are not likely to be a “core membrane.” An additional substructure was observed in the core shell which extends from the center of each polygonal subunit to the outer periphery of the RNP. The presence of substructures in the form of rings or short tubular substructures in the RNP is consistent with the hypothesis that the RNP is a hollow sphere formed by supercoiling of a single- or double-stranded helix. Multiploids were also observed in t-infected cells grown at the permissive temperature and in MuLV infected cells. We have demonstrated that the individual RNPs of a multiploid remain discrete entities. The volume of a spherical duplex is about twice that of a normal particle which suggests that it contains two complete genomes. The assembly of both normal particles and multiploids are essentially similar. Multiploids are apparently produced when two or more normal virions assemble in close proximity or assemble in succession at about the same site. The final steps in the assembly of both types of particles are preceded by the resealing of the cellular membrane. The laying down of the remaining section of the viral envelope occurs as a separate event. Elongation of the cytoplasmic strands linking the virions to the cell seems to occur before virion release. The significance of multiploids in mixed infections is discussed.

The structure and assembly of murine leukemia virus: Intracellular viral RNA

The large RNA component (70 S) of murine leukemia virus (MLV) is irreversibly converted to 38 S subunits by treatment with dimethyl sulfoxide (DMSO). According to sedimentation and gel electrophoretic analyses, the 38 S subunits of newly synthesized MLV are homogeneous in size with a molecular weight of 3.6 to 4.0 × 10 6 daltons. However, RNA subunits of virions that have been incubated at 37° for 3–6 hours are composed of a heterogeneous assortment of sizes, probably arising from the RNA degradative activity that can be detected in virions. The 70 S RNA structure found in MLV cannot be detected in MLV-infected cells. However, when cytoplasmic RNA is first treated with DMSO to convert to the 38 S form the viral RNA that is normally assembled into the 70 S structure, then RNA with an electrophoretic mobility and base composition similar to that of 38 S subunit RNA can be isolated. This cytoplasmic 38 S component amounts to 0.1–0.4% of the total cytoplasmic RNA. The 70 S RNA component found in MLV may be formed during virion assembly and release from the cell.