Assembly Of Infectious Virions Research Articles

In vitro assembly of infectious virions of double-stranded DNA phage phi 29 from cloned gene products and synthetic nucleic acids.

Up to 6 x 10(7) PFU of infectious virions of the double-stranded DNA bacteriophage phi 29 per ml were assembled in vitro, with 11 proteins derived from cloned genes and nucleic acids synthesized separately. The genomic DNA-gp3 protein conjugate was efficiently packaged into a purified recombinant procapsid with the aid of a small viral RNA (pRNA) transcript, a DNA-packaging ATPase (gp16), and ATP. The DNA-filled capsids were subsequently converted into infectious virions after the addition of four more recombinant proteins for neck and tail assembly. Electron microscopy and genome restriction mapping confirmed the identity of the infectious phi 29 virions synthesized in this system. A nonstructural protein, gp13, was indispensable for the assembly of infectious virions. The overproduced tail protein gp9 was present in solution in mostly dimeric form and was purified to homogeneity. The purified gp9 was biologically active for in vitro phi 29 assembly. Higher-order concentration dependence of in vitro phi 29 assembly on gp9 suggests that a complete tail did not form before attaching to the DNA-filled capsid, a result contrary to earlier findings for phages T4 and lambda. The work described here constitutes an extremely sensitive assay system for the analysis of components in phi 29 assembly and dissection of functional domains of structural components, enzymes, and pRNA (C.-S. Lee and P. Guo, Virology 202:1039-1042, 1995). Efficient packaging of foreign DNA in vitro and synthesis of viral particles from recombinant proteins facilitate the development of phi 29 as an in vivo gene delivery system. The finding that purified tail protein was able to incorporate into infectious virions might allow the construction of chimeric phi 29 carrying a tail fused to ligands for specific receptor of human cells.

Cleavage of p15 protein in vitro by human immunodeficiency virus type 1 protease is RNA dependent.

The human immunodeficiency virus (HIV) gag polyprotein is processed by the viral protease to yield the structural proteins of the virus. One of these structural proteins, p15, and its protease cleavage products, p7 and p6, are believed to be responsible for the viral RNA binding which is prerequisite for assembly of infectious virions. To better understand potential interactions between viral RNA, p15, and the HIV protease, we have synthesized p15 in an in vitro system and studied its processing by the viral protease. Using this system, we demonstrate that p15 synthesized in vitro is properly cleaved by the HIV protease in an RNA-dependent reaction. Mutation of cysteine residues in either zinc-binding domain of the p7 portion of p15 does not alter the RNA-dependent cleavage, but mutation of three basic residues located between the zinc-binding domains blocks HIV protease susceptibility. The results support a previously unrecognized role for the interaction of RNA and nucleocapsid-containing gag precursors that may have important consequences for virus assembly.

Adenovirus L1 52- and 55-kilodalton proteins are present within assembling virions and colocalize with nuclear structures distinct from replication centers.

Analysis of a temperature-sensitive mutant, Ad5ts369, had indicated that the adenovirus L1 52- and 55-kDa proteins (52/55-kDa proteins) are required for the assembly of infectious virions. By using monoclonal antibodies directed against bacterially produced L1 52-kDa protein, the L1 52/55-kDa proteins were found to be differentially phosphorylated forms of a single 48-kDa polypeptide. Both phosphoforms were shown to be present within all suspected virus assembly intermediates (empty capsids, 50 to 100 molecules; young virions, 1 to 2 molecules) but not within mature virions. The mobilities of these proteins in polyacrylamide gels were affected by reducing agents, indicating that the 52/55-kDa proteins may exist as homodimers within the cell and within assembling particles. Immunofluorescence analysis revealed that the 52/55-kDa proteins localize to regions within the infected nucleus that are distinct from viral DNA replication centers, indicating that replication and assembly of viral components likely occur in separate nuclear compartments. Immunoelectron microscopic studies determined that the 52/55-kDa proteins are found in close association with structures that appear to contain assembling virions. These results are consistent with an active but transient role for the L1 products in assembly of the adenovirus particle, perhaps as scaffolding proteins.

Assembly and polarized release of Punta Toro virus and effects of brefeldin A

Punta Toro virus (PTV), a member of the sandfly fever group of bunyaviruses, is assembled by budding at intracellular membranes of the Golgi complex. We have examined PTV glycoprotein transport, assembly, and release and the effects of brefeldin A (BFA) on these processes. Both the G1 and G2 proteins were transported out of the endoplasmic reticulum (ER) and retained in the Golgi complex in a stable structure, either during PTV infection or when expressed from a vaccinia virus recombinant. BFA treatment causes a rapid and dramatic change in the distribution of the G1 and G2 proteins, from a Golgi pattern to an ER pattern. The G1 and G2 proteins were found to be modified by medial but not trans Golgi network enzymes, in the presence or absence of BFA. We found that BFA blocks PTV release from cells but does not interfere with the intracellular assembly of infectious virions. Further, the BFA block of virus release is fully reversible, with high levels of virus release occurring upon removal of the inhibitor. It was also found that the release of PTV virions is polarized, occurring exclusively from the basolateral surfaces of the polarized Vero C1008 epithelial cell line.

Defect of adenovirus type 12 replication in hamster cells: absence of transcription of viral virus-associated and L1 RNAs.

The nonpermissive interaction of hamster cells with human adenovirus type 12 (Ad12) is characterized by a total block of Ad12 DNA replication and late transcription, whereas most of the early functions of Ad12 DNA can be transcribed. Ad2 can replicate in hamster cells. The replication and late transcription defects of Ad12 DNA can be complemented to a certain extent by the E1B functions of Ad2 DNA. This complementation fails, however, to lead to the synthesis of the late Ad12 proteins and to the assembly of infectious virions. It will now be demonstrated that the Ad12 L1 (late genes of group 1) and virus-associated (VA) RNAs are not transcribed in hamster cells. Synthesis of these RNAs in productively infected human cells or Ad2-infected hamster cells is readily detectable by S1 nuclease protection experiments and Northern (RNA) blotting. Similarly, the Ad2-transformed hamster cell line BHK-Ad2E1 fails to complement L1 and VA RNA syntheses after superinfection with Ad12. However, Ad12 infection of the Ad5-transformed hamster cell line BHK297-C131 leads to the transcription of the Ad12 L1 and VA segments. This difference in complementation by the two transformed hamster cell lines might be accounted for by functions in the segment of Ad5 DNA extending between map units 30 and 40 and persisting in the Ad5-transformed hamster cells or by hamster host cell functions which might be operative in cell line BHK297-C131 but not in BHK-Ad2E1 or BHK-21 hamster cells.

Alfalfa mosaic virus temperature-sensitive mutants

Mutants Bts 03 and Mts 04 of alfalfa mosaic virus (AIMV) have temperature-sensitive mutations in genomic RNAs 1 and 2, respectively. These mutants are defective in the production of viral minus-strand RNA, coat protein, and infectious virus when assayed in cowpea protoplasts at the nonpermissive temperature (30 degrees). To determine the temperature-sensitive step in the replication cycle, mutant-infected protoplasts were shifted from an incubation temperature of 25 degrees (permissive temperature) to 30 degrees at different times during a 24-hr incubation period. For both mutants an initial incubation of infected protoplasts for 6 hr at 25 degrees was sufficient to permit a normal minus-strand RNA synthesis, translation of RNA 4 into coat protein, and assembly of infectious virus during the subsequent incubation at the nonpermissive temperature. Probably, AIMV RNAs 1 and 2 encoded proteins are produced early in infection and the mutant proteins are protected from inactivation at 30 degrees once they are incorporated in a functional structure.

Alfalfa mosaic virus temperature-sensitive mutants: I. Mutants defective in viral RNA and protein synthesis

The replication in cowpea protoplasts of temperature-sensitive (ts) mutants of alfalfa mosaic virus (AIMV) was studied at the permissive (25°) and the restrictive (30°) temperature. Using the Northern blot hybridization technique, it was shown that at the restrictive temperature two RNA 1 mutants, Bts 03 and Bts 04, and two RNA 2 mutants, Mts 03 and Mts 04, were all defective in the synthesis of viral minus-strand RNA, whereas the synthesis of the plus-strand genomic RNAs 1, 2, and 3 and the subgenomic coat protein messenger, RNA 4, was relatively unimpaired. In Bts 04 inoculated protoplasts the RNA 4 produced at 30° was translated into coat protein and viral RNA was encapsidated to give infectious virus. RNA 4 in Bts 03 and Mts 04 infected protoplasts was not translated into coat protein at 30 and consequently there was no assembly of infectious virus. Protein synthesis by Mts 03 was not investigated. A1MV RNAs 1 and 2 encoded proteins are both involved in the synthesis of viral minus-strand RNA and the translation of RNA 4 and possibly other viral messengers. The results with Bts 03 and Bts 04 show that the two functions of the RNA 1 encoded protein can be mutated separately.

Host range temperature-conditional mutants in the adenovirus DNA binding protein are defective in the assembly of infectious virus

R(ts107)202 is a host range temperature-conditional mutant of adenovirus type 5. This mutant is temperature sensitive for replication and plaquing in 293 cells but is temperature independent for growth and plaquing in HeLa cells (J.C. Nicolas, F. Suarez, A. J. Levine, and M. Girard (1981) Virology 108, 521–524). The mutant was isolated in HeLa cells as a temperature-independent revertant of the H5ts107 temperature-sensitive mutant that maps in the adenovirus DNA binding protein (DBP). The reasons for the temperature conditional phenotype of this mutant in 293 cells were investigated. The mutant synthesized an unstable DBP in both HeLa and 293 cells at 39°. In 293 cells at 39°, about two- to threefold less viral DNA was synthesized by r(ts107)202 as compared to Ad5wt. R(ts107)202 infected cells at 39° produced normal (wild-type) amounts of all detectable late viral structural proteins. The mutant failed, however, to produce infectious virus or assemble virus particles in 293 cells at 39°. The altered DBP may therefore play a role in the assembly of virus particles, either directly or indirectly via an altered DNA structure. The failure of r(ts107)202 to assemble virion particles in 293 cells at 39° furthermore suggests that virus assembly is dependent upon cellular factors that differ in HeLa and 293 cell.

The effects of arginine starvation on macromolecular synthesis in infection with type 2 adenovirus: I. Synthesis and utilization of structural proteins

Although arginine deprivation inhibits the production of infectious type 2 adenovirus in KB cells, all the major structural proteins can be detected in acrylamide gel electropherograms of infected cell material, including those peptides known to be rich in arginine. However, these viral proteins synthesized during arginine deprivation are largely unused in the assembly of infectious virions which occurs after arginine is restored. With the use of a DNA inhibitor (FdUrd) and appropriate periods of arginine starvation, it can be shown that the arginine-dependent function does not occur prior to DNA synthesis. The effects of restoring arginine at intervals late in the latent period indicate that the essential function can take place within 3–4 hr prior to the appearance of mature virions, i.e., during late protein synthesis.