Interruption by Rifampin of an Early Stage in Vaccinia Virus Morphogenesis: Accumulation of Membranes Which Are Precursors of Virus Envelopes

Abstract

Assembly of vaccinia virus envelopes and immature vaccinia particles was interrupted in HeLa cells treated with rifampin ( rifampicin ). The primary action of rifampin on vaccinia morphogenesis appeared to occur during the stage of envelope formation. When envelopes and immature particles were already present, maturation could continue, even in the presence of rifampin. It was demonstrated that the trilaminar membranes of irregular contour which accumulate in the presence of rifampin are precursors of virus envelopes. When rifampin was removed under controlled conditions, synchronous transitions were observed as the precursor membranes rapidly converted into uniformly curved envelope units with a 10- to 12-nm coat on the convex surface. These experiments provided an opportunity to examine the sequence of some early events in vaccinia morphogenesis. Initially, nascent envelopes remained in clusters around dense viroplasm. Large numbers of single immature particles appeared within 10 min. Nucleation of immature particles was the first evidence of core differentiation and began within 5 to 10 min. Development of lateral bodies and modeling of the biconcave cores was observed within 30 min, and structurally mature virions were present by 2 hr after the removal of rifampin. High resolution autoradiography showed that viral deoxyribonucleic acid, which labeled with 3 H-thymidine during rifampin treatment, was incorporated by the mature vaccinia which formed after rifampin was removed. Concentration of the viral deoxyribonucleic acid in core material evidently occurred after envelope assembly, probably coincident with nucleoid formation. Cytoplasmic crystalloid bodies accumulated during rifampin treatment; they appeared morphologically identical to vaccinia nucleoids and were heavily labeled by 3 H-thymidine.

Deep-etch EM reveals that the early poxvirus envelope is a single membrane bilayer stabilized by a geodetic “honeycomb” surface coat

“…They concluded that the spike layer stabilized the crescent membrane and imposed a curvature upon it, and they stressed that when the spikes were prevented from associating with the developing IVs by the drug Rifampicin, the IV membrane became floppy and tended to close up upon itself. This latter observation has been confirmed many times, and has been highlighted by many demonstrations of just how promptly crescent formation resumes upon washout of Rifampicin-again, with the crescents invariably displaying the spike layer as soon as they begin to reform (Moss et al, 1969;Grimley et al, 1970;Nagayama et al, 1970;Pennington et al, 1970;Follett and Pennington, 1971;Grimley and Moss, 1971;Moss et al, 1971;Pennington and Follett, 1971). In a subsequent report, we will use the imaging techniques introduced here to confirm that the external lattice is indeed D13L, that D13L is indeed a trimer, and that D13L is indeed capable of polymerizing into a continuous, self-supporting honeycomb (unpublished data).…”

The Major Core Protein P4a (A10L Gene) of Vaccinia Virus Is Essential for Correct Assembly of Viral DNA into the Nucleoprotein Complex To Form Immature Viral Particles

“…Important and still unresolved issues about VV assembly are how the full complement of proteins become wrapped by the viral membrane to generate individual spherical IVs and how the genomic DNA is incorporated into these particles. Several studies suggested that the viral DNA packed into a nucleoprotein complex is taken up before the IVs are completely sealed (13,18,28,31).…”

“…These images are not frequently seen by electron microscopy during a normal infection, suggesting that this would be a very efficient and fast process. However, certain abortive infections in which VV assembly is blocked result in the accumulation of large cytoplasmic DNA crystalloids that have the appearance of densely packed, ordered fibrils (8,18,31,36,52). Such DNA crystalloids were also seen in cells infected with a conditional mutant that inducibly expresses the A32 gene and which has been described as a DNA packaging mutant (4).…”

“…After successful DNA replication and concatemer resolution (11,30,35), the viral genome is condensed and packaged as a nucleoprotein complex in the IVs (31,32). Other events implicated in virus maturation include assembly and encapsidation of a multiprotein transcription complex (69), proteolytic processing of major structural proteins (25,26,34,55,56,65), formation of a defined core (18,32), and reorganization of viral membranes (5,32). Studies with temperature-sensitive (ts) and inducible VV mutants have revealed that several proteins interacting with DNA are involved in transition from IV to IMV.…”

De novo Fatty Acid Biosynthesis Contributes Significantly to Establishment of a Bioenergetically Favorable Environment for Vaccinia Virus Infection

“…To further examine the importance of fatty acid biosynthesis to viral assembly, we performed infections in the presence of rifampicin; the reversible inhibitor rifampicin prevents the interaction of the viral proteins D13 and A17, thereby preventing the assembly of immature virions [31][32][33][34]. In the presence of rif, DNA replication and the full profile of protein expression proceed normally, but morphogenesis arrests at an early stage [35].…”