Replication

General characteristics

FV3 grows in a wide variety of tissue culture cells of piscine, amphibian, avian and mammalian origin at temperatures between 12 and 32°C. At the light microscopy level, virus assembly sites are seen as Feulgen-positive inclusion bodies in the cytoplasm of infected cells (Fig. 1) that contain viral DNA and proteins. Electron micrographs of FV3-infected cells show assembly sites as regions free of cellular structures (e.g. organelles, ribosomes, cytoskeletal filaments) that contain virus particles in various stages of assembly (Fig. 2). Like all other viruses, the time course of infectious virus production is variable, depending on the type of cells, temperature of incubation, multiplicity of infection, conditions of growth and the strain of the virus. An unusual feature of FV3 replication is that it uses both the nucleus and cytoplasm for its nucleic acid synthesis but virus assembly takes place exclusively in the cytoplasm. At 33°C, a nonpermissive temperature for virus growth, early virus-specific transcription takes place but no

Transcription (RNA polymerase II)

" Genome synthesis (unit length)

Template tor cytoplasmic DNA synthesis

Amplification of transcription

(j) Concatemer formation Transcription (?)

(5) Assembly

Figure 3 Proposed replication cycle of frog vlrus-3 (FV3). The genomes of parent FV3 particles reach the nucleus where they are transcribed during the early stages of infection. Cellular RNA polymerase II, modified by the structural protein(s) of the virus particles, is probably used for virus transcription at this stage. The parent genome in the nucleus also serves as the template for stage 1 of DNA replication. Replicative molecules are then transported to the cytoplasm where they participate in stage 2 of DNA replication. The large replicative complex produced in stage 2 is cleaved to produce mature viral DNA. Virus morphogenesis and assembly also occur in the cytoplasm.

viral DNA or infectious virions are produced. Apparently, one or more of the viral proteins are temperature sensitive and nonfunctional at this temperature. FV3 infection results in the 'shut-off' of host cell macromolecular syntheses (DNA, RNA, protein) allowing ready detection of viral DNA, RNA and protein.

DMA replication

An unusual feature of FV3 DNA replication is that it occurs in two stages that are physically compartmentalized in the nucleus and cytoplasm (Fig. 3). In the first stage, FV3 DNA replication is initiated at one or more preferred origins, probably on the linear DNA molecule in the nucleus with newly synthesized progeny DNA of genome or less than genome length. Newly synthesized viral DNA in the nucleus is not methylated. Arginine starvation of FV3-infected cells or infection of cells with a temperature sensitive mutant (is12488) at nonpermissive temperature results in the arrest of FV3 DNA replication in the first stage, suggesting that transition from the first to second stage of DNA replication is mediated by a viral protein(s). During infection, progeny DNA synthesized in the nucleus is transported to the cytoplasm where it participates in the second stage of DNA replication. Second-stage DNA replication occurs only in the cytoplasm late in infection (after 3 h) and the replicating DNA is present as a large concatemer (more than 10 times the genome size). Evidently, progeny DNA during the second stage of DNA replication consists of multiple genome length molecules (concatemers). A virus-specified DNA methyltransferase methylates the DNA after its replication in the cytoplasm. During the second stage, FV3 DNA replication is intimately associated with recombination. Progeny molecules at this stage extensively recombine which creates many more replication forks leading to a rapid increase in the rate of DNA synthesis. Since FV3 DNA is circularly permuted and terminally redundant, it greatly facilitates recombination and most likely promotes formation of a complex, branched network of replicating DNA. Inhibition of recombination greatly reduces DNA synthesis, suggesting that recombination and replication at this stage are interdependent. Pulse-chase experiments showed that concatemeric DNA serves as the precursor for production of mature viral DNA molecules.

Functions of FV3 concatemeric DNA

The production of concatemers is required for regeneration of the end of the DNA molecules in viruses that possess linear genomes. The most thoroughly studied example of such a function is phage T4, in which concatemeric DNA is cleaved and then packaged into virions via a 'headfuP mechanism. DNA packaged into phage heads through this mechanism becomes circularly permuted and terminally redundant. As mentioned earlier, FV3 concatemeric DNA is used for production of mature viral DNA. It is, therefore, reasonable to assume that FV3 may also use the 'headfuP packaging mechanism to generate circularly permuted and terminally redundant progeny molecules that are found in the infectious virions. However, experimental evidence for the 'headful' or any other mechanism of DNA packaging remains to be established.

Transcription

The genome of an incoming FV3 particle reaches the nucleus where it is transcribed during the early stages of infection. Cellular RNA polymerase II is used for transcription at this stage with a virion-associated protein required for FV3 transcription. It is not known whether this protein modifies RNA polymerase II or the FV3 DNA template for transcription.

FV3 transcripts lack polyadenylation tracts at their 3'-end. At the 5'-end, FV3 mRNAs are capped and methylated. Early viral mRNA molecules are also methylated at the internal position in adenosine residue. In contrast, although late mRNAs are terminally blocked and methylated, internal methyla-tion in this mRNA class is not detectable. Since early FV3 transcripts are synthesized in the nucleus, whereas late transcripts are synthesized in the cytoplasm, these differences in methylation of early versus late transcripts may be due to the different site of their synthesis.

Because of the complete switch-off of host cell mRNA synthesis, and lack of variable-length poly(A) tracts on FV3 mRNA, the FV3 transcript from infected cells can be resolved into 47 bands on denatured gels. Analysis of these bands indicated that FV3 transcription is temporally regulated and can be subdivided into three classes: immediate-early (IE), delayed-early (DE) and late (L). The IE class consists of those RNAs synthesized in the absence of protein synthesis and represents approximately one-third of the single-stranded genome equivalent. Depending on the cell line, IE synthesis takes place 1-2 h postinfection. An additional third of the genome is transcribed in the presence of amino acid analogue fluorophenyl-alanine or by a temperature sensitive (is) mutant, is9467, that is defective in late transcription at nonpermissive temperature. DE mRNA is normally synthesized 2-3 h postinfection. By 3-4 h postinfec tion, a complete single-stranded equivalent of the genome is transcribed.

Transcriptional mapping and sequencing of 10 FV3 genes have shown that various classes of FV3 mRNAs, as well as mRNAs within classes, are transcribed from separate promoters. FV3 genes contain no intervening sequences and no polyadenylation sites; apparently there is no post-transcriptional processing of the FV3 transcript.

Site-specific DNA methylation has a strong silencing effect on genes transcribed by eukaryotic RNA polymerase II, yet FV3 uses this enzyme to transcribe FV3 RNA from its highly methylated genome. Experimental evidence indicates that FV3 infection results in the transcription of exogenously supplied, normally silent, methylated genes in infected cells. Thus, FV3 has evolved a mechanism to permit cellular RNA polymerase II to transcribe normally silent methylated genes.

Protein synthesis

In conjunction with FV3 transcription, viral protein synthesis occurs in three phases. First, IE proteins are synthesized at the beginning of infection, then DE proteins after 2 h postinfection, and L proteins by 3— 4 h postinfection. Several viral regulatory proteins are required for transition from IE to DE to L protein synthesis. In contrast to other DNA viruses, DNA replication is not necessary for L protein synthesis. However, DNA replication increases the amount of L proteins synthesized in FV3-infected cells. FV3 protein synthesis is also regulated at the translational level. Although transcription of IE and DE genes continues concomitantly with that of L genes, their translation is greatly reduced late in infection. Experimental evidence suggests that at least one viral protein is required to inhibit the translation of IE and DE mRNA late in infection. Late FV3 mRNAs are poorly translated in vitro-, addition of a factor present in infected cells is required for efficient translation of these messages. Thus, efficient translation of late mRNAs is also mediated by viral regulatory pro-tein(s).

Assembly and release of virions

FV3 virus particles are assembled in specialized structures termed the assembly sites found exclusively in the cytoplasm of infected cells. These sites are less dense than the surrounding cytoplasm, devoid of cellular structures (e.g. organelles, cytoskeletal filaments, ribosomes), and contain assembling virions. Assembly sites are formed in two steps; in the first step, viral DNA accumulates in the cytoplasm as a spheroid mass but devoid of viral proteins (pre-

assembly site). During the second step, reorganized intermediate filaments surround preassembly sites and viral proteins accumulate into it. Cells infected with a temperature sensitive mutant (ts9467) at non-permissive temperature form preassembly sites but not mature assembly sites. The virus assembly sites are the only region of the cytoplasm at which the viral proteins and viral genomes are present in great abundance. Data suggest that viral proteins synthesized elsewhere in the cytoplasm are transported into the assembly sites. In summary, the virus assembly site is a specialized area of the cytoplasm composed of a three-dimensional filamentous matrix in which the viral proteins, viral genomes and assembling virions are suspended. It is possible that the matrix may serve as a substratum on which virus proteins interact in an ordered fashion to form icosahedral particles.

Late in infection, the surfaces of FV3-infected cells exhibit many projections through which the virus buds. These projections have been termed micro-villus-like projections because they resemble normal cellular microvilli in general morphology. At a time when the virus is released in abundance, the number of projections greatly increase and each projection possesses a series of bulges, each of which contains a single virus particle. The microvillus-like projections contain microfilaments and virus particles in the process of budding. Microfilaments play an active role in FV3 budding since microfilament-depolymer-izing drugs such as cytochalasine B and D prevent virus release. FV3 acquires its envelope during the process of budding. However, acquisition of the envelope is not necessary for virus infectivity. Intracellular virus particles, often seen as crystalline arrays in electron microscopic pictures, are infectious. Similarly, inhibition of virus budding by cytochalasine D has no effect on virus infectivity.

FV3-induced cytoskeletal changes

Soon after infection, FV3 induces dramatic organizational changes in all three classes of cytoskeletal filaments: the microtubules, the intermediate filaments and the microfilaments. In FV3-infècted cells, the microtubules appear to decrease progressively with the course of FV3 infection. Concomitant with a decrease in microtubule numbers, there is a reduction (80% reduction by 4 h postinfection) in tubulin synthesis — the constituent protein of microtubules.

In FV3-infected cells, intermediate filaments retract from the cell periphery and reorganize to surround the preassembly sites. There is increased phosphorylation of vimentin (constituent protein of intermediate filaments in fibroblasts) in FV3-infected cells before they reorganize around the preassembly sites. In

?s9467-infected cells at nonpermissive temperature, there is neither increased phosphorylation of vimentin nor reorganization of intermediate filaments around the preassembly sites. These results suggest that phosphorylation of vimentin is a prerequisite for reorganization of intermediate filaments.

In FV3-infected cells, the bundles of microfilaments (stress fibers) disappear but microfilaments underlying the microvillus-like projections are seen late in infection. The significance of the destruction of stress fibers is not known but it may be related to changes in the cell shape that occur during virus infection. Reorganization of actin into cell surface projections, however, appears to be important in virus release. Biochemical studies have shown that actin, the constituent protein of microfilaments, becomes more acidic than its counterpart in uninfected cells. It is not known whether the observed shift in actin is due to post-translational modification or whether the phenomenon represents the synthesis of a new isoform of actin. In either case, the process must occur during virus replication, since actin synthesized before infection did not show increased acidity. It is noteworthy that the changes in actin precede the formation of microvillus-like structures used in virus release.

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