Viral Replication Cycle

Attachment, entry and uncoating

Infection of the target cell is mediated by one or both of the virion glycoproteins interacting with the cellular receptor, though no receptor has yet been identified for any member of the Bunyaviridae. The relative importance of either of the glycoproteins in attachment has not been fully elucidated and may differ between the genera. For bunyaviruses, it has been suggested that G1 is the major attachment protein for vertebrate cells, whereas G2 may contain the major determinants for attachment to mosquito cells. Neutralization and hemagglutination-inhibition sites have been mapped to both glycoproteins encoded by hantaviruses and phleboviruses, suggesting that for these viruses both G1 and G2 may be involved in attachment. In common with many other enveloped viruses the Bunyaviridae can fuse cells at acidic pH; for bunyaviruses at least this is accompanied by a conformational change in Gl. Based on electron microscopic studies of phleboviruses, entry into cells is by endocytosis. It is probable that uncoating occurs when endosomes become acidified, thus initiating fusion of the viral membrane and endosomal membrane, followed by release of the nucleocapsids into the cytoplasm.

Transcription

The classical scheme for replication of a negative-strand RNA virus is that the infecting genome is first transcribed into mRNAs by the virion-associated RNA polymerase or transcriptase (Fig. 3). This process, termed primary transcription, is independent of ongoing protein synthesis. Following translation of the primary transcripts into viral proteins, the genome is replicated via a complementary full-length positive-strand RNA, the antigenome, and then further mRNA synthesis (secondary transcription) ensues. Transcriptase activity has been detected in detergent-disrupted virion preparations of representatives of most Bunyaviridae genera. The enzymatic activity was weak compared to, for example, the transcriptase of vesicular stomatitis virus, which has hampered extensive biochemical characterization of the enzyme. However, the bunyavirus transcriptase was shown to be stimulated by oligonucleotides of the (A)nG series, cap analogues (e.g. mGpppAm) and natural mRNAs such as alfalfa mosaic virus RNA 4. These appeared to act as primers for transcription. Further support for this notion was provided by sequencing studies of the 5' ends of both in vivo and in vitro synthesized mRNAs, which showed they contained an additional 10-18 nontemplated nucleotides; a cap structure was present at the 5' terminus. In vitro, an endonuclease activity which specifically cleaved methylated capped mRNAs was detected. Taken together, these data indicate that bunyavirus transcription is markedly similar to that of influenza viruses in using a 'cap-snatch' mechanism to prime transcription. In contrast to influenza viruses, bunyavirus transcription is not sensitive to actinomycin D or a-amanitin, and occurs in the cytoplasm of infected cells. The apparent reiteration of viral terminal sequences at the junction between the primer and viral sequence itself suggests that the polymerase may slip during transcription; further analysis of hantavirus RNAs suggests that this may occur during

Transcription

3' Antigenome (+) 5' 111111111111111111111111???? 3'

Replication

Replication

I Transcription

| Transcription

Negative-sense genome segment

Ambisense genome segment

Figure 3 Transcription and replication scheme of Bunyaviridae genome segments. The left panel shows the scheme for a negative-strand segment and the right panel that for an ambisense segment. The genome RNA and the positive-sense complementary RNA known as the antigenome RNA are only found as ribonucleoprotein complexes and are encapsidated by N protein (•). The mRNA species contain host-derived primer sequences at their 5' ends (■) and are truncated at the 3' end relative to the vRNA template; the mRNAs are not polyadenylated. (Adapted from Elliot RM (1997) Emerging viruses: the Bunyaviridae. Molecular Medicine 3: 572-577, with permission, Plcower Institute Press).

replication as well, and has been dubbed 'prime and realign'.

Analysis of bunyavirus primary transcription in vivo, however, appeared initially to produce results incompatible with the presence of a virion transcriptase, in that no mRNA synthesis could be detected in the presence of protein synthesis inhibitors in certain virus-cell systems. Further work showed that only short transcripts were produced in the absence of protein synthesis in vivo; subsequent gel electrophoresis analyses of the in vitro transcriptase products showed that these too were short transcripts. If the in vitro reaction was supplemented with rabbit reticulocyte lysate, however, full length RNAs were synthesized. The translational requirement was not at the level of mRNA initiation, but rather during elongation, or more precisely to prevent the transcriptase from terminating prematurely. A model to account for these observations proposes that in the absence of ribosome binding and protein translation the nascent mRNA chain and its template can base-pair, thereby preventing progression of the transcriptase enzyme. This translational requirement is not ubiquitous, however, since concurrent translation is not needed for efficient readthrough of premature termination sites in some strains of BHK cells or in the C6/36 mosquito cell line. Reconstitution and mixing experiments suggest that the translational requirement is mediated by a host cell factor, present in some BHK cell lines, which may promote interaction between the nascent mRNA and its template.

The 3' ends of the Bunyaviridae mRNAs are not coterminal with their genome templates. For the nonambisense segments, mRNAs terminate 50-100 nucleotides before the end of the template, though there does not appear to be a universal termination signal in the Bunyaviridae: for instance 3' GUUUUU 5' and 3' ACCCC 5' are two sequences that have been mapped as termination sites. The subgenomic mRNAs transcribed from ambisense S segments terminate within the noncoding intergenic sequences in the RNA; for some, but not all, viruses the intergenic region has the potential to form a stable hairpin structure, though the role of secondary structure in transcription termination is unclear. Bunyaviridae mRNAs are probably not 3' polyadenylated, but many have the potential to form stem—loop structures which may confer stability.

Genome replication

In order to replicate the negative-sense genome RNA a full-length complementary, positive-sense RNA, the antigenome, must be synthesized (Fig. 3). This molecule differs from the positive-sense mRNA in that it does not have the 5' primer sequences and the 3' end extends to the 5' terminus of the genomic RNA template. It is not known what controls the switch from transcriptive to replicative mode of the polymerase. Two events differ between transcription and replication: initiation, which does not require a primer, and readthrough of the mRNA termination signal. The difference in initiation may be because the RNA polymerase is modified by another viral or cellular protein. In the infected cell antigenomes are only found as nucleocapsids; therefore encapsidation of the nascent antigenome RNA may prevent its interaction with the template, thereby overcoming the mRNA termination signal.

Assembly and release

Maturation of the Bunyaviridae characteristically occurs at the smooth membranes in the Golgi apparatus, and hence is inhibited by monensin, a monovalent ionophore. The viral glycoproteins accumulate in the Golgi complex and cause a progressive vacuolization. However, the morphologically altered Golgi complex remains functionally active in its ability to glycosylate and transport glycoproteins destined for the plasma membrane. Using vaccinia virus recombinants it has been shown that the targetting of the Bunyaviridae glycoproteins to the Golgi is a property of the glycoproteins alone, and does not require other viral proteins or virus assembly. Electron microscopic studies revealed that viral nucleocapsids condense on the cytoplasmic side of areas of the Golgi vesicles where viral glycoproteins are present on the luminal side. The absence of a matrix-like protein in the Bunyaviridae, which for other viruses may function as a bridge between the nucleocapsid and the glycoproteins, suggests that direct transmembrane interactions between the Bunyaviridae nucleocapsid and the glycoproteins may be a prerequisite for budding. After budding into the Golgi cisternae, vesicles containing viral particles are transported to the cell surface via the exocytic pathway, eventually releasing their contents to the exterior.

There are important exceptions, however, to the above maturation scheme; Rift Valley fever phlebo-virus has been observed to bud at the surface of infected rat hepatocytes, and it appears that a characteristic of the newly described American hantaviruses which cause hantavirus pulmonary syndrome is that assembly and maturation occur at the plasma membrane.

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