Tata

FIGURE 5.5 Transcription signals in retroviral LTRs. Where the same factor binds to a number of LTRs, the same symbol has been used. The TRE elements in PTLV-1 are the Tax-responsive elements. The blue arrow marks the beginning of transcription. Note that the scale of B is twofold different from that in A. [This figure is a composite of Figs. 4, 5, 7, 8, 9, and 11 in Chapter 6 of Collins et al. (1997).]

Some fraction of the genomic RNA is exported to the cytoplasm without further processing. There it serves as mRNA for the synthesis of Gag, Gag-Pro, and/or Gag-Pro-Pol, as described below. Alternatively, it can be packaged into progeny viruses. Genomic RNA that serves as mRNA and genomic RNA that serves as the source of RNA for packaging are maintained in separate pools.

Some fraction of the genomic RNA is spliced before export to the cytoplasm. Only one spliced RNA is made in the simple retroviruses, which serves as mRNA for Env. In most retroviruses, the entire Gag-Pro-Pol region is spliced out and the initiation codon for Env is encoded in env. In the avian retroviruses, however, the upstream splice site is located within the Gag coding sequence so that Env begins with the first six codons of Gag.

The need for both spliced and unspliced versions of the viral RNA means that mechanisms must exist to ensure that both are produced and that the ratio of spliced to unspliced RNA is optimal for virus replication. In the simple retroviruses, the splice sites are suboptimal, so that not all RNA is spliced. Experiments have shown that the result is an optimal ratio of spliced to unspliced RNA. Mutations that make splicing more efficient are deleterious for virus growth and revertants quickly arise that restore the proper ratio. The second problem faced by these viruses is the need to export unspliced RNA to the cytoplasm. Eukaryotic cells have control mechanisms to ensure that RNA containing splice sites is not exported from the nucleus. It has been found that sequence elements in the unspliced RNA are required for its export, and it is assumed that these elements interact with cellular proteins that promote export. In the simian retrovirus Mason-Pfizer monkey virus, this element is called the constitutive transport element. It is 154 nucleotides long and is located in the 3' nontranslated region of the RNA. In the avian retro-viruses, an apparently unrelated element of about the same size, also present in the 3' nontranslated region, provides the same function for unspliced avian retroviral RNA. Interestingly, the avian sequence does not work in mammalian cells. The monkey virus sequence works in both mammalian and avian cells, but works better in mammalian cells. These findings are consistent with the hypothesis that these transport elements interact with cellular proteins. The inability of unspliced RNA to be exported from the nucleus is one of the reasons that the avian retroviruses will not replicate in mammalian cells.

Translation of Viral Genomic RNA

Retroviral genomic RNA is translated into two or three polyproteins that are eventually processed by the viral PR. The order of genes along the genomic RNA is gag-pro-pol, encoding the proteins Gag, Pro, and Pol. Stop codons are present between Gag and Pro, or between Pro and Pol, or in both places, as illustrated in Fig. 5.6. Termination of the polypeptide chain occurs at these stop codons most of the time during translation. These stop codons are suppressed some of the time, however, either by read-through or by frameshifing (Chapter 1), so that the amount of Pol produced is usually about 5% that of Gag. In viruses with one stop codon, the frequency of suppression is about 5%, but in viruses with two stop codons, the frequency of suppression of each stop codon is higher so that significant amounts of Pol are produced even though suppression of two stop codons is required. This means that the frequency of suppression is variable and can be controlled by changes in the sequence of the viral RNA. Because reinitiation does not occur once the chain is terminated, the polyproteins produced are Gag and/or Gag-Pro and/or Gag-Pro-Pol, depending on the positions of the stop codons (Fig. 5.6).

Pro is produced in three different ways in different retro-viruses, as illustrated in Fig. 5.6. In the avian viruses, such as ALV, there is no stop codon between Gag and Pro so that a Gag-Pro polyprotein is produced. Gag and Pro are thus produced in equal amounts. Frameshifting results in the production of a longer polyprotein, Gag-Pro-Pol. Most of the mammalian viruses also only have a single stop codon in the ORF, but it is positioned between Gag and Pro. Read-through of a UAG stop codon (murine leukemia viruses) or frameshifting (other mammalian viruses with a single stop codon) results in the longer polyprotein. The two polyproteins produced are Gag and Gag-Pro-Pol, and Pro and Pol are produced in the same low amounts. Finally, several mammalian retroviruses, for example, MMTV and PTLV-1, have two stop codons in the ORF, both of which can be suppressed by frameshifting. Thus, three polyproteins are produced, Gag, Gag-Pro, and Gag-Pro-Pol. In this case Pro is produced at intermediate levels.

Processing of these various polyproteins occurs during assembly of progeny virions. The viral Pro is an aspartate protease whose active site contains two aspartic acid residues (Chapter 1). The protease domain is functional in polypep-tides containing the Pro sequence as well as after its release by proteolysis as a small protein of about 100 residues. The enzyme is active only as a homodimer, with each chain in the dimer supplying one of the aspartic acids in the active site. Because the monomer is not active, there is a delay in processing. The high concentration of viral polyproteins that occurs in viral particles or in previral particles is required to achieve efficient dimerization of the protease and its activation. Experiments have shown that premature activation of the protease, which can be achieved by using genetic tricks, is deleterious for virus assembly. Thus, it is important that processing be delayed until assembly begins or is completed.

During processing of Gag, several different proteins are produced, some of which are quite small, whereas others are larger. The proteins produced from Gag are illustrated schematically in Fig. 5.7 for a number of retroviruses. Gag

Family Retroviridae

Was this article helpful?

0 0

Post a comment