Temporal classification and mapping of non-polyadenylated transcripts of an invertebrate iridovirus

Abstract

The temporal expression of the 54 Chilo iridescent virus (CIV) virion protein genes was investigated by combining drug treatments that inhibit protein or DNA synthesis and an RT-PCR strategy particularly suitable for non-polyadenylated mRNAs. This method generates a uniform 39 terminus by ligation of a 59-phosphorylated oligonucleotide to the 39 end of the transcript that is recognized by a complementary primer during RT-PCR. This analysis showed that CIV virion proteins are encoded by genes in all three predetermined temporal classes: 23 immediate-early, 11 delayed-early and seven late virion gene transcripts were identified and assigned to ORFs. Early transcription of many virion protein genes supports the notion that virion proteins may also play essential roles in the initial stages of infection. In addition, some of the early gene products present in the virion may reflect the intracellular path that the virus follows during infection.

Invertebrate Iridoviruses: A Glance over the Last Decade

“…The revised coding boundaries of the 215 non-overlapping proteins are listed in the Uniprot database [ 53 , 54 , 55 ]. Importantly, gene transcripts of iridovirids have no evidence of introns [ 36 , 53 , 54 ] and viral mRNAs lack poly(A) tails [ 125 ]. However, biochemical and in silico evidence suggest the existence of viral microRNAs (miRNA) that may modulate viral gene expression [ 116 ].…”

“…In IIV-6 infected cells, viral transcription occurs, in a regulated temporal gene expression cascade that can be subdivided into three temporal classes: immediate-early (IE or α), delayed-early (DE, β), and late (L, γ), with the majority of the genes expressed as IE genes [ 125 , 142 , 143 , 144 ]. Both cis -acting and trans -acting regulatory factors initiate the transcription that coincide with various stages of viral infection.…”

“…The transcripts belonging to the IE class included 022L, 104L, 118L, 123L, 155L, 179R, 209R, 232R, 261R, 268L, 295L, 312R, 361L, 380R, 415R, 439L, and 453L. These gene products are mainly involved in blocking host cell apoptosis, nucleoside metabolism, post-translational protein modifications and transcriptional activation of DE genes [ 125 , 143 ]. DE genes included 117L, 149L, 198R, 229L, 378R, 396L, 457L, which function as viral DNA polymerases, protein kinases, and transcriptional activators of late genes.…”

“…Furthermore, clear polyadenylation signals downstream of these two IIV-6 ORFs were not found [ 152 ]. To identify the transcription termination signals of IIV-6 genes, the LACE (Ligation-based amplification of cDNA ends) method was implemented to amplify the 3′ UTR regions of viral transcripts [ 125 ]. LACE showed that about half of all IIV-6 genes contained complementary TAATG and CATTA motifs in the 3′ regions of their mRNAs.…”

Determining 3'-Termini and Sequences of Nascent Single-Stranded Viral DNA Molecules during HIV-1 Reverse Transcription in Infected Cells

“…One example is 3' RACE (rapid amplification of cDNA ends)7, which relies on the polyadenylation of mRNA. In addition, adaptor ligation-based strategies employing RNA ligases were developed, which have included RLM-RACE (RNA ligase-mediated RACE)8 or LACE (ligation-based amplification of cDNA ends)9. It is important to emphasize that ligation-based amplifications are sensitive to any bias introduced by the ligation reaction itself.…”