Translation of a nonpolyadenylated viral RNA is enhanced by binding of viral coat protein or polyadenylation of the RNA

Abstract

C ircularization of the genome of RNA viruses plays an essential role in the replication and translation of viral RNAs. Formation of panhandle structures by base pairing between complementary sequences at the 5Ј and 3Јtermini of several genera of minus-strand RNA viruses has been known already for some time (1). Recently, it became clear that cyclization by RNA-RNA interactions may also play an essential role in the replication of positive-strand RNA viruses such as f laviviruses (2, 3). However, for a growing number of positive-strand RNA viruses, circularization seems to be mediated by protein bridges between the 5Ј and 3Ј termini of the viral RNA. On entry into the host cell, the genome of positive-strand RNA viruses has to serve as messenger for the translation of viral proteins including replication factors. A number of viral RNAs resemble cellular mRNAs by having a 5Ј-cap structure (m 7 GpppN) and 3Ј-poly(A) tail. In cellular mRNAs, these structures permit the formation of a protein bridge by interactions between eIF4G and eIF4E from the cap-binding complex of translation initiation factors and the poly(A) binding protein (PABP) associated with the poly(A) tail (4, 5). Picornaviruses do not have a cap structure, but the internal ribosome entry site in the 5Ј untranslated region (UTR) of the RNA binds initiation factors and a poly(rC) binding protein that mediate the interaction with PABP. Circularization of Poliovirus RNA seems to play a role both in translation and replication of the viral RNA (6, 7).The genomic RNAs of many viruses lack a poly(A) tail, and elements in the 5Ј and͞or 3Ј UTR are believed to compensate for the absence of a poly(A) tail (8-12). Recently, the rotavirus (family Reoviridae) NSP3 protein was shown to interact with eIF-4G and with the 3Ј UTR of the nonpolyadenylated viral mRNAs. Both interactions were required for efficient translation of the viral mRNA, and NSP3 was suggested to mimic the function of PABP (13). Here, we report evidence that the coat protein (CP) of alfalfa mosaic virus (AMV; genus Alfamovirus, family Bromoviridae) also may act as a functional equivalent of PABP.RNAs 1 and 2 of the tripartite AMV genome encode the replicase proteins P1 and P2, respectively. P1 contains methyltransferase-and helicase-like motifs, whereas P2 contains polymerase-like motifs. RNA 3 encodes the movement protein and CP, which is translated from a subgenomic messenger, RNA 4. The RNAs contain a 5Ј-cap structure but the 3Ј ends are not polyadenylated (14). At their 3Ј termini, RNAs 1-4 contain a homologous sequence of 145 nucleotides that can be folded into two alternative conformations: a linear array of hairpins with flanking AUGC sequences that represents a strong CP binding site or a tRNA-like structure that represents the promoter for minus-strand RNA synthesis (15). A mixture of the three genomic RNAs becomes infectious only after addition of CP or its messenger, RNA 4, to the inoculum (16). Initiation of infection requires binding of a few molecules of CP to the 3Ј termini of the g...

Degenerate In Vitro Genetic Selection Reveals Mutations That Diminish Alfalfa Mosaic Virus RNA Replication without Affecting Coat Protein Binding

“…Neeleman et al (20) reported that coat protein binding may autoregulate AMV RNA translation. This phenomenon would manifest in our experiments if the mutations inserted into genomic RNA 3 or copied into newly transcribed subgenomic coat protein mRNA (i.e., RNA 4) decreased viral RNA replication by reducing viral protein expression as a result of limited viral mRNA translation.…”

“…An amino acid mutation that disrupts coat protein binding to the viral RNA (R17A) also blocks RNA replication (37), suggesting that coat protein binding is directly linked to replication functions. The coat protein has also been reported to enhance AMV RNA translation (20); however, the mechanism is not known.…”

Tombusvirus recruitment of host translational machinery via the 3′ UTR

“…Conversely, animal flaviviruses have only a 59-cap; however, PABP interacts with an internal sequence in their 39 UTRs and this enhances translation (Polacek et al 2009). Plant ilarviruses also do not contain poly(A) tails; however, their capsid protein binds to both a 39-proximal RNA element and eIFs associated with their 59-cap, thereby mimicking the translation-enhancing functions of PABP (Neeleman et al 2001;Krab et al 2005). Tobamo-and tymoviruses contain 59-caps but possess tRNA-like structures for 39 termini (Dreher 2009).…”

Within-host Evolution of Segments Ratio for the Tripartite Genome ofAlfalfa Mosaic Virus

“…Comparing with other members of the Bromoviridae family, it has been shown that BMV and Cucumber mosaic virus (CMV) 1a protein accumulates to larger amounts than the corresponding 2a protein in purified replication complexes [35][36][37] , in spite that the SGF total for BMV was 1:2:3 for barley and 1:2:2 for wheat, suggesting that either trans elements are controlling the translation of viral RNAs or that cis-acting elements may affect the efficiency of each RNA in recruiting ribosomes. In this sense, it has been observed that AMV CP enhances the translational efficiently of viral RNAs in vivo 38 via the interaction with the 3′ termini, which adopts two alternative structures for translation (a linear array of hairpins with high affinity for CP) and replication (a pseudo-knotted structure) 39 . A similar mechanism has been reported as regulator for translation of the replication complex proteins of BMV 40 .…”