Viruses by Miguel Gonzalez

Eugene V. Koonina,1 and Bernard Mossb,1 a

National Center for Biotechnology Information, National Library of Medicine and bLaboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892

apping of mRNA is an early posttranscriptional event, unique to eukaryotes, that strongly influences subsequent processing, nuclear export, stability, and translation of mRNA. Accordingly, viruses of eukaryotes, whether they reside in the nucleus or cytoplasm, must solve the capping problem for efficient replication. In a recent issue of PNAS, Ogino et al. (1) demonstrate a distinct mechanism used by vesicular stomatitis virus (VSV), and likely other nonsegmented negative strand RNA viruses (Mononegavirales), to cap the 5′ end of mRNA. The finding is of interest from biochemical and evolutionary perspectives and exemplifies the diverse ways that viruses adapt to their hosts. A cap, consisting of a 7-methylguanosine (m7G) linked to the 5′ end of the transcript by a 5′-5′ triphosphate bridge [m7G(5′) ppp(5′)N- or m7G(5′)ppp(5′)Nm in which Nm is a 2′-O methylated nucleoside], was identified in viral and mammalian mRNA 35 years ago and was subsequently found to be ubiquitous in eukaryotes and their viruses with a few exceptions among the latter (2–4). The enzymology of cap biogenesis, originally deduced for vaccinia virus (5–8), also pertains to eukaryotes (9) (Fig. 1A). In this conventional pathway, the RNA guanylyltransferase (GTase) transfers GMP to the diphosphate end of pre-mRNA through a covalent enzymeGMP intermediate. In contrast, the VSV polyribonucleotidyltransferase (PRNTase) activity of the RNA-dependent RNA polymerase (L) protein transfers the 5′monophosphorylated pre-mRNA to GDP through a covalent enzyme-phosphorylated RNA intermediate (1) (Fig. 1B). The latter mechanism involves the formation of a phosphoamide bond to histidine instead of lysine, as occurs in the conventional system. There is also a difference in the sequence of methylations in the two systems: guanine-7-methylation precedes 2’-O-methylation in the conventional system, whereas the order is reversed with VSV (10). Extensive sequence database searches (SI Materials and Methods) confirmed the conservation of the so-called region V of the rhabodvirus L protein (11) among all Mononegavirales but failed to detect similarity between this region and any available protein sequences outside Mononegavirales. Secondary structure prediction indicated that the viral PRNTase is a globular domain with a www.pnas.org/cgi/doi/10.1073/pnas.0915061107

Conventional cap formation (i) pppN(pN)nppN(pN)n + Pi (ii) pppG + ppN(pN)n- (G5')pppN(pN)n + PPi (a) E + pppG E-pG + PPi (b) E-pG + ppN(pN)n- (G5')pppN(pN)n + E (iii) G(5')pppN(pN)n + AdoMet m7G(5')pppN(pN)n + AdoHcy m7G(5')pppNm(pN)n + AdhHcy (iv) m7G(5')pppN(pN)n + AdoMet

Alternative cap formation (i) pppG ppG + Pi (ii) ppG + pppA(pN)n- (G5')pppA(pN)n + PPi (a) E + pppA(pN)n E-pA(pN)n- + PPi (b) E-pA(pN)n- + ppG (G5')pppA(pN)n + E G(5')pppNm(pN)n + AdoHcy (iii) G(5')pppN(pN)n + AdoMet m7G(5')pppNm(pN)n + AdoHcy (iv) G(5')pppNm(pN)n + AdoMet

Fig. 1. Conventional (A) and alternative VSV (B) mechanisms of mRNA capping. Abbreviations: pppG, GTP; pppN(pN)n-, 5′ end of pre-RNA; E, enzyme; PPi, pyrophosphate; AdoMet, S-adenosylmethionine; AdoHcy; S-adenosylhomocysteine.

β-sheet core (Fig. S1); however, comparison of the predicted secondary structure of the PRNTase domain to the available protein structures failed to detect any structurally similar domains. Thus the viral PRNTase domain is so far unique to Mononegavirales, on par with the unique capping reaction of these viruses. The histidine residue that in VSV covalently binds the 5′-termini of viral mRNAs is conserved in most Mononegavirales but not in viruses of the genus Novirhabdovirus (Fig. S1). Given the low similarity between the homologous domain in Novirhabdoviruses to the PRNTase domains of other Mononegavirales, it remains unclear whether the histidine residue noted by Ogino et al. (1) in these sequences corresponds to the active histidine (Fig. S1) and, consequently, whether Novirhabdoviruses possess the same capping machinery. Given the predicted globular structure of the PRNTase domain, with a core β-sheet(s), it seems unlikely that this domain evolved de novo, from a simple, repetitive structure, as seems to be the case for many α-helical domains in eukaryotes (12). A more plausible hypothesis is that a preexisting globular domain was recruited by the common ancestor of Mononegavirales but has changed beyond recognition (with sequence analysis and fold recognition methods) in the course of adaptation to the unique capping reaction. The multiple domains of the L proteins of Mononegavirales are generally highly derived and show little sequence conservation outside this group of viruses. The exception is the MTase domain in-

volved in cap formation that shows significant similarity to ribosomal RNA MTase (13, 14). The identity of the phosphatase or GTPase that catalyzes the formation of GDP from GTP (or whether a specific enzyme is required) in Mononegavirales remains unknown. Crystal structures of L proteins of Mononegavirales or their individual domains will be key to understanding their origins and evolution. The various ways that viruses have coped with the capping problem are described in Table 1. Straightforward exploitation of the cellular capping machinery is typical of DNA viruses that replicate in the nucleus (Table 1). However, molecular strategies that make a virus independent of the cellular capping enzymes could be a clear advantage. Several such strategies have evolved independently, including internal initiation of translation on uncapped RNAs in picornaviruses and their relatives, snatching of capped oligonucleotides from host premRNAs to initiate viral transcription in segmented negativestrand RNA viruses, and recruitment of genes for the conventional, eukaryotictype capping enzymes that apparently occurred independently in widely diverse Author contributions: E.V.K. and B.M. wrote the paper. The authors declare no conflict of interest. See companion article on page 3463. 1

To whom correspondence may be addressed. E-mail: koonin@ncbi.nlm.nih.gov or bmoss@niaid.nih.gov.

This article contains supporting information online at www. pnas.org/cgi/content/full/0915061107/DCSupplemental.

PNAS | February 23, 2010 | vol. 107 | no. 8 | 3283–3284

COMMENTARY

Viruses know more than one way to don a cap

Table 1. The diversity of solutions to the capping problem among the major groups of viruses of eukaryotes Capping apparatus or alternative

Groups of viruses

Utilization of host capping apparatus. Not known whether some might modify the cellular capping machinery.

Single and double-strand DNA viruses that replicate in the nucleus of animals and plants: e.g., adenoviruses, herpesviruses, papovaviruses, parvoviruses, reverse-transcribing viruses: retro- and pararetroviruses, hepadnaviruses.

No cap, mRNAs with triphosphate 5′-termini, internal initiation of translation, 3′ translational enhancer.

Small positive-strand RNA viruses of plants including carmoviruses, tombusviruses, poleroviruses, luteoviruses. Double-stranded RNA viruses of fungi (totiviruses).

No cap, mRNAs covalently linked to a small protein, internal initiation of translation.

Small positive strand RNA viruses of the picorna-like superfamily (picornaviruses, caliciviruses, comoviruses, potyviruses). Double-stranded RNA viruses: birnaviruses.

Reutilization of host caps mediated by virus-encoded mRNA-endonuclease.

Segmented negative-strand RNA viruses: orthomyxoviruses, arenaviruses, bunyaviruses.

Virus-encoded capping apparatus,apparently host-derived: RNA triphosphatase, GTase, m7G-MTase; 2’-O-MTase.

Positive-strand RNA viruses: alpha-like superfamily, ﬂaviviruses. Doublestranded RNA viruses: reoviridae. Large Nucleocytoplasmic DNA Viruses (NCLDV: poxviruses, asfarviruses, some iridoviruses, phycodnaviruses, mimiviruses); baculoviruses, nudiviruses.

Unique virus-encoded capping apparatus: PRNTase, MTase, GTPase(?)

Mononegavirales (four families: rhabdoviruses, bornaviruses, ﬁloviruses and paramyxoviruses).

No homologs of PRNTase or MTase domains. Might use host capping apparatus or possess unique set of capping enzymes.

Ophioviridae family of negative-strand RNA viruses infecting plants. Order not assigned. The closest known relative of Mononegavirales.

groups of viruses (Table 1). The discovery of Ogino et al. (1) adds the unprecedented strategy of evolving a unique capping apparatus in Mononegavirales. The source of recruitment seems clear for the MTase domain but not for the PRNTase domain. Interestingly, ophioviruses, the

group of plant negative strand RNA viruses that is the closest relative of Mononegavirales judging by the RNA polymerase sequence, encode no homologs of the capping domains. How these viruses solve the capping problem remains uncertain. With the current rapid progress of

viral genome sequencing from diverse hosts, it will be of major interest to see whether the unique capping machinery discovered in Mononegavirales recurs in any other viruses and whether other, yet undiscovered, capping systems might exist.

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6. Barbosa E, Moss B (1978) mRNA(nucleoside-2′-)-methyltransferase from vaccinia virus. Characteristics and substrate speciﬁcity. J Biol Chem 253:7698–7702. 7. Venkatesan S, Gershowitz A, Moss B (1980) Modiﬁcation of the 5′ end of mRNA. Association of RNA triphosphatase with the RNA guanylyltransferase-RNA (guanine-7-)methyltransferase complex from vaccinia virus. J Biol Chem 255:903–908. 8. Shuman S, Hurwitz J (1981) Mechanism of mRNA capping by vaccinia virus guanylyltransferase: Characterization of an enzyme—guanylate intermediate. Proc Natl Acad Sci USA 78:187–191. 9. Shuman S (1995) Capping enzyme in eukaryotic mRNA synthesis. Prog Nucl Acid Res Mol Biol 50:101–129. 10. Rahmeh AA, Li J, Kranzusch PJ, Whelan SP (2009) Ribose 2′-O methylation of the vesicular stomatitis virus mRNA cap precedes and facilitates subsequent guanine-N-7 methylation by the large polymerase protein. J Virol 83:11043–11050.

11. Poch O, Blumberg BM, Bougueleret L, Tordo N (1990) Sequence comparison of ﬁve polymerases (L proteins) of unsegmented negative-strand RNA viruses: Theoretical assignment of functional domains. J Gen Virol 71: 1153–1162. 12. Aravind L, Iyer LM, Koonin EV (2006) Comparative genomics and structural biology of the molecular innovations of eukaryotes. Curr Opin Struct Biol 16: 409–419. 13. Bujnicki JM, Rychlewski L (2002) In silico identiﬁcation, structure prediction and phylogenetic analysis of the 2′-O-ribose (cap 1) methyltransferase domain in the large structural protein of ssRNA negative-strand viruses. Protein Eng 15:101–108. 14. Galloway SE, Richardson PE, Wertz GW (2008) Analysis of a structural homology model of the 2′-O-ribose methyltransferase domain within the vesicular stomatitis virus L protein. Virology 382:69–82.

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Koonin and Moss