The "Bridge" in the Epstein-Barr virus alkaline exonuclease protein BGLF5 contributes to shutoff activity during productive infection.
Replication of the human herpesvirus Epstein-Barr virus drastically impairs cellular protein synthesis. This shutoff phenotype results from mRNA degradation upon expression of the early lytic-phase protein BGLF5. Interestingly, BGLF5 is the viral DNase, or alkaline exonuclease, homologues of which are present throughout the herpesvirus family. During productive infection, this DNase is essential for processing and packaging of the viral genome. In contrast to this widely conserved DNase activity, shutoff is only mediated by the alkaline exonucleases of the subfamily of gammaherpesviruses. Here, we show that BGLF5 can degrade mRNAs of both cellular and viral origin, irrespective of polyadenylation. Furthermore, shutoff by BGLF5 induces nuclear relocalization of the cytosolic poly(A) binding protein. Guided by the recently resolved BGLF5 structure, mutants were generated and analyzed for functional consequences on DNase and shutoff activities. On the one hand, a point mutation destroying DNase activity also blocks RNase function, implying that both activities share a catalytic site. On the other hand, other mutations are more selective, having a more pronounced effect on either DNA degradation or shutoff. The latter results are indicative of an oligonucleotide-binding site that is partially shared by DNA and RNA. For this, the flexible "bridge" that crosses the active-site canyon of BGLF5 appears to contribute to the interaction with RNA substrates. These findings extend our understanding of the molecular basis for the shutoff function of BGLF5 that is conserved in gammaherpesviruses but not in alpha- and betaherpesviruses.
Department of Medical Microbiology, University Medical Center Utrecht, Utrecht, Netherlands.
Daniëlle
D
Horst
Wim P
WP
Burmeister
Ingrid G J
IG
Boer
Daphne
D
van Leeuwen
Marlyse
M
Buisson
Alexander E
AE
Gorbalenya
Emmanuel J H J
EJ
Wiertz
Maaike E
ME
Ressing
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D002460:Cell Line; D003851:Deoxyribonucleases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D020031:Epstein-Barr Virus Infections / Q000235:genetics* / Q000378:metabolism / Q000821:virology*; D004854:Herpesvirus 4, Human / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics / Q000502:physiology*; D006801:Humans; D008969:Molecular Sequence Data; D014176:Protein Biosynthesis; D020871:RNA Stability; D016415:Sequence Alignment; D014764:Viral Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D014779:Virus Replication
D014764:Viral Proteins; D003851:Deoxyribonucleases; C083203:deoxyribonuclease, Epstein-Barr virus