Vaccinia virus leads to ATG12–ATG3 conjugation and deficiency in autophagosome formation.
The interactions between viruses and cellular autophagy have been widely reported. On the one hand, autophagy is an important innate immune response against viral infection. On the other hand, some viruses exploit the autophagy pathway for their survival and proliferation in host cells. Vaccinia virus is a member of the family of Poxviridae which includes the smallpox virus. The biogenesis of vaccinia envelopes, including the core envelope of the immature virus (IV), is not fully understood. In this study we investigated the possible interaction between vaccinia virus and the autophagy membrane biogenesis machinery. Massive LC3 lipidation was observed in mouse fibroblast cells upon vaccinia virus infection. Surprisingly, the vaccinia virus induced LC3 lipidation was shown to be independent of ATG5 and ATG7, as the atg5 and atg7 null mouse embryonic fibroblasts (MEFs) exhibited the same high levels of LC3 lipidation as compared with the wild-type MEFs. Mass spectrometry and immunoblotting analyses revealed that the viral infection led to the direct conjugation of ATG3, which is the E2-like enzyme required for LC3-phosphoethanonamine conjugation, to ATG12, which is a component of the E3-like ATG12–ATG5-ATG16 complex for LC3 lipidation. Consistently, ATG3 was shown to be required for the vaccinia virus induced LC3 lipidation. Strikingly, despite the high levels of LC3 lipidation, subsequent electron microscopy showed that vaccinia virus-infected cells were devoid of autophagosomes, either in normal growth medium or upon serum and amino acid deprivation. In addition, no autophagy flux was observed in virus-infected cells. We further demonstrated that neither ATG3 nor LC3 lipidation is crucial for viral membrane biogenesis or viral proliferation and infection. Together, these results indicated that vaccinia virus does not exploit the cellular autophagic membrane biogenesis machinery for their viral membrane production. Moreover, this study demonstrated that vaccinia virus instead actively disrupts the cellular autophagy through a novel molecular mechanism that is associated with aberrant LC3 lipidation and a direct conjugation between ATG12 and ATG3.
Department of Pharmacology, University of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical School, Piscataway, NJ, USA.
Joseph G
JG
Moloughney
Claude E
CE
Monken
Hanlin
H
Tao
Haiyan
H
Zhang
Janice D
JD
Thomas
Edmund C
EC
Lattime
Shengkan
S
Jin
D000595:Amino Acid Sequence; D000818:Animals; D001343:Autophagy; D000071192:Autophagy-Related Protein 12; D000071187:Autophagy-Related Protein 5; D000071193:Autophagy-Related Protein 7; D000071183:Autophagy-Related Proteins; D004279:DNA, Viral / Q000378:metabolism; D005347:Fibroblasts / Q000378:metabolism / Q000821:virology; D047468:Immunoprecipitation; D008055:Lipids / Q000737:chemistry; D013058:Mass Spectrometry; D051379:Mice; D008869:Microtubule-Associated Proteins / Q000378:metabolism; D008969:Molecular Sequence Data; D041681:NIH 3T3 Cells; D010588:Phagosomes / Q000378:metabolism*; D011506:Proteins / Q000737:chemistry / Q000378:metabolism*; D044763:Ubiquitin-Conjugating Enzymes / Q000378:metabolism*; D014615:Vaccinia / Q000378:metabolism / Q000821:virology; D014616:Vaccinia virus / Q000254:growth & development / Q000378:metabolism*
C491971:Atg12 protein, mouse; C495773:Atg5 protein, mouse; C502661:Atg7 protein, mouse; D000071192:Autophagy-Related Protein 12; D000071187:Autophagy-Related Protein 5; D000071183:Autophagy-Related Proteins; D004279:DNA, Viral; D008055:Lipids; C480860:Map1lc3b protein, mouse; D008869:Microtubule-Associated Proteins; D011506:Proteins; D044763:Ubiquitin-Conjugating Enzymes; D000071193:Autophagy-Related Protein 7; C568853:Atg3 protein, mouse