Abstract:The essential Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) minor capsid protein VP80 has been recently shown to interact with the virus-triggered, nuclear F-actin cytoskeleton. A role for VP80 in virus morphogenesis has been proposed in the maturation of progeny nucleocapsids and in their egress from the virogenic stroma toward the nuclear periphery by a mechanism, which also includes F-actin filaments. We performed functional mapping of VP80 demonstrating that its highly conserved C-termin… Show more
“…Furthermore, yeast two-hybrid assays demonstrated that the 38K protein interacts with VP80 (Wu et al, 2008). The latter protein interacts with both F-actin and DNA (Marek et al, 2012). In addition, actin has been indicated to be involved in the DNA packaging since actin is transported from the cytoplasm to the nucleus (Volkman et al, 1992) and interacts with NC components (Lanier and Volkman, 1998) upon viral infection whereas inhibition of actin polymerization results in the empty NCs (Volkman, 1988).…”
Baculoviruses are a group of enveloped, double-stranded DNA insect viruses with budded (BV) and occlusion-derived (ODV) virions produced during their infection cycle. BVs are commonly described as rod shaped particles with a high apical density of protein extensions (spikes) on the lipid envelope surface. However, due to the fragility of BVs the conventional purification and electron microscopy (EM) staining methods considerably distort the native viral structure. Here, we use cryo-EM analysis to reveal the near-native morphology of two intensively studied baculoviruses, Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) and Spodoptera exigua MNPV (SeMNPV), as models for BVs carrying GP64 and F as envelope fusion protein on the surface. The now well-preserved AcMNPV and SeMNPV BV particles have a remarkable elongated, ovoid shape leaving a large, lateral space between nucleocapsid (NC) and envelope. Consistent with previous findings the NC has a distinctive cap and base structure interacting tightly with the envelope. This tight interaction may explain the partial retaining of the envelope on both ends of the NC and the disappearance of the remainder of the BV envelope in the negative-staining EM images. Cryo-EM also reveals that the viral envelope contains two layers with a total thickness of ≈ 6-7 nm, which is significantly thicker than a usual biological membrane (<4 nm) as measured by X-ray scanning. Most spikes are densely clustered at the two apical ends of the virion although some envelope proteins are also found more sparsely on the lateral regions. The spikes on the surface of AcMNPV BVs appear distinctly different from those of SeMNPV. Based on our observations we propose a new near-native structural model of baculovirus BVs.
“…Furthermore, yeast two-hybrid assays demonstrated that the 38K protein interacts with VP80 (Wu et al, 2008). The latter protein interacts with both F-actin and DNA (Marek et al, 2012). In addition, actin has been indicated to be involved in the DNA packaging since actin is transported from the cytoplasm to the nucleus (Volkman et al, 1992) and interacts with NC components (Lanier and Volkman, 1998) upon viral infection whereas inhibition of actin polymerization results in the empty NCs (Volkman, 1988).…”
Baculoviruses are a group of enveloped, double-stranded DNA insect viruses with budded (BV) and occlusion-derived (ODV) virions produced during their infection cycle. BVs are commonly described as rod shaped particles with a high apical density of protein extensions (spikes) on the lipid envelope surface. However, due to the fragility of BVs the conventional purification and electron microscopy (EM) staining methods considerably distort the native viral structure. Here, we use cryo-EM analysis to reveal the near-native morphology of two intensively studied baculoviruses, Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) and Spodoptera exigua MNPV (SeMNPV), as models for BVs carrying GP64 and F as envelope fusion protein on the surface. The now well-preserved AcMNPV and SeMNPV BV particles have a remarkable elongated, ovoid shape leaving a large, lateral space between nucleocapsid (NC) and envelope. Consistent with previous findings the NC has a distinctive cap and base structure interacting tightly with the envelope. This tight interaction may explain the partial retaining of the envelope on both ends of the NC and the disappearance of the remainder of the BV envelope in the negative-staining EM images. Cryo-EM also reveals that the viral envelope contains two layers with a total thickness of ≈ 6-7 nm, which is significantly thicker than a usual biological membrane (<4 nm) as measured by X-ray scanning. Most spikes are densely clustered at the two apical ends of the virion although some envelope proteins are also found more sparsely on the lateral regions. The spikes on the surface of AcMNPV BVs appear distinctly different from those of SeMNPV. Based on our observations we propose a new near-native structural model of baculovirus BVs.
“…To determine the viral DNA replication capacity of the AcMNPV ⌬vp1054 genome, a quantitative real-time PCR (qPCR)-based assay was carried out as previously described (16,18), in which we monitored levels of viral DNA in insect cells transfected either with Ac-⌬vp1054 bacmid or Ac-⌬vlf1 bacmid. The latter bacmid, which was constructed analogously to a published protocol (16), served as a referential genotype for a "single-cell infection phenotype" virus.…”
Section: Figmentioning
confidence: 99%
“…On the other hand, the coupling of the DNAbinding function of VP80 to virus morphogenesis has not been established yet. VP80 contains an atypical basic helix-loop-helix (bHLH) DNA-binding domain at its essential C-terminal end (18) and N-terminally located paramyosin-like motifs, likely responsible for the experimentally shown association of VP80 with host nuclear filamentous actin (F-actin) (17). Furthermore, VP80 has been previously shown to interact with 38K, another nucleocapsid-associated protein (19).…”
mentioning
confidence: 99%
“…In addition to the above-mentioned VP39, there are a number of minor, but functionally important, capsid-associated proteins (see references 1, 2, and 15 for reviews), which may play essential roles in both recognition of target DNA and its packaging. Among these capsid-associated proteins are two end-linked proteins, very late factor 1 (VLF-1) (16) and VP80 (17,18), which exhibit DNA-binding activities. VLF-1 is a site-specific recombinase and is likely implicated in postreplication processing of viral DNA preceding its packaging (16).…”
c Baculovirus VP1054 protein is a structural component of both of the virion types budded virus (BV) and occlusion-derived virus (ODV), but its exact role in virion morphogenesis is poorly defined. In this paper, we reveal sequence and functional similarity between the baculovirus protein VP1054 and the cellular purine-rich element binding protein PUR-alpha (PUR␣). The data strongly suggest that gene transfer has occurred from a host to an ancestral baculovirus. Deletion of the Autographa californica multiple nucleopolyhedrovirus (AcMNPV) vp1054 gene completely prevented viral cell-to-cell spread. Electron microscopy data showed that assembly of progeny nucleocapsids is dramatically reduced in the absence of VP1054. More precisely, VP1054 is required for proper viral DNA encapsidation, as deduced from the formation of numerous electron-lucent capsid-like tubules. Complementary searching identified the presence of genetic elements composed of repeated GGN trinucleotide motifs in baculovirus genomes, the target sequence for PUR␣ proteins. Interestingly, these GGN-rich sequences are disproportionally distributed in baculoviral genomes and mostly occurred in proximity to the gene for the major occlusion body protein polyhedrin. We further demonstrate that the VP1054 protein specifically recognizes these GGN-rich islands, which at the same time encode crucial proline-rich domains in p78/83, an essential gene adjacent to the polyhedrin gene in the AcMNPV genome. While some viruses, like human immunodeficiency virus type 1 (HIV-1) and human JC virus (JCV), utilize host PUR␣ protein, baculoviruses encode the PUR␣-like protein VP1054, which is crucial for viral progeny production.
“…S3a), which illustrated baculoviruses being trailed by actin tails during intracellular movement. According to previous virology studies [25, 26], after the fusion with acidic endosomes, baculoviruses could induce the disruption and rearrangement of the continuous filamentous actins to form tail-like actin structures, which subsequently drive baculovirus to the perinuclear region. Thus the postfusion infection event was verified at the single-virus level.Fig.…”
BackgroundQuantum dot (QD)-based single virus tracking has become a powerful tool for dissecting virus infection mechanism. However, only virus behaviors at the early stage of retrograde trafficking have been dynamically tracked so far. Monitoring of comprehensive virus retrograde transportation remains a challenge.ResultsBased on the superior fluorescence properties of QDs and their labeling of virus internal component, the dynamic interactions between baculoviruses and all key transportation-related cellular structures, including vesicles, acidic endosomes, actins, nuclear pores and nuclei, were visualized at the single-virus level. Detailed scenarios and dynamic information were provided for these critical interaction processes.ConclusionsA comprehensive model of baculovirus retrograde trafficking involving virus endocytosis, fusion with acidic endosome, translocation to nuclear periphery, internalization into nucleus, and arriving at the destination in nucleus was proposed. Thus the whole retrograde transportation of baculovirus in live host cells was elucidated at the single-virus level for the first time.Electronic supplementary materialThe online version of this article (doi:10.1186/s12951-017-0270-9) contains supplementary material, which is available to authorized users.
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