Direct Interaction of Baculovirus Capsid Proteins VP39 and EXON0 with Kinesin-1 in Insect Cells Determined by Fluorescence Resonance Energy Transfer-Fluorescence Lifetime Imaging Microscopy

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Superinfection exclusion is the ability of an established virus to interfere with a second virus infection. This effect was studied in vitro during lepidopteran-specific nucleopolyhedrovirus (genus observed following inoculation with mixtures of these two phylogenetically distant nucleopolyhedroviruses-AcMNPV and SfMNPV-but at a frequency lower than predicted, suggesting interspecific virus interference during infection or replication. The temporal window of infection is likely necessary to maintain genotypic diversity that favors virus survival but also permits dual infection by heterospecific alphabaculoviruses.IMPORTANCE Infection of a cell by more than one virus particle implies sharing of cell resources. We show that multiple infection, by closely related or distantly related baculoviruses, is possible only during a brief window of time that allows additional virus particles to enter an infected cell over a period of ca. 16 h but then blocks multiple infections as newly generated virus particles begin to leave the infected cell. This temporal window has two important consequences. First, it allows multiple genotypes to almost simultaneously infect cells within the host, thus generating genetically diverse virus particles for transmission. Second, it provides a mechanism by which different viruses replicating in the same cell nucleus can exchange genetic material, so that the progeny viruses may be a mosaic of genes from each of the parental viruses. This opens a completely new avenue of research into the evolution of these insect pathogens.

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confidence: 99%

mentioning

confidence: 99%

Superinfection Exclusion in Alphabaculovirus Infections Is Concomitant with Actin Reorganization

Beperet

Irons

Simón

et al. 2014

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“…In support of this, the depolymerization of microtubules substantially reduces the level of BV production (17,18). In addition, fluorescence resonance energy transfer by fluorescence lifetime imaging (FRET-FLIM) analysis has shown that both AC141 and VP39 (a major capsid protein) interact with the tetratricopeptide repeat (TPR) domain of Drosophila melanogaster kinesin-1 light chain (DmKLC) (18). The kinesin superfamily (KIF) is a class of motor proteins which are known to carry cargo like membranous organelles and other macromolecules anteriorly along microtubules (19).…”

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confidence: 91%

“…AcMNPV AC141, which is required for BV production, interacts with ␤-tubulin, suggesting that microtubules are involved. In support of this, the depolymerization of microtubules substantially reduces the level of BV production (17,18). In addition, fluorescence resonance energy transfer by fluorescence lifetime imaging (FRET-FLIM) analysis has shown that both AC141 and VP39 (a major capsid protein) interact with the tetratricopeptide repeat (TPR) domain of Drosophila melanogaster kinesin-1 light chain (DmKLC) (18).…”

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confidence: 98%

Trichoplusia ni Kinesin-1 Associates with Autographa californica Multiple Nucleopolyhedrovirus Nucleocapsid Proteins and Is Required for Production of Budded Virus

Biswas

Blissard

Theilmann

2016

The mechanism by which nucleocapsids of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) egress from the nucleus to the plasma membrane, leading to the formation of budded virus (BV), is not known. AC141 is a nucleocapsid-associated protein required for BV egress and has previously been shown to be associated with ␤-tubulin. In addition, AC141 and VP39 were previously shown by fluorescence resonance energy transfer by fluorescence lifetime imaging to interact directly with the Drosophila melanogaster kinesin-1 light chain (KLC) tetratricopeptide repeat (TPR) domain. These results suggested that microtubule transport systems may be involved in baculovirus nucleocapsid egress and BV formation. In this study, we investigated the role of lepidopteran microtubule transport using coimmunoprecipitation, colocalization, yeast two-hybrid, and small interfering RNA (siRNA) analyses. We show that nucleocapsid AC141 associates with the lepidopteran Trichoplusia ni KLC and kinesin-1 heavy chain (KHC) by coimmunoprecipitation and colocalization. Kinesin-1, AC141, and microtubules colocalized predominantly at the plasma membrane. In addition, the nucleocapsid proteins VP39, FP25, and BV/ODV-C42 were also coimmunoprecipitated with T. ni KLC. Direct analysis of the role of T. ni kinesin-1 by downregulation of KLC by siRNA resulted in a significant decrease in BV production. Nucleocapsids labeled with VP39 fused with three copies of the mCherry fluorescent protein also colocalized with microtubules. Yeast two-hybrid analysis showed no evidence of a direct interaction between kinesin-1 and AC141 or VP39, suggesting that either other nucleocapsid proteins or adaptor proteins may be required. These results further support the conclusion that microtubule transport is required for AcMNPV BV formation. IMPORTANCEIn two key processes of the replication cycle of the baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV), nucleocapsids are transported through the cell. These include (i) entry of budded virus (BV) into the host cell and (ii) egress and budding of nucleocapsids newly produced from the plasma membrane. Prior studies have shown that the entry of nucleocapsids involves the polymerization of actin to propel nucleocapsids to nuclear pores and entry into the nucleus. For the spread of infection, progeny viruses must rapidly exit the infected cells, but the mechanism by which AcMNPV nucleocapsids traverse the cytoplasm is unknown. In this study, we examined whether nucleocapsids interact with lepidopteran kinesin-1 motor molecules and are potentially carried as cargo on microtubules to the plasma membrane in AcMNPV-infected cells. This study indicates that microtubule transport is utilized for the production of budded virus.T he baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is an enveloped virus containing a large double-stranded circular DNA genome of approximately 134 kbp that encodes approximately 156 proteins. During the course of AcMNPV infection, nucleocapsi...

“…Part of the nucleocapsids bud out of the nucleus and traffic through the cytoplasm to the periphery. The major capsid proteins VP39 and EXON0 have been shown to interact with kinesin, a motor protein that is involved in anterograde transport in eukaryotic cells (11). The nucleocapsids finally bud through the cytoplasmic membrane that has been premodified by viral proteins to form BVs.…”

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confidence: 99%

Autographa californica Multiple Nucleopolyhedrovirus orf132 Encodes a Nucleocapsid-Associated Protein Required for Budded-Virus and Multiply Enveloped Occlusion-Derived Virus Production

Yang

Wang

Yue

et al. 2014

Autographa californica multiple nucleopolyhedrovirus orf132 (named ac132) has homologs in all genome-sequenced group I nucleopolyhedroviruses. Its role in the viral replication cycle is unknown. In this study, ac132 was shown to express a protein of around 28 kDa, which was determined to be associated with the nucleocapsids of both occlusion-derived virus and budded virus. Confocal microscopy showed that AC132 protein appeared in central region of the nucleus as early as 12 h postinfection with the virus. It formed a ring zone at the periphery of the nucleus by 24 h postinfection. To investigate its role in virus replication, ac132 was deleted from the viral genome by using a bacmid system. In the Sf9 cell culture transfected by the ac132 knockout bacmid, infection was restricted to single cells, and the titer of infectious budded virus was reduced to an undetectable level. However, viral DNA replication and the expression of late genes vp39 and odv-e25 and a reporter gene under the control of the very late gene p10 promoter were unaffected. Electron microscopy showed that nucleocapsids, virions, and occlusion bodies were synthesized in the cells transfected by an ac132 knockout bacmid, but the formation of the virogenic stroma and occlusion bodies was delayed, the numbers of enveloped nucleocapsids were reduced, and the occlusion bodies contained mainly singly enveloped nucleocapsids. AC132 was found to interact with envelope protein ODV-E18 and the viral DNA-binding protein P6.9. The data from this study suggest that ac132 possibly plays an important role in the assembly and envelopment of nucleocapsids. IMPORTANCETo our knowledge, this is the first report on a functional analysis of ac132. The data presented here demonstrate that ac132 is required for production of the budded virus and multiply enveloped occlusion-derived virus of Autographa californica multiple nucleopolyhedrovirus. This article reveals unique phenotypic changes induced by ac132 deletion on the virus and multiple new findings on ac132.