Cucumber Necrosis Virus Recruits Cellular Heat Shock Protein 70 Homologs at Several Stages of Infection

Abstract

RNA viruses often depend on host factors for multiplication inside cells due to the constraints of their small genome size and limited coding capacity. One such factor that has been exploited by several plant and animal viruses is heat shock protein 70 (HSP70) family homologs which have been shown to play roles for different viruses in viral RNA replication, viral assembly, disassembly, and cell-to-cell movement. Using next generation sequence analysis, we reveal that several isoforms of Hsp70 and Hsc70 transcripts are induced to very high levels during cucumber necrosis virus (CNV) infection of Nicotiana benthamiana and that HSP70 proteins are also induced by at least 10-fold. We show that HSP70 family protein homologs are co-opted by CNV at several stages of infection. We have found that overexpression of Hsp70 or Hsc70 leads to enhanced CNV genomic RNA, coat protein (CP), and virion accumulation, whereas downregulation leads to a corresponding decrease. Hsc70-2 was found to increase solubility of CNV CP in vitro and to increase accumulation of CNV CP independently of viral RNA replication during coagroinfiltration in N. benthamiana. In addition, virus particle assembly into virus-like particles in CP agroinfiltrated plants was increased in the presence of Hsc70-2. HSP70 was found to increase the targeting of CNV CP to chloroplasts during infection, reinforcing the role of HSP70 in chloroplast targeting of host proteins. Hence, our findings have led to the discovery of a highly induced host factor that has been co-opted to play multiple roles during several stages of the CNV infection cycle. IMPORTANCEBecause of the small size of its RNA genome, CNV is dependent on interaction with host cellular components to successfully complete its multiplication cycle. We have found that CNV induces HSP70 family homologs to a high level during infection, possibly as a result of the host response to the high levels of CNV proteins that accumulate during infection. Moreover, we have found that CNV co-opts HSP70 family homologs to facilitate several aspects of the infection process such as viral RNA, coat protein and virus accumulation. Chloroplast targeting of the CNV CP is also facilitated, which may aid in CNV suppression of host defense responses. Several viruses have been shown to induce HSP70 during infection and others to utilize HSP70 for specific aspects of infection such as replication, assembly, and disassembly. We speculate that HSP70 may play multiple roles in the infection processes of many viruses. C ucumber necrosis virus (CNV) is a positive-strand RNA virus in the genus Tombusvirus, family Tombusviridae (1). The CNV genome is monopartite and consists of ϳ4.7 kb of positive polarity single-stranded RNA. The genome contains five open reading frames (ORFs) which encode five different proteins: the auxiliary replicase factor (p33), the RNA-dependent RNA polymerase (RdRp) (p92), the coat protein (CP; p41), the movement protein (p21), and the silencing suppressor (p20) (Fig. 1A). The UAG stop codon of the p...

Evidence that Hsc70 Is Associated with Cucumber Necrosis Virus Particles and Plays a Role in Particle Disassembly

“…We wished to determine whether Nicotiana benthamiana HSP70 family homologs play a role in the disassembly of the plant virus Cucumber necrosis virus (CNV). This would extend our previous observation that HSP70 homologs play multiple roles in the CNV multiplication cycle (27).…”

“…We have previously found that HSP70 homologs play multiple roles in the CNV infection cycle and that Hsc70-2 coimmunoprecipitates with CNV CP (27). This prompted us to determine whether Hsc70-2 is present in CNV particle preparations.…”

“…It is possible that the Hsp70 associated with the vector zoospores is responsible for bringing about the conformational change required for cellular entry of CNV particles, as has been reported for rotavirus entry into cells (21). This, and our recent finding that HSP70 homologs play multiple roles in the CNV infection cycle (27), raised the possibility of the involvement of plant Hsp70 or Hsc70 homologs in the disassembly of CNV. Here we report several observations that suggest that CNV particles co-opt Hsc70-2 and/or Hsp70 for uncoating.…”

An Improved Brome mosaic virus Silencing Vector: Greater Insert Stability and More Extensive VIGS

“…Aranda et al (1996) also observed during infection with Pea seed-borne mosaic virus that one HSP70 family member, PsHCP71.2, was induced while another, PsHSC71.0, was marginally reduced in transcript level in the tissue where virus was replicating. In N. benthamiana, challenge with five RNA viruses showed significant increases in the expression of multiple HSP70 genes, many encoding proteins that resided in the cytoplasm (Chen et al, 2008;Alam and Rochon, 2015). HSP70 family members also were induced by multiple viruses in Arabidopsis (Whitham et al, 2003).…”

“…Regarding HSP70 expression and its effect on virus accumulation or plant phenotype, when one of the HSP70 family members, NbHSP70c-1, was overexpressed, the accumulation of Tobacco mosaic virus was increased (Chen et al, 2008). In Chenopodium quinoa plants heat shocked to induce HSP70 expression and then challenged with CNV, viral genomic RNA accumulation increased (Alam and Rochon, 2015). Silencing the expression of cytosolic Nicotiana HSP70 family members in N. benthamiana using the TRV-or PVXbased VIGS vectors resulted in dwarfed and crinkled leaves or white spots near major veins in developing leaves (Chen et al, 2008;Wang et al, 2009).…”