Heat Shock Protein 70 Is Related to Thermal Inhibition of Nuclear Export of the Influenza Virus Ribonucleoprotein Complex

The RNA polymerase of influenza A virus is a host range determinant and virulence factor. In particular, the PB2 subunit of the RNA polymerase has been implicated as a crucial factor that affects cell tropism as well as virulence in animal models. These findings suggest that host factors associating with the PB2 protein may play an important role during viral replication. In order to identify host factors that associate with the PB2 protein, we purified recombinant PB2 from transiently transfected mammalian cells and identified copurifying host proteins by mass spectrometry. We found that the PB2 protein associates with the cytosolic chaperonin containing TCP-1 (CCT), stress-induced phosphoprotein 1 (STIP1), FK506 binding protein 5 (FKBP5), ␣-and ␤-tubulin, Hsp60, and mitochondrial protein p32. Some of these binding partners associate with each other, suggesting that PB2 might interact with these proteins in multimeric complexes. More detailed analysis of the interaction of the PB2 protein with CCT revealed that PB2 associates with CCT as a monomer and that the CCT binding site is located in a central region of the PB2 protein. PB2 proteins from various influenza virus subtypes and origins can associate with CCT. Silencing of CCT resulted in reduced viral replication and reduced PB2 protein and viral RNA accumulation in a ribonucleoprotein reconstitution assay, suggesting an important function for CCT during the influenza virus life cycle. We propose that CCT might be acting as a chaperone for PB2 to aid its folding and possibly its incorporation into the trimeric RNA polymerase complex.Influenza A viruses, members of the family of Orthomyxoviridae, contain a segmented RNA genome of negative polarity. The genomic RNA segments together with the three subunits of the viral RNA-dependent RNA polymerase (PB1, PB2, and PA protein) and the nucleoprotein (NP) form viral ribonucleoprotein complexes (vRNPs). The PB1 subunit is the polymerase itself, while the PB2 and PA subunits are involved in the generation of 5Ј capped RNA primers through binding to and endonucleolytic cleavage of host pre-mRNAs (8,10,11,41,61). After the virus enters the cell via endocytosis, vRNPs are released into the cytoplasm and transported into the nucleus. In the nucleus, vRNPs catalyze the synthesis of viral mRNAs and complementary RNAs (cRNA) which, in turn, are used as templates for the synthesis of vRNAs. The newly formed vRNPs in association with other viral proteins (M1 and nonstructural protein 2/nuclear export factor [NS2/NEP]) are transported into the cytoplasm and subsequently to the cell membrane, where the assembly process takes place, followed by the release of progeny virions by budding (44).The PB1, PB2, and PA proteins are synthesized in the cytoplasm whereupon PB1 and PA form a dimeric complex that is transported into the nucleus. In the nucleus the dimer assembles with the PB2 subunit, which is transported separately (7,14). RanBP5 was identified as a factor that is involved in the import of the PB1-PA dimer into the nucleus ...

Section: Discussionsupporting

confidence: 61%

mentioning

confidence: 99%

Association of the Influenza Virus RNA Polymerase Subunit PB2 with the Host Chaperonin CCT

Fislová

Thomas

Graef

et al. 2010

“…An increase of its expression confers to cells a protection against rotavirus (2), vesicular stomatitis virus (9), respiratory syncytial virus (41), and influenza virus (16) infections. In the latter case, it has been reported that Hsp70, which interacts with the ribonucleoprotein complex, interferes with the polymerase activity and negatively regulates viral transcription and replication (16,22). Interestingly, Hsp70 was found to have both positive and negative regulatory effects on the RNA replication of flock house virus, a nodavirus (39), highlighting the complexity of this particular virus-chaperone interaction.…”

mentioning

confidence: 99%

Hsp70 Protein Positively Regulates Rabies Virus Infection

Lahaye

Vidy

Fouquet

et al. 2012

126

105

The Hsp70 chaperone plays a central role in multiple processes within cells, including protein translation, folding, intracellular trafficking, and degradation. This protein is implicated in the replication of numerous viruses. We have shown that rabies virus infection induced the cellular expression of Hsp70, which accumulated in Negri body-like structures, where viral transcription and replication take place. In addition, Hsp70 is present in both nucleocapsids purified from infected cells and in purified virions. Hsp70 has been shown to interact with the nucleoprotein N. The downregulation of Hsp70, using specific chaperone inhibitors, such as quercetin or RNA interference, resulted in a significant decrease of the amount of viral mRNAs, viral proteins, and virus particles. These results indicate that Hsp70 has a proviral function during rabies virus infection and suggest that Hsp70 is involved in at least one stage(s) of the viral life cycle, such as viral transcription, translation, and/or production. The mechanism by which Hsp70 controls viral infection will be discussed.

“…Various mechanisms are involved. For instance, influenza virus cannot replicate above 41°C because Hsp70 inhibits nuclear export of the ribonucleoprotein complex (16). Hsp70 also blocks Sendai virus infection at 41°C, by disrupting HN protein assembly (17).…”

Section: Discussionmentioning

confidence: 99%

The Role of Aldehyde Dehydrogenase and Hsp70 in Suppression of White Spot Syndrome Virus Replication at High Temperature

Lin

Hung

Leu

et al. 2011

High temperature (32 to 33°C) has been shown to reduce mortality in white spot syndrome virus (WSSV)-infected shrimps, but the mechanism still remains unclear. Here we show that in WSSV-infected shrimps cultured at 32°C, transcriptional levels of representative immediate-early, early, and late genes were initially higher than those at 25°C. However, neither the IE1 nor VP28 protein was detected at 32°C, suggesting that high temperature might inhibit WSSV protein synthesis. Two-dimensional gel electrophoresis analysis revealed two proteins, NAD-dependent aldehyde dehydrogenase (ALDH) and the proteasome alpha 4 subunit (proteasome ␣4), that were markedly upregulated in WSSV-infected shrimps at 32°C. Reverse transcription-PCR (RT-PCR) analysis of members of the heat shock protein family also showed that hsp70 was upregulated at 32°C. When aldh, proteasome ␣4, and hsp70 were knocked down by double-stranded RNA interference and shrimps were challenged with WSSV, the aldh and hsp70 knockdown shrimps became severely infected at 32°C, while the proteasome ␣4 knockdown shrimps remained uninfected. Our results therefore suggest that ALDH and Hsp70 both play an important role in the inhibition of WSSV replication at high temperature.White spot syndrome virus (WSSV) is a notorious pathogen in shrimp aquaculture. It has caused huge economic losses since it was first reported in 1992 in East Asia (6), and the virus is presently endemic in many parts of the world. WSSV is an ovoid to rod-shaped, enveloped, double-stranded DNA virus which belongs to the genus "Whispovirus" of the family Nimaviridae (35). The destructiveness of WSSV is partly due to its wide host range, which includes shrimp, crayfish, crabs, lobsters, and copepods (2, 9). In shrimps, mortality can reach 100% in 3 to 10 days after WSSV infection (20).Shrimps are exothermic animals, and unlike the case for endotherms, the body temperature of exotherms is directly susceptible to change depending on environmental conditions. Shrimp culture records show that outbreaks of WSSV are less likely to occur during warm seasons (29, 39), and there is other evidence suggesting that high temperature might protect shrimps from WSSV. Vidal et al. (34) reported that temperatures of 32 to 33°C can significantly reduce mortality in WSSVinfected shrimps. Other research has suggested that the water temperature needs to be maintained at 33°C for only 12 or 18 h per day in order to delay or reduce mortality in WSSV-infected shrimps (26). However, even though the use of warm water for shrimp cultivation has generally been accepted as beneficial by the shrimp culture industry and is already applied in some commercial shrimp farms, the exact mechanisms that underlie this phenomenon are still unknown. Granja et al. (12) proposed that high temperature might increase cell apoptosis and that this would help to prevent the replication of WSSV. However, apoptosis is no longer thought to be a principal protective factor against WSSV in shrimps (10, 40). Others have suggested that high tempera...