Search

As an important neurotropic enterovirus, enterovirus 71 (EV71) is occasionally associated with severe neurological diseases and high mortality rates in infants and young children. Understanding the interaction between host factors and EV71 will play a vital role in developing antivirals and optimizing vaccines. Here, we performed a genome-wide CRISPR-Cas9 knockout screen and revealed that scavenger receptor class B member 2 (SCARB2), solute carrier family 35 member B2 (SLC35B2), and beta-1,3-glucuronyltransferase 3 (B3GAT3) are essential in facilitating EV71 replication. Subsequently, the exploration of molecular mechanisms suggested that the knockout of SLC35B2 or B3GAT3, not SCARB2, led to a remarkable decrease in the binding of EV71 to cells and internalization into cells. Furthermore, we found that the infection efficiency for EV71 was positively correlated with the level of host cell sulfation, not simply with the amount of heparan sulfate, suggesting that an unidentified sulfated protein(s) must contribute to EV71 infection. In support of this idea, we screened possible sulfated proteins among the proteinous receptors for EV71 and confirmed that SCARB2 could uniquely interact with both tyrosyl protein sulfotransferases in humans. We then performed mass spectrometric analysis of SCARB2, identifying five sites with tyrosine sulfation. The function verification test indicated that there were more than five tyrosine-sulfated sites on SCARB2. Finally, we constructed a model for EV71 entry in which both heparan sulfate and SCARB2 are regulated by SLC35B2 and act cooperatively to support viral binding, internalization, and uncoating. Taken together, this is the first time that we performed the pooled CRISPR-Cas9 genetic screening to investigate the interplay of host cells and EV71. Furthermore, we found that a novel host factor, SLC35B2, played a dual role in regulating the overall sulfation comprising heparan sulfate sulfation and protein tyrosine sulfation, which are critical for EV71 entry. IMPORTANCE As the most important nonpolio neurotropic enterovirus lacking specific treatments, EV71 can transmit to the central nervous system, leading to severe and fatal neurological complications in infants and young children. The identification of new factors that facilitate or inhibit EV71 replication is crucial to uncover the mechanisms of viral infection and pathogenesis. To date, only a few host factors involved in EV71 infection have been characterized. Herein, we conducted a genome-wide CRISPR-Cas9 functional knockout (GeCKO) screen for the first time to study EV71 in HeLa cells. The screening results are presented as a ranked list of candidates, including 518 hits in the positive selection that facilitate EV71 replication and 1,044 hits in the negative selection that may be essential for cell growth and survival or for suppressing EV71 infection. We subsequently concentrated on the top three hits in the positive selection: SCARB2, SLC35B2, and B3GAT3. The knockout of any of these three genes confers strong resistance against EV71 infection. We confirmed that EV71 infection is codependent on two receptors, heparan sulfate and SCARB2. We also identified a host entry factor, SLC35B2, indirectly facilitating EV71 infection through regulation of the host cell sulfation, and determined a novel posttranslational modification, protein tyrosine sulfation existing in SCARB2. This study revealed that EV71 infectivity exhibits a significant positive correlation with the level of cellular sulfation regulated by SLC35B2. Due to the sulfation pathway being required for many distinct viruses, including but not limited to EV71 and respiratory syncytial virus (RSV), which were tested in this study, SLC35B2 represents a target of broad-spectrum antiviral therapy.

Human parainfluenza virus type 3 (HPIV3) belongs to the Paramyxoviridae family. Its three internal viral proteins, the nucleoprotein (N), the phosphoprotein (P), and the polymerase (L), form the ribonucleoprotein (RNP) complex, which encapsidates the viral genome and associates with the matrix protein (M) for virion assembly. We previously showed that the M protein expressed alone is sufficient to assemble and release virus-like particles (VLPs) and a mutant with the L305A point mutation in the M protein (M L305A ) has a VLP formation ability similar to that of wild-type M protein. In addition, recombinant HPIV3 (rHPIV3) containing the M L305A mutation (rHPIV3-M L305A ) could be successfully recovered. In the present study, we found that the titer of rHPIV3-M L305A was at least 10-fold lower than the titer of rHPIV3. Using VLP incorporation and coimmunoprecipitation assays, we found that VLPs expressing the M protein (M-VLPs) can efficiently incorporate N and P via an N-M or P-M interaction and M L305A -VLPs had an ability to incorporate P via a P-M interaction similar to that of M-VLPs but were unable to incorporate N and no longer interacted with N. Furthermore, we found that the incorporation of P into M L305A -VLPs but not M-VLPs was inhibited in the presence of N. In addition, we provide evidence that the C-terminal region of P is involved in its interaction with both N and M and N binding to the C-terminal region of P inhibits the incorporation of P into M L305A -VLPs. Our findings provide new molecular details to support the idea that the N-M interaction and not the P-M interaction is critical for packaging N and P into infectious viral particles. IMPORTANCE Human parainfluenza virus type 3 (HPIV3) is a nonsegmented, negative-sense, single-stranded RNA virus that belongs to the Paramyxoviridae family and can cause lower respiratory tract infections in infants and young children as well as elderly or immunocompromised individuals. However, no effective vaccine has been developed or licensed. We used virus-like particle (VLP) incorporation and coimmunoprecipitation assays to determine how the M protein assembles internal viral proteins. We demonstrate that both nucleoprotein (N) and phosphoprotein (P) can incorporate into M-VLPs and N inhibits the M-P interaction via the binding of N to the C terminus of P. We also provide additional evidence that the N-M interaction but not the P-M interaction is critical for the regulation of HPIV3 assembly. Our studies provide a more complete characterization of HPIV3 virion assembly and substantiation that N interaction with M regulates internal viral organization.

Paramyxovirus particles, like other enveloped virus particles, are formed by budding from membranes of infected cells, and matrix (M) proteins are critical for this process. To identify the M protein important for this process, we have characterized the budding of the human parainfluenza virus type 3 (HPIV3) M protein. Our results showed that expression of the HPIV3 M protein alone is sufficient to initiate the release of virus-like particles (VLPs). Electron microscopy analysis confirmed that VLPs are morphologically similar to HPIV3 virions. We identified a leucine (L302) residue within the C terminus of the HPIV3 M protein that is critical for M protein-mediated VLP production by regulating the ubiquitination of the M protein. When L302 was mutated into A302, ubiquitination of M protein was defective, the release of VLPs was abolished, and the membrane binding and budding abilities of M protein were greatly weakened, but the M L302A mutant retained oligomerization activity and had a dominant negative effect on M protein-mediated VLP production. Furthermore, treatment with a proteasome inhibitor also inhibited M protein-mediated VLP production and viral budding. Finally, recombinant HPIV3 containing the M L302A mutant could not be rescued. These results suggest that L302 acts as a critical regulating signal for the ubiquitination of the HPIV3 M protein and virion release. IMPORTANCE Human parainfluenza virus type 3 (HPIV3) is an enveloped virus with a nonsegmented negative-strand RNA genome. It can cause severe respiratory tract diseases, such as bronchiolitis, pneumonia, and croup in infants and young children. However, no valid antiviral therapy or vaccine is currently available. Thus, further elucidation of its assembly and budding will be helpful in the development of novel therapeutic approaches. Here, we show that a leucine residue (L302) located at the C terminus of the HPIV3 M protein is essential for efficient production of virus-like particles (VLPs). Furthermore, we found L302 regulated M protein-mediated VLP production via regulation of M protein ubiquitination. Recombinant HPIV3 containing the M L302A mutant is growth defective. These findings provide new insight into the critical role of M protein-mediated VLP production and virion release of a residue that does not belong to L domain and may advance our understanding of HPIV3 viral assembly and budding.

The nucleoprotein (N) and phosphoprotein (P) interaction of nonsegmented negative-strand RNA viruses is essential for viral replication; this includes N 0 -P (N 0 , free of RNA) interaction and the interaction of N-RNA with P. The precise site(s) within N that mediates the N-P interaction and the detailed regulating mechanism, however, are less clear. Using a human parainfluenza virus type 3 (HPIV3) minigenome assay, we found that an N mutant (N L478A ) did not support reporter gene expression. Using in vivo and in vitro coimmunoprecipitation, we found that N L478A maintains the ability to form N L478A 0 -P, to self-assemble, and to form N L478A -RNA but that N L478A -RNA does not interact with P. Using an immunofluorescence assay, we found that N-P interaction provides the minimal requirement for the formation of cytoplasmic inclusion bodies, which contain viral RNA, N, P, and polymerase in HPIV3-infected cells. N L478A was unable to form inclusion bodies when coexpressed with P, but the presence of N rescued the ability of N L478A to form inclusion bodies and the transcriptional function of N L478A , thereby suggesting that hetero-oligomers formed by N and N L478A are functional and competent to form inclusion bodies. Furthermore, we found that N L478A is also defective in virus growth. To our knowledge, we are the first to use a paramyxovirus to identify a precise amino acid within N that is critical for N-RNA and P interaction but not for N 0 -P interaction for the formation of inclusion bodies, which appear to be bona fide sites of RNA synthesis.

Viral inclusion bodies (IBs), or replication factories, are unique structures generated by viral proteins together with some cellular proteins as a platform for efficient viral replication, but little is known about the mechanism underlying IB formation and fusion. Our previous study demonstrated that the interaction between the nucleoprotein (N) and phosphoprotein (P) of human parainfluenza virus type 3 (HPIV3), an enveloped virus with great medical impact, can form IBs. In this study, we found that small IBs can fuse with each other to form large IBs that enhance viral replication. Furthermore, we found that acetylated α-tubulin interacts with the N-P complex and colocalizes with IBs of HPIV3 but does not interact with the N-P complex of human respiratory syncytial virus or vesicular stomatitis virus and does not colocalize with IBs of human respiratory syncytial virus. Most importantly, enhancement of α-tubulin acetylation using the pharmacological inhibitor trichostatin A (TSA), RNA interference (RNAi) knockdown of the deacetylase enzymes histone deacetylase 6 (HDAC6) and sirtuin 2 (SIRT2), or expression of α-tubulin acetyltransferase 1 (α-TAT1) resulted in the fusion of small IBs into large IBs and effective viral replication. In contrast, suppression of acetylation of α-tubulin by overexpressing HDAC6 and SIRT2 profoundly inhibited the fusion of small IBs and viral replication. Our findings offer previously unidentified mechanistic insights into the regulation of viral IB fusion by acetylated α-tubulin, which is critical for viral replication. IMPORTANCE Inclusion bodies (IBs) are unique structures generated by viral proteins and some cellular proteins as a platform for efficient viral replication. Human parainfluenza virus type 3 (HPIV3) is a nonsegmented single-stranded RNA virus that mainly causes lower respiratory tract disease in infants and young children. However, no vaccines or antiviral drugs for HPIV3 are available. Therefore, understanding virus-host interactions and developing new antiviral strategies are increasingly important. Acetylation on lysine (K) 40 of α-tubulin is an evolutionarily conserved modification and plays an important role in many cellular processes, but its role in viral IB dynamics has not been fully explored. To our knowledge, our findings are the first to show that acetylated α-tubulin enhances viral replication by regulating HPIV3 IB fusion.

Hepatitis C virus (HCV) is a major cause of chronic liver diseases worldwide, often leading to the development of hepatocellular carcinoma (HCC). Constitutive activation of the Ras/Raf/MEK pathway is responsible for approximately 30% of cancers. Here we attempted to address the correlation between activation of this pathway and HCV replication. We showed that knockdown of Raf1 inhibits HCV replication, while activation of the Ras/Raf/MEK pathway by V12, a constitutively active form of Ras, stimulates HCV replication. We further demonstrated that this effect is regulated through attenuation of the interferon (IFN)-JAK-STAT pathway. Activation of the Ras/Raf/MEK pathway downregulates the expression of IFN-stimulated genes (ISGs), attenuates the phosphorylation of STAT1/2, and inhibits the expression of interferon (alpha, beta, and omega) receptors 1 and 2 (IFNAR1/2). Furthermore, we observed that HCV infection activates the Ras/Raf/MEK pathway. Thus, we propose that during HCV infection, the Ras/Raf/MEK pathway is activated, which in turn attenuates the IFN-JAK-STAT pathway, resulting in stimulation of HCV replication.

The nucleocapsid (N) protein of nonsegmented negative-strand (NNS) RNA viruses, when expressed in eukaryotic cells, aggregates and forms nucleocapsid-like complexes with cellular RNAs. The phosphoprotein (P) has been shown to prevent such aggregation by forming a soluble complex with the N protein free from cellular RNAs (designated N 0 ). The N 0 -P complex presumably mediates specific encapsidation of the viral genome RNA. The precise mechanism by which the P protein carries out this function remains unclear. Here, by using a series of deleted and truncated mutant forms of the P protein of vesicular stomatitis virus (VSV), Indiana serotype, we present evidence that the N-terminal 11 to 30 amino acids (aa) of the P protein are essential in keeping the N protein soluble. Furthermore, glutathione S-transferase fused to the N-terminal 40 aa by itself is able to form the N 0 -P complex. Interestingly, the N-terminal 40-aa stretch failed to interact with the viral genome N-RNA template whereas the C-terminal 72 aa of the P protein interacted specifically with the latter. With an in vivo VSV minigenome transcription system, we further show that a deletion mutant form of P( P1-10) lacking the N-terminal 10 aa which is capable of forming the N 0 -P complex was unable to support VSV minigenome transcription, although it efficiently supported transcription in vitro in a transcriptionreconstitution reaction when used as purified protein. However, the same mutant protein complemented minigenome transcription when expressed together with a transcription-defective P deletion mutant protein containing N-terminal aa 1 to 210 (PIIIII). Since the minigenome RNA needs to be encapsidated before transcription ensues, it seems that the entire N-terminal 210 aa are required for efficient genome RNA encapsidation. Taking these results together, we conclude that the N-terminal 11 to 30 aa are required for N 0 -P complex formation but the N-terminal 210 aa are required for genome RNA encapsidation. Vesicular stomatitis virus (VSV) is a negative-strand RNA virus belonging to the family Rhabdoviridae. Its genome RNA is enwrapped in a helical form with the nucleocapsid (N) protein and packaged within the virion together with the RNAdependent RNA polymerase large protein (L) and the phosphoprotein (P). During cell infection, the associated RNA polymerase transcribes the genome RNA into five mRNAs that are subsequently translated into the glycoprotein (G), the matrix (M) protein, and the N, P, and L proteins. The N protein is an RNA-binding protein since it forms a high-mo

The phosphoprotein (P protein) of vesicular stomatitis virus (VSV) is an essential subunit of the viral RNA-dependent RNA polymerase complex and plays a central role in viral transcription and replication. Using both the yeast two-hybrid system and coimmunoprecipitation assays, we confirmed the self-association of the P protein of Indiana serotype (Pind) and heterotypic interaction between Pind and the P protein of New Jersey serotype (Pnj). Furthermore, by using various truncation and deletion mutants of Pind, the self-association domain of the Pind protein was mapped to amino acids 161 to 210 within the hinge region. The self-association domain of Pind protein is not required for its binding to nucleocapsid and large proteins. We further demonstrated that the self-association domain of Pind protein is essential for VSV transcription in a minireplicon system and that a synthetic peptide spanning amino acids 191 to 210 in the self-association domain of Pind protein strongly inhibited the transcription of the VSV genome in vitro in a dose-dependent manner. These results indicated that the self-association domain of Pind protein plays a critical role in VSV transcription.

Using a C-terminal domain (PCT) of the measles virus (MV) phosphoprotein (P protein) as bait in a yeast two-hybrid screen, a cDNA identical to the recently described human p53-induced-RING-H2 (hPIRH2) cDNA was isolated. A glutathione S -transferase-hPIRH2 fusion protein expressed in bacteria was able to pull down P protein when mixed with an extract from P-expressing HeLa cells in vitro, and myc-tagged hPIRH2 could be reciprocally coimmunoprecipitated with MV P protein from human cells. Additionally, immunoprecipitation experiments demonstrated that hPIRH2-myc, MV P, and nucleocapsid (N) proteins form a ternary complex. The hPIRH2 binding site was mapped to the C-terminal X domain region of the P protein by using a yeast two-hybrid assay. The PCT binding site was mapped on hPIRH2 by using a novel yeast two-hybrid tagged PCR approach and by coimmunoprecipitation of hPIRH2 cysteine mutants and mouse/human PIRH2 chimeras. The hPIRH2 C terminus could mediate the interaction with MV P which was favored by the RING-H2 motif. When coexpressed with an enhanced green fluorescent protein-tagged hPIRH2 protein, MV P alone or in a complex with MV N was able to redistribute hPIRH2 to outside the nucleus, within intracellular aggregates. Finally, MV P efficiently stabilized hPIRH2-myc expression and prevented its ubiquitination in vivo but had no effect on the stability or ubiquitination of an alternative ubiquitin E3 ligase, Mdm2. Thus, MV P protein is the first protein from a pathogen that is able to specifically interact with and stabilize the ubiquitin E3 ligase hPIRH2 by preventing its ubiquitination.