Zika virus (ZIKV), a member of the Flaviviridae family, has recently emerged as an important human pathogen with increasing economic and health impact worldwide. Because of its teratogenic nature and association with the serious neurological condition Guillain-Barré syndrome, a tremendous amount of effort has focused on understanding ZIKV pathogenesis. To gain further insights into ZIKV interaction with host cells, we investigated how this pathogen affects stress response pathways. While ZIKV infection induces stress signaling that leads to phosphorylation of eIF2␣ and cellular translational arrest, stress granule (SG) formation was inhibited. Further analysis revealed that the viral proteins NS3 and NS4A are linked to translational repression, whereas expression of the capsid protein, NS3/NS2B-3, and NS4A interfered with SG formation. Some, but not all, flavivirus capsid proteins also blocked SG assembly, indicating differential interactions between flaviviruses and SG biogenesis pathways. Depletion of the SG components G3BP1, TIAR, and Caprin-1, but not TIA-1, reduced ZIKV replication. Both G3BP1 and Caprin-1 formed complexes with capsid, whereas viral genomic RNA stably interacted with G3BP1 during ZIKV infection. Taken together, these results are consistent with a scenario in which ZIKV uses multiple viral components to hijack key SG proteins to benefit viral replication.IMPORTANCE There is a pressing need to understand ZIKV pathogenesis in order to advance the development of vaccines and therapeutics. The cellular stress response constitutes one of the first lines of defense against viral infection; therefore, understanding how ZIKV evades this antiviral system will provide key insights into ZIKV biology and potentially pathogenesis. Here, we show that ZIKV induces the stress response through activation of the UPR (unfolded protein response) and PKR (protein kinase R), leading to host translational arrest, a process likely mediated by the viral proteins NS3 and NS4A. Despite the activation of translational shutoff, formation of SG is strongly inhibited by the virus. Specifically, ZIKV hijacks the core SG proteins G3BP1, TIAR, and Caprin-1 to facilitate viral replication, resulting in impaired SG assembly. This process is potentially facilitated by the interactions of the viral RNA with G3BP1 as well as the viral capsid protein with G3BP1 and Caprin-1. Interestingly, expression of capsid proteins from several other flaviviruses also inhibited SG formation. Taken together, the present study provides novel insights into how ZIKV modulates cellular stress response pathways during replication.

Similar to all other viruses, human immunodeficiency virus type 1 (HIV-1) depends heavily on cellular factors for its successful replication. In this study we have investigated the interaction of HIV-1 integrase (IN) with several host nuclear import factors using co-immunoprecipitation assays. Our results indicate that IN interacts specifically with host importin 7 (Imp7) in vivo, but does not interact with importin 8 (Imp8) or importin ␣ (Rch1). To carry out a successful infection, human immunodeficiency virus type 1 (HIV-1) 4 takes advantage of various host cellular proteins and cellular pathways. The interaction between cellular proteins and viral components takes place during various steps of the HIV-1 life cycle, including viral DNA nuclear import. The most striking feature of HIV-1 is its ability to replicate in non-dividing cells. This feature depends on the ability of the virus to transport its cDNA, as part of a large preintegration complex (PIC), from the cytoplasm to the nucleus by an active and energy-dependent process (1-3). However, the mechanism by which the PIC translocates across the nuclear membrane into the nucleus of non-dividing cells is still not fully understood. It has been shown that three HIV-1 PIC-associated proteins including MAp17, IN, and Vpr possess karyophilic properties, and contribute to nuclear translocation of viral PICs. This action is accomplished through their interactions with karyophilic cellular proteins, thereby directing the PIC through the nuclear pore complex (4 -10). In addition, a cis-acting element named the central DNA flap, which is located in the 3Ј region of the pol gene sequence, was also shown to contribute to HIV-1 nuclear import in both dividing and non-dividing cells (11)(12)(13)(14). Nuclear import of proteins in mammalian cells can be mediated by several distinct pathways. The importin ␣/␤ heterodimer meditates nuclear import of proteins harboring a classical nuclear localization signal, which either contains a cluster of basic amino acids or two basic clusters separated by 10 -20 amino acids (bipartite nuclear localization signal) (for reviews see Refs. 15 and 16). Also, importin ␤ (Imp␤) was shown to bind to and import HIV-1 proteins, such as HIV-1 Tat, Rev, and HTLV Rex, independently of importin ␣ (Imp␣) (17-21). Similarly, transportin, an Imp␤-related receptor, imports its substrates (heterogeneous nuclear ribonucleoproteins) by directly binding to the glycine-rich M9 domain of the protein (22,23). Based on the similarity to Imp␤, several other nuclear import factors, including Imp7 and Imp8, have also been identified (24). Imp7 is one of several cellular importins that bind to and mediate nuclear import of ribosomal proteins in mammalian cells, and it was also found to translocate other proteins, such as glucocorticoid receptor and histone H1 into the nucleus (18,(25)(26)(27). In the case of histone H1, Jakel et al. (27) have demonstrated that two receptors, Imp␤ and Imp7,

Flaviviruses are significant human pathogens that have an enormous impact on the global health burden. Currently, there are very few vaccines against or therapeutic treatments for flaviviruses, and our understanding of how these viruses cause disease is limited. Evidence suggests that the capsid proteins of flaviviruses play critical nonstructural roles during infection, and therefore, elucidating how these viral proteins affect cellular signaling pathways could lead to novel targets for antiviral therapy. We used affinity purification to identify host cell proteins that interact with the capsid proteins of West Nile and dengue viruses. One of the cellular proteins that formed a stable complex with flavivirus capsid proteins is the peroxisome biogenesis factor Pex19. Intriguingly, flavivirus infection resulted in a significant loss of peroxisomes, an effect that may be due in part to capsid expression. We posited that capsid protein-mediated sequestration and/or degradation of Pex19 results in loss of peroxisomes, a situation that could result in reduced early antiviral signaling. In support of this hypothesis, we observed that induction of the lambda interferon mRNA in response to a viral RNA mimic was reduced by more than 80%. Together, our findings indicate that inhibition of peroxisome biogenesis may be a novel mechanism by which flaviviruses evade the innate immune system during early stages of infection. IMPORTANCERNA viruses infect hundreds of millions of people each year, causing significant morbidity and mortality. Chief among these pathogens are the flaviviruses, which include dengue virus and West Nile virus. Despite their medical importance, there are very few prophylactic or therapeutic treatments for these viruses. Moreover, the manner in which they subvert the innate immune response in order to establish infection in mammalian cells is not well understood. Recently, peroxisomes were reported to function in early antiviral signaling, but very little is known regarding if or how pathogenic viruses affect these organelles. We report for the first time that flavivirus infection results in significant loss of peroxisomes in mammalian cells, which may indicate that targeting of peroxisomes is a key strategy used by viruses to subvert early antiviral defenses. Flaviviruses are arthropod-transmitted pathogens that infect hundreds of millions of people each year. Dengue virus (DENV) is the etiological agent of the most common mosquitoborne disease in the world, dengue fever (reviewed in reference 1). The related flavivirus West Nile virus (WNV) is the most important vector-transmitted pathogen in North America. Despite their medical significance, there are no DENV/WNV-specific vaccines or antiviral therapies that are approved for use in humans. Understanding how these viruses take advantage of and manipulate host cells may provide the foundation for therapies that target virushost interactions.Recent studies identified flavivirus capsid proteins as critical components of the virus-host interface. T...

Recent findings suggest that in addition to its role in packaging genomic RNA, the West Nile virus (WNV) capsid protein is an important pathogenic determinant, a scenario that requires interaction of this viral protein with host cell proteins. We performed an extensive multitissue yeast two-hybrid screen to identify capsid-binding proteins in human cells. Here we describe the interaction between WNV capsid and the nucleolar RNA helicase DDX56/NOH61. Coimmunoprecipitation confirmed that capsid protein binds to DDX56 in infected cells and that this interaction is not dependent upon intact RNA. Interestingly, WNV infection induced the relocalization of DDX56 from the nucleolus to a compartment in the cytoplasm that also contained capsid protein. This phenomenon was apparently specific for WNV, as DDX56 remained in the nucleoli of cells infected with rubella and dengue 2 viruses. Further analyses showed that DDX56 is not required for replication of WNV; however, virions secreted from DDX56-depleted cells contained less viral RNA and were 100 times less infectious. Together, these data suggest that DDX56 is required for assembly of infectious WNV particles.

HIV-associated neurocognitive disorders (HAND) represent a spectrum neurological syndrome that affects up to 25% of patients with HIV/AIDS. Multiple pathogenic mechanisms contribute to the development of HAND symptoms including chronic neuroinflammation and neurodegeneration. Among the factors linked to development of HAND is altered expression of host cell microRNAs (miRNAs) in brain. Here, we examined brain miRNA profiles among HIV/AIDS patients with and without HAND. Our analyses revealed differential expression of 17 miRNAs in brain tissue from HAND patients. A subset of the upregulated miRNAs (miR-500a-5p, miR-34c-3p, miR-93-3p and miR-381-3p), are predicted to target peroxisome biogenesis factors (PEX2, PEX7, PEX11B and PEX13). Expression of these miRNAs in transfected cells significantly decreased levels of peroxisomal proteins and concomitantly decreased peroxisome numbers or affected their morphology. The levels of miR-500a-5p, miR-34c-3p, miR-93-3p and miR-381-3p were not only elevated in the brains of HAND patients, but were also upregulated during HIV infection of primary macrophages. Moreover, concomitant loss of peroxisomal proteins was observed in HIV-infected macrophages as well as in brain tissue from HIV-infected patients. HIV-induced loss of peroxisomes was abrogated by blocking the functions of the upregulated miRNAs. Overall, these findings point to previously unrecognized miRNA expression patterns in the brains of HIV patients. Targeting peroxisomes by up-regulating miRNAs that repress peroxisome biogenesis factors may represent a novel mechanism by which HIV-1 subverts innate immune responses and/or causes neurocognitive dysfunction.

West Nile virus (WNV) is a blood-borne pathogen that causes systemic infections and serious neurological disease in human and animals. The most common route of infection is mosquito bites and therefore, the virus must cross a number of polarized cell layers to gain access to organ tissue and the central nervous system. Resistance to trans-cellular movement of macromolecules between epithelial and endothelial cells is mediated by tight junction complexes. While a number of recent studies have documented that WNV infection negatively impacts the barrier function of tight junctions, the intracellular mechanism by which this occurs is poorly understood. In the present study, we report that endocytosis of a subset of tight junction membrane proteins including claudin-1 and JAM-1 occurs in WNV infected epithelial and endothelial cells. This process, which ultimately results in lysosomal degradation of the proteins, is dependent on the GTPase dynamin and microtubule-based transport. Finally, infection of polarized cells with the related flavivirus, Dengue virus-2, did not result in significant loss of tight junction membrane proteins. These results suggest that neurotropic flaviviruses such as WNV modulate the host cell environment differently than hemorrhagic flaviviruses and thus may have implications for understanding the molecular basis for neuroinvasion.

Although flaviviruses encode their own helicases, evidence suggests that cellular helicases are also required for replication and/or assembly of these viruses. By and large, the mechanisms of action for viral and cellular helicases are not known. Moreover, in some cases, enzymatic activity is not even required for their roles in virus biology. Recently, we showed that expression of the host nucleolar helicase DDX56 is important for infectivity of West Nile virus (WNV) particles. In the present study, we demonstrate that the helicase activity of this enzyme is essential for its role in assembly of infectious WNV virions. Over-expression of the capsid-binding region of DDX56 also reduces infectivity of WNV suggesting that interaction of DDX56 and capsid protein is an important step in the virion assembly pathway. To our knowledge, this is the first study showing that enzymatic activity of a cellular helicase is critical for infectivity of flaviviruses.

Zika virus (ZIKV) has emerged as an important human pathogen that can cause congenital defects in the fetus and neurological conditions in adults. The interferon (IFN) system has proven crucial in restricting ZIKV replication and pathogenesis. The canonical IFN response is triggered by the detection of viral RNA through RIG-I like receptors followed by activation of the adaptor protein MAVS on mitochondrial membranes. Recent studies have shown that a second organelle, peroxisomes, also function as a signaling platforms for the IFN response. Here, we investigated how ZIKV infection affects peroxisome biogenesis and antiviral signaling. We show that ZIKV infection depletes peroxisomes in human fetal astrocytes, a brain cell type that can support persistent infection. The peroxisome biogenesis factor PEX11B was shown to inhibit ZIKV replication, likely by increasing peroxisome numbers and enhancing downstream IFN-dependent antiviral signaling. Given that peroxisomes play critical roles in brain development and nerve function, our studies provide important insights into the roles of peroxisomes in regulating ZIKV infection and potentially neuropathogenesis.