Modeling reveals the strength of weak interactions in stacked-ring assembly.

Cells employ many large macromolecular machines for the execution and regulation of processes that are vital for cell and organismal viability. Interestingly, cells cannot synthesize these machines as functioning units. Instead, cells synthesize the molecular parts that must then assemble into the functional complex. Many important machines, including chaperones such as GroEL and proteases such as the proteasome, comprise protein rings that are stacked on top of one another. While there is some experimental data regarding how stacked-ring complexes such as the proteasome self-assemble, a comprehensive understanding of the dynamics of stacked-ring assembly is currently lacking. Here, we developed a mathematical model of stacked-trimer assembly and performed an analysis of the assembly of the stacked homomeric trimer, which is the simplest stacked-ring architecture. We found that stacked rings are particularly susceptible to a form of kinetic trapping that we term "deadlock," in which the system gets stuck in a state where there are many large intermediates that are not the fully assembled structure but that cannot productively react. When interaction affinities are uniformly strong, deadlock severely limits assembly yield. We thus predicted that stacked rings would avoid situations where all interfaces in the structure have high affinity. Analysis of available crystal structures indicated that indeed the majority-if not all-of stacked trimers do not contain uniformly strong interactions. Finally, to better understand the origins of deadlock, we developed a formal pathway analysis and showed that, when all the binding affinities are strong, many of the possible pathways are utilized. In contrast, optimal assembly strategies utilize only a small number of pathways. Our work suggests that deadlock is a critical factor influencing the evolution of macromolecular machines and provides general principles for understanding the self-assembly efficiency of existing machines.

MicroRNA-25/93 induction by Vpu as a mechanism for counteracting MARCH1-restriction on HIV-1 infectivity in macrophages.

IMPORTANCE: In order to efficiently produce infectious viral particles, HIV must counter several restrictions exerted by host cell antiviral proteins. MARCH1 is a member of the MARCH protein family that restricts HIV infection by limiting the incorporation of viral envelope glycoproteins into nascent virions. Here, we identified two regulatory RNAs, microRNAs-25 and -93, induced by the HIV-1 accessory protein Vpu, that downregulate MARCH1 mRNA. We also show that Vpu induces these cellular microRNAs in macrophages by hijacking the cellular ß-catenin pathway. The notion that HIV-1 has evolved a mechanism to counteract MARCH1 restriction on viral infectivity underlines the importance of MARCH1 in the host antiviral response.

Evolution of naturally arising SARS-CoV-2 defective interfering particles.

Defective interfering (DI) particles arise during virus propagation, are conditional on parental virus for replication and packaging, and interfere with viral expansion. There is much interest in developing DIs as anti-viral agents. Here we characterize DI particles that arose following serial passaging of SARS-CoV-2 at high multiplicity of infection. The prominent DIs identified have lost ~84% of the SARS-CoV-2 genome and are capable of attenuating parental viral titers. Synthetic variants of the DI genomes also interfere with infection and can be used as conditional, gene delivery vehicles. In addition, the DI genomes encode an Nsp1-10 fusion protein capable of attenuating viral replication. These results identify naturally selected defective viral genomes that emerged and stably propagated in the presence of parental virus.

The HIV-1 Accessory Protein Vpu Downregulates Peroxisome Biogenesis.

Human immunodeficiency virus type 1 (HIV-1) establishes lifelong infections in humans, a process that relies on its ability to thwart innate and adaptive immune defenses of the host. Recently, we reported that HIV-1 infection results in a dramatic reduction of the cellular peroxisome pool. Peroxisomes are metabolic organelles that also function as signaling platforms in the innate immune response. Here, we show that the HIV-1 accessory protein Vpu is necessary and sufficient for the depletion of cellular peroxisomes during infection. Vpu induces the expression of four microRNAs that target mRNAs encoding proteins required for peroxisome formation and metabolic function. The ability of Vpu to downregulate peroxisomes was found to be dependent upon the Wnt/ß-catenin signaling pathway. Given the importance of peroxisomes in innate immune signaling and central nervous system function, the roles of Vpu in dampening antiviral signaling appear to be more diverse than previously realized. Finally, our findings highlight a potential role for Wnt/ß-catenin signaling in peroxisome homeostasis through modulating the production of biogenesis factors.IMPORTANCE People living with HIV can experience accelerated aging and the development of neurological disorders. Recently, we reported that HIV-1 infection results in a dramatic loss of peroxisomes in macrophages and brain tissue. This is significant because (i) peroxisomes are important for the innate immune response and (ii) loss of peroxisome function is associated with cellular aging and neurodegeneration. Accordingly, understanding how HIV-1 infection causes peroxisome depletion may provide clues regarding how the virus establishes persistent infections and, potentially, the development of neurological disorders. Here, we show that the accessory protein Vpu is necessary and sufficient for the induction of microRNAs that target peroxisome biogenesis factors. The ability of Vpu to downregulate peroxisome formation depends on the Wnt/ß-catenin pathway. Thus, in addition to revealing a novel mechanism by which HIV-1 uses intracellular signaling pathways to target antiviral signaling platforms (peroxisomes), we have uncovered a previously unknown link between the Wnt/ß-catenin pathway and peroxisome homeostasis.

Interplay between Zika Virus and Peroxisomes during Infection.

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.

Targeted Elimination of Peroxisomes During Viral Infection: Lessons from HIV and Other Viruses.

Peroxisomes are membrane-bound organelles that are best known for their roles in lipid metabolism. Mounting evidence indicates that they are also important nodes for antiviral signaling. While research over the past few decades has revealed effective viral strategies to block antiviral signalling pathways from the plasma membrane, mitochondria and/or the nucleus, until recently, very little was known about how viruses interfere with peroxisome-based antiviral signaling. In this essay, we review how viruses use a variety of strategies to interfere with peroxisome biogenesis, a phenomenon that has implications for evasion of the host immune system as well as pathogenesis.

MicroRNAs upregulated during HIV infection target peroxisome biogenesis factors: Implications for virus biology, disease mechanisms and neuropathology.

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.

Zika Virus Hijacks Stress Granule Proteins and Modulates the Host Stress Response.

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 eIF2α 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.

Flavivirus Infection Impairs Peroxisome Biogenesis and Early Antiviral Signaling.

UNLABELLED: 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. IMPORTANCE: RNA 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.

The helicase activity of DDX56 is required for its role in assembly of infectious West Nile virus particles.

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.

West Nile virus infection causes endocytosis of a specific subset of tight junction membrane proteins.

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.

The capsid-binding nucleolar helicase DDX56 is important for infectivity of West Nile virus.

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.

Contribution of the C-terminal region within the catalytic core domain of HIV-1 integrase to yeast lethality, chromatin binding and viral replication.

BACKGROUND: HIV-1 integrase (IN) is a key viral enzymatic molecule required for the integration of the viral cDNA into the genome. Additionally, HIV-1 IN has been shown to play important roles in several other steps during the viral life cycle, including reverse transcription, nuclear import and chromatin targeting. Interestingly, previous studies have demonstrated that the expression of HIV-1 IN induces the lethal phenotype in some strains of Saccharomyces cerevisiae. In this study, we performed mutagenic analyses of the C-terminal region of the catalytic core domain of HIV-1 IN in order to delineate the critical amino acid(s) and/or motif(s) required for the induction of the lethal phenotype in the yeast strain HP16, and to further elucidate the molecular mechanism which causes this phenotype. RESULTS: Our study identified three HIV-1 IN mutants, V165A, A179P and KR186,7AA, located in the C-terminal region of the catalytic core domain of IN that do not induce the lethal phenotype in yeast. Chromatin binding assays in yeast and mammalian cells demonstrated that these IN mutants were impaired for the ability to bind chromatin. Additionally, we determined that while these IN mutants failed to interact with LEDGF/p75, they retained the ability to bind Integrase interactor 1. Furthermore, we observed that VSV-G-pseudotyped HIV-1 containing these IN mutants was unable to replicate in the C8166 T cell line and this defect was partially rescued by complementation with the catalytically inactive D64E IN mutant. CONCLUSION: Overall, this study demonstrates that three mutations located in the C-terminal region of the catalytic core domain of HIV-1 IN inhibit the IN-induced lethal phenotype in yeast by inhibiting the binding of IN to the host chromatin. These results demonstrate that the C-terminal region of the catalytic core domain of HIV-1 IN is important for binding to host chromatin and is crucial for both viral replication and the promotion of the IN-induced lethal phenotype in yeast.

Interaction of human immunodeficiency virus type 1 integrase with cellular nuclear import receptor importin 7 and its impact on viral 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 alpha (Rch1). In contrast, another HIV-1 karyophilic protein MAp17, which is capable of binding Rch1, fails to interact with Imp7, suggesting that IN and Map17 may interact with different cellular pathways during HIV-1 replication. Genetic analysis revealed that the C-terminal domain of IN is the region responsible for interaction between IN with Imp7, and an IN mutant (K240A,K244A/R263A,K264A) disrupted the Imp7 binding ability of the protein, indicating that both regions ((235)WKGPAKLLWKG and (262)RRKAK) within the C-terminal domain of IN are required for efficient IN/Imp7 interaction. Using a vesicular stomatitis virus G glycoprotein pseudotyped HIV single-cycle replication system, we showed that the IN/Imp7 interaction-deficient mutant was unable to mediate viral replication and displayed impairment at both viral reverse transcription and nuclear import steps. Moreover, transient knockdown of Imp7 in both HIV-1 producing and target cells resulted in a 2.5-3.5-fold inhibition of HIV infection. Altogether, our results indicate that HIV-1 IN specifically interacts with Imp7, and this viral/cellular protein interaction contributes to efficient HIV-1 infection.