A Tripartite Complex HIV-1 Tat-Cyclophilin A-Capsid Protein Enables Tat Encapsidation That Is Required for HIV-1 Infectivity.

HIV-1 Tat is a key viral protein that stimulates several steps of viral gene expression. Tat is especially required for the transcription of viral genes. Nevertheless, it is still not clear if and how Tat is incorporated into HIV-1 virions. Cyclophilin A (CypA) is a prolyl isomerase that binds to HIV-1 capsid protein (CA) and is thereby encapsidated at the level of 200 to 250 copies of CypA/virion. Here, we found that a Tat-CypA-CA tripartite complex assembles in HIV-1-infected cells and allows Tat encapsidation into HIV virions (1 Tat/1 CypA). Biochemical and biophysical studies showed that high-affinity interactions drive the assembly of the Tat-CypA-CA complex that could be purified by size exclusion chromatography. We prepared different types of viruses devoid of transcriptionally active Tat. They showed a 5- to 10 fold decrease in HIV infectivity, and conversely, encapsidating Tat into ΔTat viruses greatly enhanced infectivity. The absence of encapsidated Tat decreased the efficiency of reverse transcription by ~50% and transcription by more than 90%. We thus identified a Tat-CypA-CA complex that enables Tat encapsidation and showed that encapsidated Tat is required to initiate robust viral transcription and thus viral production at the beginning of cell infection, before neosynthesized Tat becomes available. IMPORTANCE The viral transactivating protein Tat has been shown to stimulate several steps of HIV gene expression. It was found to facilitate reverse transcription. Moreover, Tat is strictly required for the transcription of viral genes. Although the presence of Tat within HIV virions would undoubtedly favor these steps and therefore enable the incoming virus to boost initial viral production, whether and how Tat is present within virions has been a matter a debate. We here described and characterized a tripartite complex between Tat, HIV capsid protein, and the cellular chaperone cyclophilin A that enables efficient and specific Tat encapsidation within HIV virions. We further showed that Tat encapsidation is required for the virus to efficiently initiate infection and viral production. This effect is mainly due to the transcriptional activity of Tat.

Interaction of a Densovirus with Glycans of the Peritrophic Matrix Mediates Oral Infection of the Lepidopteran Pest Spodoptera frugiperda.

The success of oral infection by viruses depends on their capacity to overcome the gut epithelial barrier of their host to crossing over apical, mucous extracellular matrices. As orally transmitted viruses, densoviruses, are also challenged by the complexity of the insect gut barriers, more specifically by the chitinous peritrophic matrix, that lines and protects the midgut epithelium; how capsids stick to and cross these barriers to reach their final cell destination where replication goes has been poorly studied in insects. Here, we analyzed the early interaction of the Junonia coenia densovirus (JcDV) with the midgut barriers of caterpillars from the pest Spodoptera frugiperda. Using combination of imaging, biochemical, proteomic and transcriptomic analyses, we examined in vitro, ex vivo and in vivo the early interaction of the capsids with the peritrophic matrix and the consequence of early oral infection on the overall gut function. We show that the JcDV particle rapidly adheres to the peritrophic matrix through interaction with different glycans including chitin and glycoproteins, and that these interactions are necessary for oral infection. Proteomic analyses of JcDV binding proteins of the peritrophic matrix revealed mucins and non-mucins proteins including enzymes already known to act as receptors for several insect pathogens. In addition, we show that JcDV early infection results in an arrest of N-Acetylglucosamine secretion and a disruption in the integrity of the peritrophic matrix, which may help viral particles to pass through. Finally, JcDV early infection induces changes in midgut genes expression favoring an increased metabolism including an increased translational activity. These dysregulations probably participate to the overall dysfunction of the gut barrier in the early steps of viral pathogenesis. A better understanding of early steps of densovirus infection process is crucial to build biocontrol strategies against major insect pests.

Cyclophilin A enables specific HIV-1 Tat palmitoylation and accumulation in uninfected cells.

Most HIV-1 Tat is unconventionally secreted by infected cells following Tat interaction with phosphatidylinositol (4,5) bisphosphate (PI(4,5)P2) at the plasma membrane. Extracellular Tat is endocytosed by uninfected cells before escaping from endosomes to reach the cytosol and bind PI(4,5)P2. It is not clear whether and how incoming Tat concentrates in uninfected cells. Here we show that, in uninfected cells, the S-acyl transferase DHHC-20 together with the prolylisomerases cyclophilin A (CypA) and FKBP12 palmitoylate Tat on Cys31 thereby increasing Tat affinity for PI(4,5)P2. In infected cells, CypA is bound by HIV-1 Gag, resulting in its encapsidation and CypA depletion from cells. Because of the lack of this essential cofactor, Tat is not palmitoylated in infected cells but strongly secreted. Hence, Tat palmitoylation specifically takes place in uninfected cells. Moreover, palmitoylation is required for Tat to accumulate at the plasma membrane and affect PI(4,5)P2-dependent membrane traffic such as phagocytosis and neurosecretion.

Unconventional secretion of viral proteins.

Although largely less numerous and characterized than bacterial secreted effectors, several viral virulence factors are secreted by virus infected cells. However, their mode of secretion only starts to be studied at the molecular level. Several of these viral effectors are secreted using an unconventional secretion pathway, i.e. despite the lack of signal sequence. We here review recent results illustrating the diversity of these pathways. In the case of HIV-1 proteins Tat and matrix (p17) proteins, secretion directly takes place at the plasma membrane level following binding to PI(4,5)P2. The secretion of HTLV-I Tax was found to partly rely on exocytic pathway intermediates. The secretion pathways of VP22 of Herpes simplex virus type I and VP40 of the Ebola virus are less well characterized but VP40 can be recruited to the plasma membrane by PI(4,5)P2 that thus appears as a key partner enabling the unconventional secretion of many viral proteins. Several studies indicated that circulating retroviral transactivating proteins Tat and Tax are involved in the development of AIDS and HTLV-I associated myelopathy/tropical spastic paraparesis, respectively.