Human immunodeficiency virus type 1 (HIV-1) remains a deadly infectious disease despite existing antiretroviral therapies. A comprehensive understanding of the specific mechanisms of viral infectivity remains elusive and currently limits the development of new and effective therapies. Through in-depth proteomic analysis of HIV-1 virions, we discovered the novel post-translational modification of highly conserved residues within the viral matrix and capsid proteins to the dehydroamino acids, dehydroalanine and dehydrobutyrine. We further confirmed their presence by labeling the reactive alkene, characteristic of dehydroamino acids, with glutathione via Michael addition. Dehydroamino acids are rare, understudied, and have been observed mainly in select bacterial and fungal species. Until now, they have not been observed in HIV proteins. We hypothesize that these residues are important in viral particle maturation and could provide valuable insight into HIV infectivity mechanisms.

HIV-1 infectivity requires incorporation of the Gag-Pol (GP) precursor polyprotein into virions during the process of virus particle assembly. Mechanisms dictating GP incorporation into assembling virions are poorly defined, with GP levels in virions traditionally thought to solely reflect relative levels of Gag and GP expressed in cells, dictated by the frequency of a −1 programmed ribosomal frameshifting (PRF) event that occurs in gag-pol mRNAs.

HIV-1 generates unspliced (US), partially spliced (PS), and completely spliced (CS) classes of RNAs, each playing distinct roles in viral replication. Elucidating their host protein ‘interactomes’ is crucial to understanding virus-host interplay. Here, we present HyPR-MSSV for isolation of US, PS, and CS transcripts from a single population of infected CD4+ T-cells and mass spectrometric identification of their in vivo protein interactomes. Analysis revealed 212 proteins differentially associated with the unique RNA classes, including preferential association of regulators of RNA stability with US and PS transcripts and, unexpectedly, mitochondria-linked proteins with US transcripts. Remarkably, >80 of these factors screened by siRNA knockdown impacted HIV-1 gene expression. Fluorescence microscopy confirmed several to co-localize with HIV-1 US RNA and exhibit changes in abundance and/or localization over the course of infection. This study validates HyPR-MSSV for discovery of viral splice variant protein interactomes and provides an unprecedented resource of factors and pathways likely important to HIV-1 replication.

HIV-1 generates unspliced (US), partially spliced (PS), and completely spliced (CS) classes of RNAs; each playing distinct roles in viral replication. Elucidating their host protein “interactomes” is crucial to understanding virus-host interplay. Here, we present HyPR-MSSV for isolation of US, PS, and CS transcripts from a single population of infected CD4+ T-cells and mass spectrometric identification of their in vivo protein interactomes. Analysis revealed 212 proteins differentially associated with the unique RNA classes; including, preferential association of regulators of RNA stability with US- and PS-transcripts and, unexpectedly, mitochondria-linked proteins with US-transcripts. Remarkably, >80 of these factors screened by siRNA knock-down impacted HIV-1 gene expression. Fluorescence microscopy confirmed several to co-localize with HIV-1 US RNA and exhibit changes in abundance and/or localization over the course of infection. This study validates HyPR-MSSV for discovery of viral splice variant protein interactomes and provides an unprecedented resource of factors and pathways likely important to HIV-1 replication.

Humans are infected with two distinct strains (Type 1 (T1) and Type 2 (T2)) of Epstein-Barr virus (EBV) that differ substantially in their EBNA2 and EBNA 3A/B/C latency genes and the ability to transform B cells in vitro. While most T1 EBV strains contain the "prototype" form of the BZLF1 immediate-early promoter ("Zp-P"), all T2 strains contain the "Zp-V3" variant, which contains an NFAT binding motif and is activated much more strongly by B-cell receptor signalling. Whether B cells infected with T2 EBV are more lytic than cells infected with T1 EBV is unknown. Here we show that B cells infected with T2 EBV strains (AG876 and BL5) have much more lytic protein expression compared to B cells infected with T1 EBV strains (M81, Akata, and Mutu) in both a cord blood-humanized (CBH) mouse model and EBV-transformed lymphoblastoid cell lines (LCLs). Although T2 LCLs grow more slowly than T1 LCLs, both EBV types induce B-cell lymphomas in CBH mice. T1 EBV strains (M81 and Akata) containing Zp-V3 are less lytic than T2 EBV strains, suggesting that Zp-V3 is not sufficient to confer a lytic phenotype. Instead, we find that T2 LCLs express much higher levels of activated NFATc1 and NFATc2, and that cyclosporine (an NFAT inhibitor) and knockdown of NFATc2 attenuate constitutive lytic infection in T2 LCLs. Both NFATc1 and NFATc2 induce lytic EBV gene expression when combined with activated CAMKIV (which is activated by calcium signaling and activates MEF2D) in Burkitt Akata cells. Together, these results suggest that B cells infected with T2 EBV are more lytic due to increased activity of the cellular NFATc1/c2 transcription factors in addition to the universal presence of the Zp-V3 form of BZLF1 promoter.

ABSTRACT HIV-1 RNA genomes interact with diverse RNA binding proteins (RBPs) in the cytoplasm including antiviral factor APOBEC3G (A3G) that, in the absence of viral Vif proteins, is packaged into virions. Where and when HIV-1-A3G interactions are initiated for packaging inside the cell is unknown, and the relative contributions of genome vs. Gag capsid proteins to this process remains controversial. Here we visualized A3G responses to HIV-1 infection over an entire replication cycle using long-term (up to 72 h) live single cell imaging. We show that Vif-deficient HIV-1 dramatically shifts A3G and a second RNA surveillance factor, MOV10, from the cytoplasm to virus particle assembly sites with little to no net discernible effects on general markers of cytoplasmic processing bodies (DCP1A), stress granules (TIA-1), or a marker of the nonsense-mediated decay machinery (UPF1). Using a new live cell RNA-protein interaction assay based on RNA tethering (the i n- c ell RNA-protein i nteraction p rotocol, or IC-IP), we provide evidence that A3G- and MOV10- genome interactions are selective, strong, occur in presence or absence of Gag, and are initiated in the cytosol soon if not immediately after genome nuclear export. Finally, although Gag is sufficient to package A3G into virions even in the absence of genomes, single virion imaging indicates that selective A3G-genome interactions promote much more consistent per virion delivery of A3G to assembly sites. Collectively, these studies suggest a paradigm for early, strong, and persistent cytosolic detection of select HIV-1 RNA signatures by A3G, MOV10 and other host RBPs that are enriched in virions. IMPORTANCE Host-pathogen interactions determine the success of viral replication. While extensive work has identified many interactions between HIV-1 and cellular factors, our understanding of where these interactions in cells occur during the course of infection is incomplete. Here, we show that multiple RNA-binding proteins (including the antiviral restriction factor, APOBEC3G, and MOV10 helicase) bind HIV-1 RNA genomes in the cytoplasm and co-traffic with them into progeny virions. Furthermore, we show that these interactions with HIV-1 RNA occur in the absence of Gag and are sufficiently strong to recruit these to otherwise non-native subcellular locales. Together, these data begin to illuminate the intracellular trafficking pathways shared by host RNA binding proteins and the viral RNAs they preferentially bind.

HIV-1 RNA genomes interact with diverse RNA binding proteins in the cytoplasm including antiviral factor APOBEC3G (A3G) that, in the absence of viral Vif proteins, is packaged into virions. When and where genome-A3G interactions are initiated in the host cell is unknown. Here we use quantitative long-term (>24 h) live cell fluorescence video microscopy and a new in-cell RNA-protein interaction assay (the “IC-IP”) to describe subcellular viral and A3G trafficking behaviors over the entire HIV-1 productive phase. Among other findings, we demonstrate that genome-A3G interactions are initiated in the cytosol soon if not immediately after genome nuclear export; that A3G-genome interactions are sufficiently strong so that tethering either factor to membranes inhibits trafficking of the reciprocal binding partner; and that selective recognition of genomes promotes consistent delivery of A3G to sites of virion assembly. Further elucidation of RNA signature(s) detected by A3G may inform development of RNA-targeted antivirals.

ABSTRACT Human immunodeficiency virus (HIV) replication is strongly dependent upon a programmed ribosomal frameshift. Here we investigate the relationships between the thermodynamic stability of the HIV type 1 (HIV-1) RNA frameshift site stem-loop, frameshift efficiency, and infectivity, using pseudotyped HIV-1 and HEK293T cells. The data reveal a strong correlation between frameshift efficiency and local, but not overall, RNA thermodynamic stability. Mutations that modestly increase the local stability of the frameshift site RNA stem-loop structure increase frameshift efficiency 2-fold to 3-fold in cells. Thus, frameshift efficiency is determined by the strength of the thermodynamic barrier encountered by the ribosome. These data agree with previous in vitro measurements, suggesting that there are no virus- or host-specific factors that modulate frameshifting. The data also indicate that there are no sequence-specific requirements for the frameshift site stem-loop. A linear correlation between Gag-polymerase (Gag-Pol) levels in cells and levels in virions supports the idea of a stochastic virion assembly mechanism. We further demonstrate that the surrounding genomic RNA secondary structure influences frameshift efficiency and that a mutation that commonly arises in response to protease inhibitor therapy creates a functional but inefficient secondary slippery site. Finally, HIV-1 mutants with enhanced frameshift efficiencies are significantly less infectious, suggesting that compounds that increase frameshift efficiency by as little as 2-fold may be effective at suppressing HIV-1 replication. IMPORTANCE HIV, like many retroviruses, utilizes a −1 programmed ribosomal frameshift to generate viral enzymes in the form of a Gag-Pol polyprotein precursor. Thus, frameshifting is essential for viral replication. Here, we utilized a panel of mutant HIV strains to demonstrate that in cells, frameshifting efficiency is correlated with the stability of the local thermodynamic barrier to ribosomal translocation. Increasing the stability of the frameshift site RNA increases the frameshift efficiency 2-fold to 3-fold. Mutant viruses with increased frameshift efficiencies have significantly reduced infectivity. These data suggest that this effect might be exploited in the development of novel antiviral strategies.