SARS-CoV-2

This presentation about the biology of Coronaviruses was presented virtually to the RNA Biology Journal Club at the University of Massachusetts Medical School on April 7, 2020

Closing the net on retroviruses

Structural studies reveal how an antiviral factor forms a molecular net to restrict retroviruses including HIV-1

TRIM5 and the Regulation of HIV-1 Infectivity

The past ten years have seen an explosion of information concerning host restriction factors that inhibit the replication of HIV-1 and other retroviruses. Among these factors is TRIM5, an innate immune signaling molecule that recognizes the capsid lattice as soon as the retrovirion core is released into the cytoplasm of otherwise susceptible target cells. Recognition of the capsid lattice has several consequences that include multimerization of TRIM5 into a complementary lattice, premature uncoating of the virion core, and activation of TRIM5 E3 ubiquitin ligase activity. Unattached, K63-linked ubiquitin chains are generated that activate the TAK1 kinase complex and downstream inflammatory mediators. Polymorphisms in the capsid recognition domain of TRIM5 explain the observed species-specific differences among orthologues and the relatively weak anti-HIV-1 activity of human TRIM5. Better understanding of the complex interaction between TRIM5 and the retrovirus capsid lattice may someday lead to exploitation of this interaction for the development of potent HIV-1 inhibitors

The retroviral restriction factor TRIM5α

Retroviruses are obligate intracellular parasites that have coevolved with their hosts for millions of years. It is therefore not surprising that retroviruses take advantage of numerous host factors during their life cycle. In addition to positive cellular factors that are of use to the virus, host cells have also evolved intracellular proteins to antagonize the retroviral replication cycle. Such inhibitory cellular factors have been called retroviral restriction factors. Recently, several such restriction factors have been cloned, including Friend virus susceptibility factor 1, apolipoprotein B mRNA-editing enzyme catalytic proteins 3F and 3G, and ZAP. Here, we review the explosion of publications from the past 2 years concerning TRIM5, a host factor that potently inhibits HIV-1 and other retroviruse

Gammaretroviral pol sequences act in cis to direct polysome loading and NXF1/NXT-dependent protein production by gag-encoded RNA

BACKGROUND: All retroviruses synthesize essential proteins via alternatively spliced mRNAs. Retrovirus genera, though, exploit different mechanisms to coordinate the synthesis of proteins from alternatively spliced mRNAs. The best studied of these retroviral, post-transcriptional effectors are the trans-acting Rev protein of lentiviruses and the cis-acting constitutive transport element (CTE) of the betaretrovirus Mason-Pfizer monkey virus (MPMV). How members of the gammaretrovirus genus translate protein from unspliced RNA has not been elucidated. RESULTS: The mechanism by which two gammaretroviruses, XMRV and MLV, synthesize the Gag polyprotein (Pr65Gag) from full-length, unspliced mRNA was investigated here. The yield of Pr65Gag from a gag-only expression plasmid was found to be at least 30-fold less than that from an otherwise isogenic gag-pol expression plasmid. A frameshift mutation disrupting the pol open reading frame within the gag-pol expression plasmid did not decrease Pr65Gag production and 398 silent nucleotide changes engineered into gag rendered Pr65Gag synthesis pol-independent. These results are consistent with pol-encoded RNA acting in cis to promote Pr65Gag translation. Two independently-acting pol fragments were identified by screening 17 pol deletion mutations. To determine the mechanism by which pol promoted Pr65Gag synthesis, gag RNA in total and cytoplasmic fractions was quantitated by northern blot and by RT-PCR. The pol sequences caused, maximally, three-fold increase in total or cytoplasmic gag mRNA. Instead, pol sequences increased gag mRNA association with polyribosomes ~100-fold, a magnitude sufficient to explain the increase in Pr65Gag translation efficiency. The MPMV CTE, an NXF1-binding element, substituted for pol in promoting Pr65Gag synthesis. A pol RNA stem-loop resembling the CTE promoted Pr65Gag synthesis. Over-expression of NXF1 and NXT, host factors that bind to the MPMV CTE, synergized with pol to promote gammaretroviral gag RNA loading onto polysomes and to increase Pr65Gag synthesis. Conversely, Gag polyprotein synthesis was decreased by NXF1 knockdown. Finally, overexpression of SRp20, a shuttling protein that binds to NXF1 and promotes NXF1 binding to RNA, also increased gag RNA loading onto polysomes and increased Pr65Gag synthesis. CONCLUSION: These experiments demonstrate that gammaretroviral pol sequences act in cis to recruit NXF1 and SRp20 to promote polysome loading of gag RNA and, thereby license the synthesis of Pr65Gag from unspliced mRNA

Cyclophilin A promotes HIV-1 reverse transcription but its effect on transduction correlates best with its effect on nuclear entry of viral cDNA

BACKGROUND: The human peptidyl-prolyl isomerase Cyclophilin A (CypA) binds HIV-1 capsid (CA) and influences early steps in the HIV-1 replication cycle. The mechanism by which CypA regulates HIV-1 transduction efficiency is unknown. Disruption of CypA binding to CA, either by genetic means or by the competitive inhibitor cyclosporine A (CsA), reduces the efficiency of HIV-1 transduction in some cells but not in others. Transduction of certain cell types increases significantly when CypA binding to particular HIV-1 CA mutants, i.e., A92E, is prevented. Previous studies have suggested that this cell type-specific effect is due to a dominant-acting, CypA-dependent restriction factor. RESULTS: Here we investigated the mechanism by which CypA regulates HIV-1 transduction efficiency using 27 different human cell lines, 32 HeLa subclones, and several previously characterized HIV-1 CA mutants. Disruption of CypA binding to wild-type CA, or to any of the mutant CAs, caused a decrease in HIV-1 reverse transcription in all the cell lines analyzed here. This block to reverse transcription, though, did not correlate with cell type-specific effects on transduction efficiency. The level of 2-LTR circles, a marker for nuclear transport of the viral cDNA that results from reverse transcription, correlated closely with effects on infectivity. No correlation was observed between the cell type-specific effects on infectivity and the steady-state CypA protein levels in these cells. Instead, as indicated by a fate-of-capsid assay, CsA released the HIV-1 CA core from an apparent state of hyperstabilization, in a cell type-specific manner. CONCLUSION: These data demonstrate that, while CypA promotes reverse transcription under all conditions tested here, its effect on HIV-1 infectivity correlates more closely with effects on nuclear entry of the viral cDNA. The data also support the hypothesis that a cell-type specific CypA-dependent restriction factor blocks HIV-1 replication by delaying CA core uncoating and hindering nuclear entry

Vpx rescues HIV-1 transduction of dendritic cells from the antiviral state established by type 1 interferon

<p>Abstract</p> <p>Background</p> <p>Vpx is a virion-associated protein encoded by SIV_SM, a lentivirus endemic to the West African sooty mangabey (<it>Cercocebus atys</it>). HIV-2 and SIV_MAC, zoonoses resulting from SIV_SMtransmission to humans or Asian rhesus macaques (<it>Macaca mulatta</it>), also encode Vpx. In myeloid cells, Vpx promotes reverse transcription and transduction by these viruses. This activity correlates with Vpx binding to DCAF1 (VPRBP) and association with the DDB1/RBX1/CUL4A E3 ubiquitin ligase complex. When delivered experimentally to myeloid cells using VSV G-pseudotyped virus-like particles (VLPs), Vpx promotes reverse transcription of retroviruses that do not normally encode Vpx.</p> <p>Results</p> <p>Here we show that Vpx has the extraordinary ability to completely rescue HIV-1 transduction of human monocyte-derived dendritic cells (MDDCs) from the potent antiviral state established by prior treatment with exogenous type 1 interferon (IFN). The magnitude of rescue was up to 1,000-fold, depending on the blood donor, and was also observed after induction of endogenous IFN and IFN-stimulated genes (ISGs) by LPS, poly(I:C), or poly(dA:dT). The effect was relatively specific in that Vpx-associated suppression of soluble IFN-β production, of mRNA levels for ISGs, or of cell surface markers for MDDC differentiation, was not detected. Vpx did not rescue HIV-2 or SIV_MACtransduction from the antiviral state, even in the presence of SIV_MACor HIV-2 VLPs bearing additional Vpx, or in the presence of HIV-1 VLPs bearing all accessory genes. In contrast to the effect of Vpx on transduction of untreated MDDCs, HIV-1 rescue from the antiviral state was not dependent upon Vpx interaction with DCAF1 or on the presence of DCAF1 within the MDDC target cells. Additionally, although Vpx increased the level of HIV-1 reverse transcripts in MDDCs to the same extent whether or not MDDCs were treated with IFN or LPS, Vpx rescued a block specific to the antiviral state that occurred after HIV-1 cDNA penetrated the nucleus.</p> <p>Conclusion</p> <p>Vpx provides a tool for the characterization of a potent, new HIV-1 restriction activity, which acts in the nucleus of type 1 IFN-treated dendritic cells.</p

HERV-H RNA is abundant in human embryonic stem cells and a precise marker for pluripotency

BACKGROUND: Certain post-translational modifications to histones, including H3K4me3, as well as binding sites for the transcription factor STAT1, predict the site of integration of exogenous gamma-retroviruses with great accuracy and cell-type specificity. Statistical methods that were used to identify chromatin features that predict exogenous gamma-retrovirus integration site selection were exploited here to determine whether cell type-specific chromatin markers are enriched in the vicinity of endogenous retroviruses (ERVs). RESULTS: Among retro-elements in the human genome, the gamma-retrovirus HERV-H was highly associated with H3K4me3, though this association was only observed in embryonic stem (ES) cells (p < 10(-300)) and, to a lesser extent, in induced pluripotent stem (iPS) cells. No significant association was observed in nearly 40 differentiated cell types, nor was any association observed with other retro-elements. Similar strong association was observed between HERV-H and the binding sites within ES cells for the pluripotency transcription factors NANOG, OCT4, and SOX2. NANOG binding sites were located within the HERV-H 5(′)LTR itself. OCT4 and SOX2 binding sites were within 1 kB and 2 kB of the 5(′)LTR, respectively. In keeping with these observations, HERV-H RNA constituted 2% of all poly A RNA in ES cells. As ES cells progressed down a differentiation pathway, the levels of HERV-H RNA decreased progressively. RNA-Seq datasets showed HERV-H transcripts to be over 5 kB in length and to have the structure 5(′)LTR-gag-pro-3(′)LTR, with no evidence of splicing and no intact open reading frames. CONCLUSION: The developmental regulation of HERV-H expression, the association of HERV-H with binding sites for pluripotency transcription factors, and the extremely high levels of HERV-H RNA in human ES cells suggest that HERV-H contributes to pluripotency in human cells. Proximity of HERV-H to binding sites for pluripotency transcription factors within ES cells might be due to retention of the same chromatin features that determined the site of integration of the ancestral, exogenous, gamma-retrovirus that gave rise to HERV-H in the distant past. Retention of these markers, or, alternatively, recruitment of them to the site of the established provirus, may have acted post-integration to fix the provirus within the germ-line of the host species. Either way, HERV-H RNA provides a specific marker for pluripotency in human cells

Vpx rescue of HIV-1 from the antiviral state in mature dendritic cells is independent of the intracellular deoxynucleotide concentration

BACKGROUND: SIVMAC/HIV-2 Vpx recruits the CUL4A-DCAF1 E3 ubiquitin ligase complex to degrade the deoxynucleotide hydrolase SAMHD1. This increases the concentration of deoxynucleotides available for reverse transcription in myeloid cells and resting T cells. Accordingly, transduction of these cells by SIVMAC requires Vpx. Virus-like particles containing SIVMAC Vpx (Vpx-VLPs) also increase the efficiency of HIV-1 transduction in these cells, and rescue transduction by HIV-1, but not SIVMAC, in mature monocyte-derived dendritic cells (MDDCs). Differences in Vpx mechanism noted at that time, along with recent data suggesting that SAMHD1 gains additional restriction capabilities in the presence of type I IFN prompted further examination of the role of Vpx and SAMHD1 in HIV-1 transduction of mature MDDCs. RESULTS: When challenged with Vpx-VLPs, SAMHD1 was degraded in MDDCs even after cells had been matured with LPS, though there was no increase in deoxynucleotide levels. Steady-state levels of HIV-1 late reverse transcription products in mature MDDCs were increased to the same extent by either Vpx-VLPs or exogenous nucleosides. In contrast, only Vpx-VLPs increased the levels of 2-LTR circles and proviral DNA in myeloid cells. These results demonstrate that exogenous nucleosides and Vpx-VLPs both increase the levels of HIV-1 cDNA in myeloid cells, but only Vpx-VLPs rescue 2-LTR circles and proviral DNA in myeloid cells with a previously established antiviral state. Finally, since trans-acting Vpx-VLPs provide long-lasting rescue of HIV-1 vector transduction in the face of the antiviral state, and exogenous nucleosides do not, exogenous nucleosides were used to achieve efficient transduction of MDDCs by vectors that stably encode Vprs and Vpxs from a collection of primate lentiviruses. Vpr from SIVDEB or SIVMUS, Vpx from SIVMAC251 or HIV-2, but not SIVRCM, degraded endogenous SAMHD1, increased steady-state levels of HIV-1 cDNA, and rescued HIV-1 from the antiviral state in MDDCs. CONCLUSION: Inhibition of deoxynucleotide hydrolysis by promoting SAMHD1 degradation is not the only mechanism by which Vpx rescues HIV-1 in MDDCs from the antiviral state. Vpx has an additional effect on HIV-1 transduction of these cells that occurs after completion of reverse transcription and acts independently of deoxynucleotide levels