Retroviruses are obligate intracellular parasites that carry the information necessary for replication within their genomes. The three polyproteins, Gag, Pol, and Env, encoded by all retroviruses, function to generate progeny virions inside the host cell. Formation of new viral particles requires detailed instructions contained within the Gag polyprotein. Herein we describe our investigation into assembly of the Mason-Pfizer monkey virus (M-PMV). During retrovirus assembly, the transition from immature to a fully infectious mature particle is associated with the operation of molecular switches that trigger dramatic conformational changes of the Gag proteins. A dominant maturation switch that stabilizes the immature capsid lattice is located in the C-terminus of the capsid (CA) protein in many retroviral Gags. The HIV-1 Gag contains a stretch of five amino acid residues termed the 'clasp motif', important for the organization of the hexameric subunits that provide stability to the overall immature HIV-1 shell. Sequence alignment of the CA C-terminal domains (CTDs) of the HIV-1 and M-PMV highlighted a spacer-like domain in M-PMV that may provide comparable function. In the present study we report an examination of the role of the clasp motif in the M-PMV life cycle. Our results demonstrate that claps motif mutants display major defects in virion assembly and release, and even larger defects in infectivity. Our data identifies the clasp motif as a fundamental contributor to CA-CTD interactions necessary for efficient viral infection. The retroviral life cycle, unlike that of any other viral family, leads to the obligate integration of a proviral DNA into the host genome of somatic cells and in some cases even into the germ line. This remarkable feature of the Retroviridae family of viruses accounts for their extraordinary persistence through time and widespread abundance among vertebrate hosts. Because retroviral infection can have serious consequences to the host, there is great selective pressure to evolve strong networks that act to control incoming viruses. In the second study presented here, we report a novel cofactor of an antiviral system, Riplet, which operates to augment HIV-1 restriction by ZAP. The zinc-finger antiviral protein (ZAP) is an interferon-stimulated gene (ISG) with potent intrinsic antiviral activity. ZAP inhibits replication of retroviruses including MLV and HIV-1, as well as alphaviruses, filoviruses, hepatitis B virus, etc. ZAP operates at the post-transcriptional stage, reducing the number of viral transcripts available for translation in the cytoplasm, although additional pathways might be at play. The exact mechanisms by which ZAP restricts viral replication are not fully understood. ZAP lacks enzymatic activity and utilizes other cellular proteins to suppress viral replication. TRIM25 and the nuclease KHNYN have been identified as ZAP cofactors, but its activity may well involve other cellular proteins. Here we identify Riplet, a protein known to play a central role in the activation of the retinoic acid-inducible gene I (RIG-I), as a novel ZAP cofactor that acts to augment ZAP’s antiviral activity. Our data demonstrates that Riplet significantly augments ZAP-mediated restriction of HIV-1. Additionally, we show that Riplet interacts with ZAP via its PRY/SPRY domain and that the ubiquitin ligase activity of Riplet is not required to stimulate ZAP-mediated inhibition. Moreover, we show that Riplet interacts with TRIM25 suggesting that both Riplet and TRIM25 may operate synergistically to augment ZAP-mediated inhibition of HIV-1. The intracellular tropism of viruses is determined by a diverse combination of host proteins that allow infection to proceed efficiently. To achieve successful infection the virus needs the contribution of numerous cellular factors that assist at various steps of the life cycle. Conversely, replication requires resistance to species-specific restriction factors that act to suppress virus infection. The replication of M-PMV has been found to be highly restricted in mouse cell lines. The mechanism underlying the restriction of M-PMV replication in mouse cells has not been characterized. In the third study presented here, we examined this potent post-entry block and performed an unbiased genome-wide CRISPR-Cas9 screen, selecting for knock-out of host factors that relieved the block. Our data identified several candidate genes that encode proteins involved in virus trafficking and innate immune activation.

Retroviruses are capable of infecting diverse vertebrates, and successful infection requires intimate interaction between virus and the host cell. During an infection, retroviral particles must bind specifically to cell surface receptors on the target cell, cross the plasma membrane, reverse-transcribe their RNA genome into double stranded DNA, find their way to the nucleus, enter the nucleus and integrate its DNA into host chromosomes. Following integration, expression of viral mRNA ensues, followed by viral mRNA export into the cytoplasm, translation of viral mRNA into proteins, and assembly of new virions that will egress from the host cell. We now appreciate that at many steps of this complex process, the virus must hijack the cellular machinery to replicate. At the same time, the host cell mobilizes a variety of cellular defense mechanisms to suppress viral infection. This thesis investigates various aspects of virus-host interactions. I will first describe the involvement of cellular transcriptional repressor protein ErbB3 binding protein 1 (EBP1) in facilitating transcriptional shutdown of Moloney murine leukemia virus (MLV) gene expression in mouse embryonic cells. Next, I describe a novel means of regulating the activity of Yin Yang 1 (YY1), a cellular transcription factor regulating retroviral gene expression, through post-translational modifications. I show that YY1 is a target of tyrosine phosphorylation by Src family kinases. Phosphorylation of YY1 impairs its ability to bind DNA and RNA, thereby downregulating its activity as a transcription factor on retroviral and cellular promoters. Apart from studying retroviral gene expression, I have also investigated intrinsic cellular defenses against retroviral infection. This is exemplified by our finding that mouse cells are intrinsically resistant to infection by betaretroviruses such as Mason-Pfizer monkey virus (M-PMV). The block against M-PMV occurs after reverse transcription and prior to viral nuclear entry. Finally, I will present ongoing work examining the fate of viral DNAs following infection, focusing on the kinetics of its association with cellular core histones and viral structural proteins. Together, this work provides critical insights into numerous aspects of the virus-host interactions.

Integrase catalyzes insertion of a retroviral genome into the host chromosome. Following reverse transcription, integrase binds specifically to the ends of the duplex retroviral DNA, endonucleolytically cleaves two nucleotides from each 3'-end (the processing activity), and inserts these ends into the host DNA (the joining activity) in a concerted manner. Additionally, it has been observed that integrase can catalyze the removal of inserted viral ends (the disintegration activity) in vitro. Presteady-state experiments were performed using synapsed substrates to probe the processing reaction and a disintegration substrate to determine the number of protomers in a functional multimeric complex. In single-turnover studies, a novel "splicing" reaction was observed that revealed complications with accurate quantification of enzymatic activity using the synapsed substrates. The splicing reaction was further used to gain insight into the selection of nucleophiles and electrophiles at the binding site. To reduce the complexity introduced by the integrase-catalyzed splicing reaction, 5'-5' reverse-polarity synapsed substrates were designed that were not susceptible to the splicing reaction and that allowed direct comparison of LTR ends simultaneously bound at the active site. Analysis of the presteady-state assays using these reverse-polarity substrates revealed that the concurrent binding of the biologically relevant U3/U5 combination of viral ends facilitates maximal activity of the processing reaction. A disintegration substrate was used in presteady-state active site titrations to determine a reaction stoichiometry of four integrase protomers per one substrate molecule for the disintegration reaction. A tetrameric active complex was then confirmed using atomic force microscopy to image integrase-DNA complexes during the first catalytic turnover. The observed increase of the tetramer population in the presence of substrate DNA demonstrates that the binding of the disintegration substrate induces assembly of the active tetramer and suggests that tetramer assembly may be an integral and dynamic component of the catalytic pathway. / Graduation date: 2003

Diss. (sammanfattning) Uppsala : Uppsala universitet, 2007. / Härtill 6 uppsatser.

Après intégration dans le génome cellulaire de l’hôte, l’expression des rétrovirus dépend d’éléments agissant en cis localisés dans la longue répétition terminale 5’ (LTR5’) et la région leader, de facteurs de transcription cellulaires et viraux agissant en trans ainsi que de l’organisation chromatinienne du provirus intégré. Notre laboratoire a précédemment identifié dans le génome du rétrovirus HIV-1 (Human Immunodeficiency Virus type 1) une région intragénique importante (nt 4079-6026, où nt +1 est le début de U3 dans le LTR5’) composée du fragment 5103, du site hypersensible aux nucléases SH7 et du fragment 5105. Lors de ce travail, nous avons caractérisé physiquement et fonctionnellement différents sites de liaison pour des facteurs de transcription cellulaires localisés dans la région intragénique du virus HIV-1, dont trois sites de liaison pour le facteur inductible AP-1, dans des expériences de retard de migration sur gel et de transfection transitoire. Nous avons montré l’importance de ces trois sites AP-1 pour la réplication virale au niveau transcriptionnel dans des expériences d’infection et d’immunoprécipitation de la chromatine. De plus, nous avons caractérisé l’activité transcriptionnelle associée à la région intragénique du virus HIV-1. D’autre part, la structure nucléosomale du provirus intégré et les modifications épigénétiques associées jouent un rôle crucial pour l’expression des rétrovirus. La répression transcriptionnelle du rétrovirus oncogène BLV (Bovine Leukemia Virus) lui permet d’échapper au système immunitaire de son hôte bovin et favorise ainsi l’apparition de tumeurs. Dans ce contexte, nous avons montré que la méthylation de l’ADN au niveau du promoteur viral permet le maintient de la latence transcriptionnelle. En effet, la méthylation des dinucleotides CpGs localisés dans le LTR5‘ empêche le recrutement in vivo des facteurs de transcription activateurs CREB/CREM/ATF. Nous avons également montré que l’activation transcriptionnelle de l’expression du BLV par la combinaison PMA/ionomycine s’accompagne d’un remodelage chromatinien rapide mais transitoire au niveau du promoteur viral par des expériences de marquage indirect des extrémités et d’immunoprécipitation de chromatine. Nous avons ensuite démontré l’importance du site de liaison pour le facteur de transcription PU.1 et de la E-box 4 qui lie USF-1/-2, tous deux localisés dans la région dont l’accessibilité aux nucléases s’accroît après traitement des cellules, pour l’activation transcriptionnelle de l’expression virale par cette combinaison d’inducteurs. En conclusion, notre travail devrait permettre une meilleure compréhension des mécanismes transcriptionnels et épigénétiques régulant l’expression des rétrovirus HIV-1 et BLV. / Doctorat en Sciences / info:eu-repo/semantics/nonPublished