The lentiviral capsid core, which encapsulates the viral RNA genome, is delivered into the target cell cytoplasm during the viral entry process. In the cytoplasm, the conical core undergoes morphological changes, which are termed uncoating. Proper uncoating has been shown to be critical for the infectivity of the lentivirus HIV-1. In addition, the HIV-1 capsid protein is critical for the process of nuclear import of the preintegration complex (PIC). The lentivirus equine infectious anemia virus (EIAV) shares many similarities to HIV-1, including similarities in the capsid protein. In particular, both HIV-1 and EIAV capsid contain a proline-rich loop region in the amino terminal domain of capsid between helices 4 and 5. The host cellular factor cyclophilin A binds this loop in HIV-1 and is critical for proper uncoating. We hypothesized that this helix 4-5 loop was also critical for EIAV infectivity at some early step in the viral infection cycle. We created a panel of amino acid substitution mutations in this loop region. Some of the mutations resulted in severely deleterious effects on EIAV infectivity. Some mutations caused a slight increase in infectivity. The deleterious mutations did not affect uncoating or reverse transcription but appeared to block nuclear import of the PIC. Those mutations in which infectivity was slightly increased did not exhibit significantly different phenotypes from wild-type EIAV at any of the stages examined. The results of this study lend further support to the role of capsid as a determinant of nuclear import and suggest that viral and cellular factors critical to HIV-1 import may also be applicable to EIAV. Future research should focus on identifying the causes of the defects in nuclear import observed for some mutants, as well as attempt to identify the reason for the infectivity increase in others. In addition, inclusion of EIAV in future studies of nuclear import involving HIV-1 can broaden the scope of the data to lentiviruses in general rather than HIV-1 in particular.

The host restriction factor TRIM5α mediates a post-entry, pre-integration block to retroviral infection that depends upon recognition of the viral capsid by the TRIM5α PRYSPRY domain. The two predominant alleles of rhesus macaque TRIM5α (rhTRIM5αQ and rhTRIM5αTFP) restrict HIV 1, but cannot restrict the macaque-adapted virus SIVmac239. To investigate how TRIM5α recognizes retroviral capsids, we exploited the differential sensitivities of these two viruses to identify gain-of-sensitivity mutations in SIVmac239, and we solved the structure of the SIVmac239 capsid N-terminal domain. When mapped onto this structure, single amino acid substitutions affecting both alleles were in the β-hairpin. In contrast, mutations specifically affecting rhTRIM5αTFP surround a highly conserved patch of amino acids that is unique to capsids of primate lentiviruses. This "patch" sits at the junction between the binding sites of multiple cellular cofactors (cyclophilin A, Nup-358 cyclophilin A-like domain, Nup-153 and CPSF6). Differential restriction of these alleles is due to a Q/TFP polymorphism in the first variable loop (V1) within the PRYSPRY domain. Q reflects the ancestral state (present in the last common ancestor of Old World primates) and has remained unmodified in all but one lineage of African monkeys, the Cercopithecinae. While Q-alleles can be found among some Cercopithecinae primates, in others Q has been replaced by a G or overwritten by a two amino acid insertion (giving rise to TFP in macaques). In one lineage, the Q to G substitution was later followed by an adjacent 20 amino acid duplication. We found that these modifications in TRIM5α specifically impart the ability to restrict Cercopithecinae SIVs without altering β-hairpin recognition. At least twice Cercopithecinae TRIM5αs independently evolved to target the same conserved patch of amino acids in capsid. Based on these findings, we propose that the β-hairpin is a retrovirus associated molecular pattern widely exploited by TRIM5α proteins, while recognition of the cofactor binding region was driven by the emergence of the ancestors of modern Cercopithecinae SIVs. Distribution on the Cercopithecinae phylogenetic tree indicates that selection for these changes in TRIM5α V1 began 11-16 million years ago, suggesting that primate lentiviruses are at least as ancient.

Thesis (Ph. D.)--University of Washington, 2004. / Vita. Includes bibliographical references (leaves 72-83).

Thesis (Ph. D.)--University of Washington, 2002. / Vita. Includes bibliographical references (leaves 104-125).

Thesis advisor: Welkin E. Johnson / For lentiviruses such as HIV-1, the viral capsid protein (CA) plays a crucial role in replication by facilitating active transport across nuclear pore complexes (NPCs). Nucleoporin Nup358/RanBP2 – a large, multidomain protein that comprises the main component of cytoplasmic NPC filaments – was previously identified as a potential cofactor for HIV-1 nuclear entry, and its C-terminal cyclophilin-like domain (Nup358Cyp) is able to interact with the CA of both HIV-1 and HIV-2. The importance of this interaction to viral replication is unclear though as certain cell-culture experiments suggest CA interaction with Nup358Cyp is dispensable for viral replication, and the CA of several other lentiviruses like SIVmac do not appear to interact with the Nup358Cyp domain. However, we have found that CA interaction with Nup358 is widely conserved among primate lentiviruses and is maintained by natural selection. The exception, SIVmac, likely reflects an evolutionary trade-off allowing escape from rhesus macaque TRIM5Cyp. Together, our observations are strong evidence that the interaction between viral CA and the Nup358Cyp domain must be biologically relevant in vivo. Specifically, by comparing interactions between multiple SIVsm/HIV-2 lineage CAs and several primate orthologs of Nup358, we identified interspecies differences in the Nup358Cyp domain that affect the CA interaction, but only when assayed in conjunction with the preceding Ran-binding domain 4 (Nup358R4). We next found that selection preserves the interaction during cross-species transmission, resulting in adaptation to differences between the Nup358Cyp homologs of the reservoir and spillover hosts. For example, SIVsm CA does not interact with human Nup358R4-Cyp, while HIV-2 CA interacts with both the human and sooty mangabey orthologs. We confirmed these distinct interaction phenotypes in an extended set of SIVsm/HIV-2 CAs, and mapped the difference to a single position – residue 3173 – in the Nup358Cyp domain. The differing ability to interact with human Nup358R4-Cyp is due to residue 85 in the CA 4-5 loops; most SIVsm strains encode a glutamine at position 85, whereas most HIV-2 strains encode an isoleucine. Reciprocal swaps reverse the interaction phenotypes, such that the SIVsm Q85I CA mutant strongly interacts with human Nup358R4-Cyp, while HIV-2 I85Q CA mutant does not. This difference also correlates with differences in single- and multi-cycle infectivity on human cell lines and levels of nuclear import in HeLa cells. Together, these results indicate that HIV-2 adapted to human Nup358 during emergence in humans. We also examined the ability of our CA panel to interact with Cyclophilin A. While all HIV-2 CA interact with CypA, the ability to interact varied among the other SIVsm CA tested, and was absent for SIVpbj. Thus, conservation of CA interaction with Nup358Cyp does not correlate to the ability to interact with CypA, and is not simply a consequence of maintaining the CA-CypA interaction. / Thesis (PhD) — Boston College, 2020. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.

Gp12 est une protéine qui se fixe symétriquement au centre de chacun des 60 hexamères de la capside icosaédrique du bactériophage SPP1. La protéine produite dans un système d’expression hétérologue se lie à la capside de particules virales dont le gène codant gp12 a été inactivé. Cette interaction a lieu spécifiquement avec des capsides qui ont subi le processus d’expansion et encapsulé l'ADN viral.L'analyse de la séquence de gp12 montre la présence d'un motif (GXY)n retrouvé dans des protéines de type collagène. Nous avons démontré que gp12 est un trimère allongé en solution. Ce trimère s'avère sensible à la collagénase VII qui coupe la protéine gp12 dans un site spécifique du motif (GXY)8. Le profil de dichroïsme circulaire de gp12 porte aussi la signature d'une protéine de type collagène. La fixation de gp12 sur la capside virale conduit à une augmentation de 20°C de sa stabilité thermique. Gp12 peut être dénaturée-dissociée et puis renaturée-reassociée sous l'effet de la température. Le trimer de gp12 et sa forme dénaturée se fixent à la capside de SPP1 mais avec des profils d’interaction différents. Ces propriétés permettent d’utiliser gp12 comme un ligand réversible de la capside phagique en fonction de la température. Gp12 a une organisation modulaire avec un motif collagène qui sépare les modules amino et carboxyl-terminaux. Des protéines avec une organisation similaire sont codées par des gènes adjacents à celui codant pour la protéine majoritaire de la capside dans des prophages de Bacilli, suggérant une fonction similaire à gp12. Leurs modules ont une taille variable. Une recherche de protéines procaryotes et virales avec des segments collagène a montré qu’elles sont abondantes parmi les bactéries et les virus. Le motif est rare parmi les archées et leurs virus. Ces résultats montrent l’importance des protéines avec des séquences de type collagène dans le monde non-eucaryote et du développement de leur étude biochimique et fonctionnelle. / Gp12 is a protein found distributed symmetrically at the surface of the icosahedral capsid from bacteriophage SPP1. Recombinant gp12 binds to phage particles whose gene coding for gp12 was disrupted. This interaction occurs specifically with capsids that undergone expansion and packaged DNA.The gp12 protein sequence is marked by the presence of a stretch of 8 repeats of a GXY motif, which is the sequence signature of collagen. Our results showed that gp12 is an elongated trimer in solution. The trimer is sensitive to collagenase VII that cuts the gp12 protein inside the collagen motif. Its circular dichroism profile has also the signature of a collagen-like protein. Binding of gp12 to SPP1 capsids increases its thermal stability by 20°C. Gp12 is denatured and dissociated reversibly by temperature shift. The gp12 trimer and its denatured form bind to SPP1 capsids but with a different interaction behavior. These properties allow to use gp12 as thermo-switchable SPP1 capsid binder. Gp12 has a modular organization with a central collagen motif that connects the amino and carboxyl termini. Proteins with a similar organization that are encoded by genes adjacent to the gene coding for the major capsid protein were identified in prophages of Bacilli, suggesting a function similar to gp12. Their modules have a variable length.A pangenome-wide search for collagen-like proteins in prokaryotes and viruses shows that they are abundant among bacteria and viruses. In contrast, this motif is rare is archaea and their viruses. Our analysis highlights the importance of collagen-like proteins in the non-eukaryotic world and supports the interest to develop their biochemical and structural study.

No description available.

The rubella virus (RUB) capsid protein (C) is a multifunctional phosphoprotein with roles beyond encapsidation. It is able to rescue a large lethal deletion of the Q domain in the P150 replicase gene at a step in replication before detectable viral RNA synthesis, indicating a common function shared by RUB C and the Q domain. The goal of this dissertation was to use constructs containing the N-terminal 88 amino acids of RUB C, the region previously defined as the minimal region required for the rescue of Q domain mutants, to elucidate the function of RUB C in Q domain rescue and viral RNA synthesis. In the first specific aim, the rescue function of 1-88 RUB C and the importance of an arginine-rich cluster, R2, within 1-88 RUB C for rescue were confirmed. Rescue was not correlated with intracellular localization or phosphorylation status of RUB C. In the second specific aim, the involvement of RUB C in early events post-transfection with RUB RNA was analyzed. RUB C specifically protected RUB transcripts early post-transfection and protection required R2. However, it was concluded the protection observed was due to the encapsidation function of RUB C and not related to Q domain rescue. No differences in the translation of the RUB nonstructural proteins in the presence or absence of RUB C were observed. Interactions of RUB C with host cell proteins were analyzed. Although the interaction of RUB C with cellular p32 required the R2 cluster, both wild type (does not require RUB C for replication) and RQQ (requires RUB C for replication) Q domain bound p32, indicating interaction with this binding partner is not the basis of rescue. Using a human protein array phosphatidylinositol transfer protein alpha isoform (PITPα) was found to interact with RUB C but not its R2 mutant. However, co-immunoprecipitation experiments revealed that this protein binds both forms of RUB C. Although the mechanism behind the rescue of the RUB P150 Q domain by RUB C remains unknown, we propose a model that RUB C plays a role in generation of the virus replication complex in infected cells.

No description available.

High-risk human papillomaviruses (HPVs) cause 5% of all human cancers worldwide. The HPV capsid consists of 72 disulfide-linked pentamers of major capsid protein L1 and up to 72 molecules of minor capsid protein L2. The viral genome (vDNA) is 8KB circular dsDNA, condensed with histones and complexed with L2. HPV infection requires the virion particle to get access to basal layer keratinocytes, binding and entry of the cells, uncoating, and transport of the viral genomes to the host cell nucleus. During infection, L2 is important for transport of the viral genome from membrane bound vesicular compartments, through the cytosol and into the host cell nucleus. Previous work has identified a conserved disulfide bond between Cys22 and Cys28, which is necessary for HPV16 infection. We hypothesize that endosomal reduction of this disulfide might be important for L2 conformational changes that allow a hydrophobic transmembrane-like region in L2 to span across endosomal membranes, exposing sorting adaptor binding motifs within L2 to the cytosol. Prior research suggests that cytosolic glutathione (GSH) redox potential is important for reduction of disulfide-linked proteins within the lumen of endosomes. This is achieved by endosomal influx of cytosolic reduced cysteine, where it can reduce disulfide bonds in lumenal proteins. Cytosolic GSH regenerates the pool of reduced cysteine needed to maintain endosomal redox potential. Here we studied the relationship between cytosolic GSH and HPV16 infection. siRNA knockdown of critical enzymes of the GSH biosynthesis pathway or the endosomal cystine efflux pump cystinosin caused partial abrogation of HPV16 infection. Likewise, inhibition of the GSH biosynthesis pathway with L-buthionine sulfoximine (L-BSO) blocked HPV16 infection in multiple cell types, suggesting that cytosolic GSH redox may be important for HPV16 infection. Further studies have revealed that the decrease of HPV16 infection is not because of defects in binding, entry, L2 cleavage or capsid uncoating, but rather is due to inefficient cytosolic translocation of L2/viral genome from the trans-Golgi network (TGN). Contrary to our initial hypothesis, we show that L2 is able to span the endosomal membrane and direct TGN localization in the presence of BSO. Lack of cytosolic GSH causes L2/viral genome to become trapped in the TGN lumen. This suggests that there are redox-sensitive viral or cellular factors necessary for L2/viral genome translocation at the TGN. Future research will focus on the redox state of the Cys22-Cys28 disulfide bond during infection of normal and GSH-depleted cells.