京都大学 / 新制・課程博士 / 博士(医学) / 甲第24524号 / 医博第4966号 / 新制||医||1065(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 齊藤 博英, 教授 萩原 正敏, 教授 山崎 渉 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

No description available.

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

Human endogenous retroviruses are the remnants of ancient retroviral infections present within our genome. These molecular fossils show similarities with present day exogenous retroviruses but act as typical Mendelian elements that are passed vertically between generations. Despite being repeatedly linked to a number of autoimmune diseases and disorders, no conclusive proof has been identified. Rheumatoid arthritis (RA) is one such disease which has been associated with an increase in HERV expression, compared to controls. In order to elucidate a clear role for HERVs in RA pathogenesis, autoantigens implicated in disease pathogenesis were scanned for sequence homology to retroviral genes. Such epitopes would induce antibodies cross reactive with host proteins, resulting in disease. Short peptides mimicking these regions were synthesised and the prevalence of anti-HERV antibodies was determined in RA patients and disease controls. Additionally, a novel real-time Polymerase Chain Reaction (PCR) assay was developed to accurately quantify levels of HERV-K (HML-2) gag expression, relative to normalised levels of housekeeping gene expression. Both serological and molecular assays showed significant increases in HERV-K (HML-2) activity in RA patients compared to disease controls with CD4+ lymphocytes harbouring the highest activity. The real-time assay was also used to determine whether factors within the synovium could modulate HERVs, resulting in their upregulation. Exogenous viral protein expression and pro-inflammatory cytokines were shown to exert a significant modulatory effect over HERV-K (HML-2) transcription. From this data, it is clear that RA patients have increased levels of HERV-K (HML-2) gag activity compared to controls. Despite this it is likely that factors within the synovium such as exogenous viral expression and pro-inflammatory cytokines also influence HERV-K (HML-2) transcription possibly contributing to a role of bystander activation, i.e. being influenced by external factors, rather than actively contributing to disease processes. The exact role of HERVs in RA pathology remains elusive; however this research proposes several mechanisms by which HERV-K (HML-2) may contribute to disease.

Thesis advisor: Welkin Johnson / Endogenous Retroviruses (ERVs) are “fossilized” retroviruses of a once exogenous retrovirus located in the genome of extant vertebrates. Retroviral infection results in a provirus integration into the host genome. An infection of a germline cell could lead to the provirus potentially being inherited by the offspring of the infected individual. Once in the genome, the provirus becomes subject to evolutionary processes and can become either lost or fixed in a population, remaining as “fossils” long after the exogenous retrovirus has gone extinct23. Notably, 8% of the human genome consists of ERVs30. Human Endogenous Retrovirus Type K (HERV-K)(HML-2) family is of particular interest. HERV-K integrations are as old as 30-35 million years, endogenizing before the separation of humans and Old World Monkeys. However, there are human specific insertions, some as young as 150,000 – 250,000 years, making them the youngest insertion in the human genome. There are over 90 insertions in the human genome; the bulk is shared by all humans44,47. Transcripts of HERV-K genes are upregulated in multiple cancer and tumor cell lines 14,39,46, as well as in HIV-1 infected patients 7,11,29. Just as there are human specific insertions of ERV-K, there are also Old World Monkey specific insertions44. I have identified an intact endogenous retroviral envelope open reading frame on chromosome 12 of the rhesus macaque genome. This viral envelope-encoding sequence, which I refer to as rhERV-K env, retains all the canonical features of a retroviral Env protein. An alignment between rhERV-K env and a consensus sequence of HERV-K, HERV-Kcon env, shows a 70% amino acid sequence identity. For experimental purposes, reconstructed HERV-K envelopes have been incorporated into virions of Human Immunodeficiency virus (HIV-1)19,26,49, Murine Leukemia Virus (MLV)12, and Vesicular stomatitis Virus (VSV)26,41,49. While these approaches have illuminated some aspects of HERV-K Env-mediated entry, to date a cell-surface receptor has not been identified for any ERV-K Env. This could be due to its low infectivity levels12,26,49, its seemingly broad cell tropism limiting identification of null cell lines26,49, or possibly the HERV-K consensus reconstructions are not an accurate representation of the progenitor HERV-K virus. I am interested in understanding how the ERV-K retrovirus accessed the human germline (some 150,000 – 250,000 years ago). To do this, I focused specifically on the envelope proteins of HERV-K and rhERV-K, with the goal of analyzing the ERV-K entry process. The identification and inclusion of rhERV-K Env in this study is meant to circumvent the possibility that the previously described consensus reconstructions of human HERV-K Env are not representative, and may also provide a means to compare the endogenization process in the human/ape and old-world monkey lineages. I focused on developing two systems for single-cycle infection, one based on Mason-Pfizer Monkey Virus (MPMV) (which has not been done before), and a second based on MLV, which has previously been reported on. MPMV, like HERV-K, is a betaretrovirus, and I reasoned that possibly using a betaretrovirus would overcome some of the low-infectivity issues associated with prior attempts using HIV and MLV. To develop a system for examining function of the ERV-K Env proteins, I addressed 3 issues: 1. Are the HERV-K Env and rhERV-K Env proteins expressed and properly processed? 2. Can they be incorporated into virions of a heterologous virus? 3. Are ERV-K pseudotyped virions infectious? I have answered these questions in the following thesis. / Thesis (MS) — Boston College, 2017. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.

Endogenous viral elements, or viral genomic fossils, have proven extremely valuable in the study of the macroevolution of viruses, providing important, and otherwise unobtainable, insights into the ancient origin of viruses, and how their ancestors might have co-evolved with their hosts in the distant past. This type of investigation falls within the realm of paleovirology—the study of ancient viruses. Investigations of extant viruses and paleovirological analyses, however, often give conflicting results, especially those concerning viral evolutionary rates and timescales. Reconciling these two types of analyses is a necessary step towards a better understanding of the overall long-term evolutionary dynamics of viruses. The main study system of this thesis is foamy viruses (FVs). FVs are characterised by their stable co-speciation history with their hosts, allowing their evolutionary dynamics to be modelled and investigated over various timescales. This unique evolutionary feature makes FVs one of the best subjects for connecting recent and ancient viral evolution. The work here reports the discovery of several endogenous mammalian FVs, and examines how mammalian FVs co-evolve with their hosts. Analyses reveal a co-diversifying history of the two that could be dated back to the basal radiation of eutherians more than 100 million years ago. However, a small number of ancient FV cross-species transmissions could still be found, mostly involving New World monkey FVs. Based on this extended FV-mammal co-speciation pattern, this thesis investigates the long-term evolutionary rate dynamics of FVs, and shows that the rate estimates of FV evolution appear to decrease continuously as the rate measurement timescale increases, following a power-law decay function. The work presented here also shows that this so-called 'time-dependent rate phenomenon' is in fact a pervasive evolutionary feature of all viruses, and surprisingly, the rate estimates of evolution of all viruses seem to decay at the same speed, decreasing by approximately half for every 3-fold increase in the measurement timescale. Based on this power-law rate-decay pattern, we could infer evolutionary timescales of modern-day lentiviruses that are consistent with paleovirological analyses for the first time. Finally, this thesis reports the discovery of basal FV-like endogenous retroviruses (FLERVs) in amphibian and fish genomes. Phylogenetic analyses reveal that the progenitors of ray-finned fish FLERVs co-diversify broadly with their fish hosts, but also suggest that there might have been several ancient viral cross-class transmissions, involving lobe-finned fish, shark, and frog FLERVs. Again, by using the power-law rate-decay model, analyses in this thesis suggest that this major retroviral clade has an ancient Ordovician marine origin, originating together with their jawed vertebrate hosts more than 450 million years ago. This finding implies that the origin of retroviruses as a whole must be in the early Paleozoic Era, if not earlier. The results presented here bridge ancient and recent viral evolution.

Made available in DSpace on 2016-04-28T20:16:25Z (GMT). No. of bitstreams: 1 2008- Daniel de Barros Macieira.pdf: 1044606 bytes, checksum: 797036e36833a30609c06a68880f37c8 (MD5) Previous issue date: 2008-02-28 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior / Infectious diseases present high morbidity among domestic cats and a delayed or misdiagnosis may result in patient death. The association between feline immunodeficiency (FIV) and/or feline leukemia (FeLV) viruses infections with hemoplasmas has great importance in veterinary practice, since agents may act synergically increasing risks for infected animals. To date, there are no studies in Brazil critically examining whether or not hemoplasma infections are associated with retroviral infection in cat. Thus, the aim of this study was to determine the prevalence and risk factors for Mycoplasma haemofelis (Mhf) and Candidatus Mycoplasma haemominutum (Mhm) infections in domestic cats tested for FIV and FeLV with a commercial ELISA kit. Based on serologic testing, cats were grouped as i) FIV-positive (n=25); ii) FeLV-positive (n=39); iii) FIV/FeLV-positive (n=8); and iv) FIV/FeLV-negative (n=77), or base-comparison group. CBCs were followed by DNA extraction, species-specific PCR (16S rRNA gene) for Mhf and Mhm and Southern blotting for all animals. Among all animals tested for retroviral infection (n=149), 16,8%, 26,2% and 5,4% were seropositive for FIV, FeLV and both retroviruses respectively. Associated factors to retroviral infections included an increased mean age (FIV, p<0,001), neutered status and history of fights/bites (FIV, p=0,033 and <0,001 respectively); presence of fleas (FeLV, p=0,018); history of blood transfusions and roaming outdoors (FIV/FeLV, p=0,028 and 0,033 respectively). Among evaluated clinical signs, anorexia and pale mucous (FIV, p=0,004 and 0,034 respectively); lethargy/depression, jaundice and tachycardia (FeLV, p=0,033, 0,036 and 0,003 respectively) were identified. Among CBC findings, anemia was related to FIV (p=0,012) and FeLV (p<0,001) infections, whereas thrombocytopenia was only related to FIV infection (p=0,015). Animals with FIV or double FIV and FeLV infections had a reduced WBC count (p=0,021 and 0,012 respectively). Mhf DNA was found in 4.0%, 2.6%, 12.5% and 7.8% of the cats from groups i, ii, iii and iv respectively, while 32.0%, 5.1%, 50.0% and 5.2% of these animals had a Mhm infection. Host factors that were associates to the presence of hemoplasma DNA included the retroviral status (p=0,001), history of fights/bites (p=0,003), roaming outdoors (p=0,001), living with other cats (p=0,008) and pale mucous (p=0,033). The logistic regression model used herein showed that cats with FIV (OR=4,25; p=0,009) and both FIV and FeLV (OR=7,56; p=0,014) were at greater risk of being hemoplasma infected than retroviral negative cats, mainly due to Mhm infection (OR=8,59, p=0,001 and OR=18,25, p=0,001 respectively). Among pure-breed cats, FIV-positive status was associated with hemoplasma infection (OR 45,0; p=0,001). / As doen?as infecto-contagiosas possuem uma alta morbidade entre os gatos dom?sticos e um diagn?stico impreciso ou retardado pode resultar no ?bito do paciente. A associa??o entre infec??es pelos v?rus da imunodefici?ncia (FIV) e/ou leucemia (FeLV) felina com hemoplasmas possui grande import?ncia na cl?nica m?dica, uma vez que os agentes podem agir de modo sin?rgico elevando os riscos para os animais acometidos. Devido ao fato de inexistirem no Brasil estudos que analisem criticamente a associa??o entre estes retrov?rus e os hemoplasmas, o presente trabalho teve como objetivo determinar a preval?ncia e poss?veis fatores associados para as infec??es por Mycoplasma haemofelis (Mhf) e Candidatus M. haemominutum (Mhm) em gatos dom?sticos, testados para a presen?a de FIV e FeLV, atrav?s de um kit comercial baseado na metodologia de ELISA. Com base no teste sorol?gico, os gatos foram agrupados como i) FIV-positivos (n=25); ii) FeLV-positivos (n=39); iii) FIV/FeLV-positivos (n=8); e iv) FIV/FeLV-negativos (n=77), ou grupo de refer?ncia para as compara??es. Hemogramas foram seguidos da extra??o/purifica??o de DNA, rea??o em cadeia da polimerase esp?cie-espec?fica para a detec??o de Mhf e Mhm e Southern Blot/hibridiza??o (SB) para todos os animais. Entre os 149 animais testados, 16,8%, 26,2% e 5,4% foram soropositivos para FIV, FeLV e ambos os retrov?rus respectivamente. Fatores associados ? infec??o por retrov?rus inclu?ram idade m?dia mais elevada (FIV, p<0,001), condi??o reprodutiva castrado e hist?rico de brigas/mordidas (FIV, p=0,033 e <0,001 respectivamente); presen?a de pulgas (FeLV, p=0,018); transfus?o de sangue e acesso ao ambiente externo (FIV/FeLV, p=0,028 e 0,033 respectivamente). Dentre as altera??es cl?nicas avaliadas, anorexia e mucosas p?lidas (FIV, p=0,004 e 0,034 respectivamente); letargia/depress?o, icter?cia e taquicardia (FeLV, p=0,033, 0,036 e 0,003 respectivamente) foram identificadas. Dentre os par?metros hematol?gicos a anemia esteve associada a presen?a de FIV (p=0,012) e FeLV (p<0,001) enquanto a trombocitopenia esteve apenas associada a infec??es por FIV (p=0,015). Animais com FIV ou com dupla infec??o (FIV e FeLV) tiveram uma contagem de leuc?citos reduzida (p=0,021 e 0,012 respectivamente). O DNA de Mhf foi achado em 4,0%, 2,6%, 12,5% e 7,8% dos gatos dos grupos i, ii, iii e iv, respectivamente, enquanto 32,0% 5,1%, 50,0% e 5,2% desses animais tinham infec??es por Mhm. Fatores inerentes ao hospedeiro que apresentaram associa??o com a presen?a do DNA de hemoplasmas inclu?ram o estado retroviral (p=0,001), hist?rico de brigas/mordidas (p=0,003), acesso ao ambiente externo (p=0,001), coabitar com outros gatos (p=0,008) e mucosas p?lidas (p=0,033). O modelo de regress?o log?stica usado mostrou que gatos com FIV (OR=4,25, p=0,009) e animais duplamente infectados para FIV e FeLV (OR=7,56, p=0,014) estavam em maior risco de serem infectados por hemoplasmas do que animais negativos para ambos os retrov?rus, principalmente em virtude de infec??es por Mhm (OR=8,59, p=0,001 e OR=18,25, p=0,001 respectivamente). Entre os animais de ra?a definida, a positividade para FIV estava associada com infec??es para hemoplasmas (OR=45, p=0,001).

A imunidade inata na interface materno-fetal é um dos mecanismos de proteção essenciais na resposta antiviral, sobretudo, na infecção por HIV-1. Neste trabalho, avaliamos a influência da infecção materna por HIV-1 na expressão de fatores antivirais, nas moléculas do complexo inflamassoma e de retrovírus endógenos (HERV), em células mononucleares (CMN) maternas, células do cordão umbilical (recém-natos, RN), colostro e no tecido placentário. Os resultados mostraram que no vilo das placentas de mães infectadas há um aumento na expressão de mRNA dos fatores antivirais, como IFN tipo I e III, DAMPs damage-associated molecular patterns e HERVs. Entretanto, na análise proteica, essas diferenças não se confirmam, indicando que o sistema imune inato é capaz de reconhecer o vírus, ou ainda, o dano causado pela infecção, mas controla a produção exacerbada da proteína. Sob estímulo de agonista de TLRs Toll-like receptor, em CMNs a expressão de HERVs não se altera entre RNs. Além disto, avaliando os níveis de &#223-quimiocinas, CCL5 e CCL3, e de IFN-&#967 nos sobrenadantes das culturas de CMNs, a ativação por LPS foi capaz de diminuir a produção de CCL3 e CCL5 nas CMNs de mães infectadas por HIV em relação às mães controles, contudo o CL097 promoveu níveis similares às mães do grupo controle. Nos RNs, enquanto os níveis de CCL5 são inferiores aos de adultos, os níveis de CCL3 são semelhantes. Já o ligante TLR7/8 foi capaz de restaurar a secreção de IFN-&#945 no grupo infectado por HIV-1. Além disso, no vilo das placentas das mães infectadas por HIV, há intensa modificação na expressão de mRNA dos fatores analisados, sejam antivirais, como IFN tipo I (IFN-&#945), tipo III (IFN-&#955), fatores relacionados ao dano celular (DAMPs e seus receptores). Entre os DAMPs, um aumento da expressão de S100A9 e HMGB1 e seus receptores RAGE, TLR4 e TLR9 foi observado nos vilos de placentas de mães infectadas por HIV-1, contudo, os níveis séricos de HMGB1 estão diminuídos em mães infectadas e RN expostos. Quanto aos níveis séricos de citocinas, foram observados níveis reduzidos de HMGB1, IL-6 e IL-1&#223 nos RNs expostos, o que evidencia um controle do estado inflamatório na exposição ao HIV-1. Também observamos presença de níveis séricos de HERV-W, livre ou em exossomas, em ambos os grupos analisados. Já no colostro, não encontramos diferenças nas análises de fatores inflamatórios e HERVs indicando que, nesse compartimento, a infecção não altera os padrões de expressão desses alvos. A vigência do estado antiviral e a supressão do ambiente inflamatório podem equilibrar a resposta imune placentária, promovendo a homeostase para o desenvolvimento do feto e de proteção à infecção por HIV-1 nos neonatos. / Innate immunity at the maternal-fetal interface is one of the main protection mechanisms in the antiviral response, especially in HIV-1 infection. In this work, we present the influence of maternal HIV-1 infection on the expression of antiviral factors, inflammasome molecular complex and human endogenous retroviruses (HERV), in maternal mononuclear cells (MNCs), umbilical cord cells (newborns, NB), colostrum and placental tissue. The results show that in infected mothers cells has an increase in mRNA expression of antiviral factors, such as IFN type I and III, DAMPs (damage-associated molecular patterns) and HERVs. However, in protein analysis, these differences are not confirmed, indicating that the immune system is able to detect the virus, or even the damage caused by the infection, but controls the exacerbated protein production. Under stimulation of the TLR (Toll-like receptor) agonist, CMNs do not change among the RNs. In addition, by evaluating the levels of &#223-chemokines, CCL5 and CCL3, and of IFN-&#945 in the supernatants of CMNs cultures, LPS activation was able to decrease the production of CCL3 and CCL5 in CMNs of HIV-infected mothers compared to control mothers, nevertheless CL097 promoted similar levels between HIV-infected mothers and control group. In RNs, while CCL5 levels are lower than in adults, CCL3 levels are similar. TLR7/8 agonist was able to restore IFN- secretion in the HIV-infected group. In contrast, the TLR7/8 agonist was able to restore IFN- secretion in HIV-infected group. In addition, in placental villi, there is intense modification in the mRNA expression of the analyzed factors, whether they are antiviral, such as IFN type I (IFN-&#945), type III (IFN-&#955), related to cell damage (DAMPs and their receptors). Among DAMPs, increased expression of S100A9 and HMGB1 and their receptors RAGE, TLR4 and TLR9 was observed in placental villi of HIV-infected mothers, however, serum HMGB1 levels are decreased in infected-mothers and exposed-newborns. About the cytokines serum levels, reduced levels of HMGB1, IL-6 and IL-1&#223 were observed in the exposed-NBs, which evidences an inflammatory status control in HIV-1 exposure. We also observed the presence of free HERV-W or exosomes levels in serum in both groups analyzed. In colostrum, we did not find differences in inflammatory factors and HERVs analysis indicating that, in this compartment, the infection does not alter the expression patterns of these targets. The effectiveness of antiviral status and suppression of the inflammatory environment can balance the placental immune response, promoting homeostasis for fetal development and protection of HIV-1 infection in neonates.

The initial focus of this PhD project was on comparative gene mapping. Comparative gene mapping is facilitated by consensus PCR primers which amplify homologous gene fragments in many species. As a part of an international co-ordinated programme of comparative mapping in pigs, 47 CATS (Comparative Anchor Tagged Sequence) consensus primer pairs for loci located on human chromosomes 9, 10, 20, and 22, were used for amplifying homologous loci in pigs. After optimization of PCR conditions, 23 CATS products have confirmed by comparison with homologous sequences in GenBank. A French somatic cell hybrid panel was used to physically map the 6 porcine CATS products distinguishable from rodent background product, namely ADRA1A, ADRA2A, ARSA, GNAS1, OXT and TOP1. Of these, the map location of ADRA1A and OXT showed inconsistency with the previously recognised conserved relationship between human and pig. The other four loci mapped to positions consistent with known syntenic relationships. Despite low levels of polymorphism, frequently indistinguishable rodent and porcine products in somatic hybrids and some confusion of identity of gene family members, these CATS primers have made a useful contribution to the porcine-human comparative map. The focus of the project then changed to genetic and molecular characterisation of endogenous retroviruses in pigs and their relatives. Pigs are regarded as a potentially good source of organs and tissues for transplantation into humans. However, porcine endogenous retroviruses have emerged as a possible problem as they can infect cultured human cells. Two main types of pig retrovirus, determined by envelope protein, PERV-A and PERV-B, are widely distributed in different pig breeds and a third less common type, PERV-C, has also been recognised. Endogenous retroviruses were analyzed from the Westran (Westmead transplantation) inbred line of pig, specially bred for biomedical research. Thirty-one 1.8 kb env PCR product clones were sequenced after preliminary screening with the restriction enzymes KpnI and MboI. Five recombinant clones between A and B were identified. 55% of clones (17/31) sequenced had stop codons within the envelope protein-encoding region, which would prevent the retrovirus from making full-length envelope protein recognizable by cell-surface receptors of the virus. The endogenous viruses were physically mapped in Westran pigs by FISH (Fluorescence In Situ Hybridisation) using PERV-A and PERV-B envelope clones as probes. Preliminary FISH data suggest that there are at least 22 PERVs (13 PERV-A and 9 PERV-B) and the chromosomal locations of these in the Westran strain are quite different from European Large White pigs. The sequences and mapping results of inbred Westran pig suggest that there are relatively few PERV integration sites compared with commercial pigs and further that a large proportion of clones are defective due to premature stop codons in the envelope gene. To investigate the relationship of endogenous retroviruses in peccaries and pigs, a set of degenerate primers was used to amplify peccary retroviral sequences. The sequences of two putative retroviral clones showed close homology, albeit with a 534 bp deletion, to mouse and pig retroviral sequences. Also, four non-target sequences were amplified from peccary with the degenerate retroviral primers. They are a part of the peccary cofilin gene, a SINE, and a sequence containing a microsatellite. The peccary endogenous retroviral sequences are significant in that they are the first such sequences reported in peccary species and repudiate old claims in the literature that peccaries do not have C-type retroviral sequences.

The most common gene transfer vehicle used in gene therapy protocols are mammalian virus vectors. Specifically, retroviruses are one of the most common viral vectors used since they are able to permanently integrate their transgene into the host cell genome, providing, in principal, to a long-term cure. The potential applications of gene therapies are vast, ranging from monogenic disorders such as cystic fibrosis to complex gene disorders such as cancer. However, the application of such therapies in clinical settings has been limited partially because of inefficient gene delivery of the therapeutic gene to diseased cells. Furthermore, safety concerns of accidently altering the genetic expression in healthy bystander cells or nearby tissue has increased the interest in creating targeted viral vectors which infect only the diseased cells without infecting others. Thus, the success of gene therapy will depend on identifying and understanding the parameters critical for virus entry into cells, including factors that facilitate virus absorption onto the cell surface, virus binding, and fusion. The objective of this thesis was to understand the role of lipids in binding and infection, and to investigate the use of lipid-based conjugates to alter the surface of virus particles.