Human endogenous retroviruses in the primate lineage and their influence on host genomes

Primates emerged about 60 million years ago. Since that time various primate-targeting retroviruses have integrated in the germ line of primate species, and some drifted to fixation. After germ line fixation, continued activity of proviruses resulted in intragenomic spread of so-called endogenous retroviruses (ERVs). Variant ERVs emerged, amplified in the genome and profoundly altered genome structures and potentially functionality. Importantly, ERVs are genome modifiers of exogenous origin. The human genome contains about 8% of sequences of retroviral origin. The human ERVs (HERVs) comprise many distinct families that amplified to copy numbers of up to several thousand. We review here the evolution of several well-characterized HERV families in the human lineage since initial germ line fixation. It is apparent that endogenous retroviruses profoundly affected the genomes of species in the evolutionary lineage leading to Homo sapiens.     

Differential sensitivities of retroviruses to integrase strand transfer inhibitors

Integrase inhibitors are emerging anti-human immunodeficiency virus (HIV) drugs, and multiple retroviruses and transposable elements were evaluated here for susceptibilities to raltegravir (RAL) and elvitegravir (EVG). All viruses, including primate and nonprimate lentiviruses, a Betaretrovirus, a Gammaretrovirus, and the Alpharetrovirus Rous sarcoma virus (RSV), were susceptible to inhibition by RAL. EVG potently inhibited all lentiviruses and intermediately inhibited Betaretrovirus and Gammaretrovirus infections yet was basically ineffective against RSV. Substitutions based on HIV type 1 (HIV-1) resistance changes revealed that integrase residue Ser150 contributed significantly to the resistance of RSV. The drugs intermediately inhibited intracisternal A-particle retrotransposition but were inactive against Sleeping Beauty transposition and long interspersed nucleotide element 1 (LINE-1) retrotransposition.     

Pseudotypes of vesicular stomatitis virus with the envelope properties of mammalian and primate retroviruses.

By employing improved techniques it has been possible to produce and characterize a representative spectrum of mammalian and primate retrovirus pseudotypes of vesicular stomatitis virus (VSV). Selection of appropriate cell lines for both the production and subsequent detection of the VSV pseudotypes has been the most important factor in permitting their demonstration. The host range for penetration of these retrovirus pseudotypes of VSV has been defined and found to differ from that reported for the replication of the corresponding retroviruses. Additionally, retroviruses having an identical host range for replication were distinguishable by differences in their host range for penetration, implying that restriction of replication may be occurring by different mechanisms. Studies of the plaque-forming efficiency of retrovirus pseudotypes of VSV in cell lines nonpermissive for replication of the corresponding retroviruses permitted a distinction to be made between the restriction of replication occurring as a consequence of postpenetration events and that occurring as a consequence of a block of penetration itself. The demonstration of primate retrovirus pseudotypes of VSV permits the use of VSV as a probe for the detection of this group of viruses.     

Identification of a new viral protein containing CAp30 and NCp10 sequences in murine and feline leukemia retroviruses.

Because Pr65gag is in part located in the nucleus and contains a putative bipartite nuclear targeting signal, we investigated the cellular location and structure of P55gag, a gag-encoded polyprotein known to lack the nucleocapsid (NC) protein NCp10. P55gag was found to be restricted to the cytoplasm of Moloney murine leukemia virus-infected cells. Of interest, P55gag was produced in cells infected by a viral protease deletion mutant and by a recombinant murine sarcoma virus known to lack the protease gene. Surprisingly, our structural and immunological studies indicated that P55gag also lacks carboxy-terminal residues of CAp30. During the course of studying P55gag, we detected a new viral protein within purified virus particles that contained NCp10 tryptic peptide sequences and a CAp30 tryptic peptide lacking in P55gag. This viral protein, which we have named nucleocapsid-related protein (NCRP), also contained antigenic epitopes present in CAp30 and NCp10. P55gag- and NCRP-like proteins were also observed in AKV murine leukemia virus and feline leukemia virus systems. The precise site of cleavage within Pr65gag that produces P55gag and NCRP is unknown but lies upstream of the CAp30-NCp10 junction within the carboxy-terminal domain of CAp30. The existence of a form of NCp10 containing carboxy-terminal CAp30 sequences raises interesting possibilities about its functional role in genomic RNA packaging and/or viral RNA dimerization.     

A sodium-dependent neutral-amino-acid transporter mediates infections of feline and baboon endogenous retroviruses and simian type D retroviruses

The type D simian retroviruses cause immunosuppression in macaques and have been reported as a presumptive opportunistic infection in a patient with AIDS. Previous evidence based on viral interference has strongly suggested that the type D simian viruses share a common but unknown cell surface receptor with three type C viruses: feline endogenous virus (RD114), baboon endogenous virus, and avian reticuloendotheliosis virus. Furthermore, the receptor gene for these viruses has been mapped to human chromosome 19q13.1-13.2. We now report the isolation and characterization of a cell surface receptor for this group of retroviruses by using a human T-lymphocyte cDNA library in a retroviral vector. Swiss mouse fibroblasts (NIH 3T3), which are naturally resistant to RD114, were transduced with the retroviral library and then challenged with an RD114-pseudotyped virus containing a dominant selectable gene for puromycin resistance. Puromycin selection yielded 12 cellular clones that were highly susceptible to a beta-galactosidase-encoding lacZ(RD114) pseudotype virus. Using PCR primers specific for vector sequences, we amplified a common 2.9-kb product from 10 positive clones. Expression of the 2.9-kb cDNA in Chinese hamster ovary cells conferred susceptibility to RD114, baboon endogenous virus, and the type D simian retroviruses. The 2.9-kb cDNA predicted a protein of 541 amino acids that had 98% identity with the previously cloned human Na+-dependent neutral-amino-acid transporter Bo. Accordingly, expression of the RD114 receptor in NIH 3T3 cells resulted in enhanced cellular uptake of L-[3H]alanine and L-[3H]glutamine. RNA blot (Northern) analysis suggested that the RD114 receptor is widely expressed in human tissues and cell lines, including hematopoietic cells. The human Bo transporter gene has been previously mapped to 19q13.3, which is closely linked to the gene locus of the RD114 receptor.     

Differential expression in human and mouse cells of human immunodeficiency virus pseudotyped by murine retroviruses.

Expression of cell surface CD4 influences susceptibility of cells to human immunodeficiency virus (HIV) infection; however, some CD4-positive human and mouse cells are still resistant to HIV infection. To search for mechanisms of resistance to HIV independent of CD4 expression, HIV expression was studied in human and mouse cells normally resistant to HIV infection by introducing infectious virus by transfection of HIV DNA or infection with HIV pseudotyped with amphotropic or polytropic murine leukemia viruses. The results indicated that even when barriers to viral entry were bypassed, mouse NIH 3T3 cells and Dunni cells still showed a marked reduction in number of cells expressing HIV compared with the human cells studied, although the intensity of immunostaining of individual positive mouse cells was indistinguishable from that seen on permissive human cell lines. CD4 expression in mouse cells or human brain or skin cells did not influence the number of HIV foci observed after transfection with HIV DNA or infection with pseudotyped HIV. These results suggested that in addition to a block in the usual HIV fusion and entry process, CD4-positive mouse cells differed from human cells in exhibiting partial resistance to HIV infection which acted at a postpenetration step in the infection cycle. This resistance was partially overcome when mouse cells were infected by direct exposure to human lymphocytes producing HIV pseudotyped by amphotropic murine leukemia virus.     

A journey with T cells, primate/human retroviruses and other persisting human T-cell tropic viruses

A study of the growth of primate/human T cells led to mechanisms for temporary laboratory culture of these cells (discovery of interleukin-2) and also their continuous culture (by immortalization after infection with human T-cell lymphotropic virus type 1 or 2 (HTLV-1 or 2)). Cultures of lymphocytes also led us to isolate five persisting T-tropic viruses: 1. the Hall's Island strain of gibbon ape leukemia virus, 2. HTLV-1, 3. HTLV-2, 4. human immunodeficiency virus and 5. human herpes virus-6 (HHV-6). This report is a brief synopsis of the discoveries of the first human retroviruses, the HTLV.     

灵长类动物中TRIMCyp融合基因模式及对逆转录病毒复制的限制作用

TRIM5-CypA融合基因(TRIMCyp)是一种独特的TRIM5基因形式。迄今已发现新大陆猴中包括鹰猴在内的夜猴属所有代表种,以及在北平顶猴、巽他平顶猴、食蟹猴、印度恒河猴和熊猴等旧大陆猴中均存在这种基因融合现象,但在新大陆猴与旧大陆猴中的TRIMCyp融合基因的基因融合模式和表达剪接方式不同。新大陆猴TRIMCyp融合基因是由CypA假基因的cDNA序列通过LINE-1逆转座子介导的逆转座方式插入至TRIM5α基因的第7和第8外显子之间的内含子中形成,而旧大陆猴TRIMCyp融合基因则是由CypA假基因的cDNA序列以相似的逆转座方式插入至TRIM5基因的3’非翻译区(untranslatedregions,UTR)形成。TRIMCyp融合基因在不同灵长类动物中的存在比例、基因型、TRIMCyp融合蛋白的表达以及对逆转录病毒的限制活性均有所差异。鹰猴和平顶猴的TRIMCyp融合基因研究较多,鹰猴TRIMCyp融合蛋白可能以与TRIM5α相似机制限制HIV-1的感染,而平顶猴TRIMCyp融合蛋白则丧失了限制HIV-1的作用。这两个功能截然不同的融合基因为TRIM5α作用机制研究提供了难得的实验材料,也为建立HIV-1感染的新型灵长类动物艾滋病模型奠定了科学依据。该文综述了TRIMCyp融合基因在灵长类动物中的分布、存在形式及其限制逆转录病毒复制的作用机制等方面的研究情况。     

The regulation of genes and genomes by small RNAs

A recent Keystone Symposium on 'MicroRNAs and siRNAs: Biological Functions and Mechanisms' was organized by David Bartel and Shiv Grewal (and was held in conjunction with 'RNAi for Target Validation and as a Therapeutic', organized by Stephen Friend and John Maraganore). The 'MicroRNAs and siRNAs' meeting brought together scientists working on diverse biological aspects of small regulatory RNAs, including microRNAs, small interfering RNAs (siRNAs) and Piwi-interacting RNAs (piRNAs and rasiRNAs). Among the themes discussed were the diversity of small regulatory RNAs and their developmental functions, their biogenesis, the identification of their regulatory targets, their mechanisms of action, and their roles in the elaboration of multicellular complexity.     

Gene transfer, speciation, and the evolution of bacterial genomes.

Studies in microbial evolution have focused on the origin and vertical transmission of genetic variation within populations experiencing limited recombination. Genomic analyses have highlighted the importance of horizontal genetic transfer in shaping the composition of microbial genomes, providing novel metabolic capabilities, and catalyzing the diversification of bacterial lineages.     

Comparative analysis of the genomes of feline leukemia viruses.

The genomes of several strains of feline leukemia virus (FeLV) were compared by two-dimensional polyacrylamide gel electrophoresis of the large RNase T1-resistant oligonucleotides of the 70S RNA. Differences between each strain of FeLV tested were detected by this method. We estimate that the degree of sequence identity between the viruses is: FeLV A (Glasgow-1) to FeLV B (Snyder-Theilen), 52%; FeLV A (Glasgow-1) to FeLV C(Sarma), 66%; FeLV B(Snyder-Theilen) to FeLV C (Sarma), 37%. The fingerprints of two independent isolates of FeLV strains of subgroup A (Glasgow-1 and Rickard) were detectably different. We conclude that the RNase T1 oligonucleotide fingerprint pattern provides a useful tool for identification of FeLV strains.     

Protein coding potential of retroviruses and other transposable elements in vertebrate genomes

We suggest an annotation strategy for genes encoded by retroviruses and transposable elements (RETRA genes) based on a set of marker protein domains. Usually RETRA genes are masked in vertebrate genomes prior to the application of automated gene prediction pipelines under the assumption that they provide no selective advantage to the host. Yet, we show that about 1000 genes in four vertebrate gene sets analyzed contain at least one RETRA gene marker domain. Using the conservation of genomic neighborhood (synteny), we were able to discriminate between RETRA genes with putative functionality in the vertebrates and those that probably function only in the context of mobile elements. We identified 35 such genes in human, along with their corresponding mouse and rat orthologs; which included almost all known human genes with similarity to mobile elements. The results also imply that the vast majority of the remaining RETRA genes in current gene sets are unlikely to encode vertebrate functions. To automatically annotate RETRA genes in other vertebrate genomes, we provide as a tool a set of marker protein domains and a manually refined list of domesticated or ancestral RETRA genes for rescuing genes with vertebrate functions.     

Structural studies of retroviruses: characterization of oligomeric complexes of murine and feline leukemia virus envelope and core components formed upon cross-linking.

To examine the protein proximity and subunit organization of type C retroviruses, preparations of AKR murine leukemia virus were treated with bifunctional cross-linking reagents and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The cross-linked components obtained were characterized by immunoprecipitation with monospecific antisera against purified viral proteins, followed by SDS-PAGE analysis both before and after cleavage of the cross-links. With these procedures, complexes of both viral envelope and core components were identified. The major envelope subunit obtained was a large (apparent molecular weight of 450,000 to 500,000), glycosylated complex, composed of four to six gp70-p15(E) subunits. This complex was detected over a 100-fold range of cross-linker concentration and thus seems to represent a particularly stable viral substructure. The cross-linked complexes of the core proteins consisted of oligomers of p30 dimers, suggesting that the p30 dimer is a basic structural unit of the viral core. When virion preparations, which had previously been disrupted with the nonionic detergent Nonidet P-40, were cross-linked, the envelope complex was still observed, indicating that this structure is stable in the presence of Nonidet P-40. A similar envelope structure was observed for feline leukemia virus, suggesting that such a complex may be a conserved feature of oncornavirus structure.     

Differential transmission of the Cucumis organellar genomes

 Although plants generally show maternal transmission of the organellar genomes, previous research has demonstrated that the mitochondrial (mt) genome of cucumber is paternally transmitted. In this study, we identified RFLPs in the organellar genomes of melon, squash, and watermelon to establish organellar DNA transmission. Serial dilutions of DNA demonstrated that our hybridizations revealed the presence of a polymorphic cytoplasm when it represented at least 1% of the DNA sample. At this level of sensitivity, the chloroplast genomes of melon, squash, and watermelon were maternally transmitted. The mitochondrial genomes of squash and watermelon were maternally transmitted; however, melon, like cucumber, showed paternal transmission of the mitochondrial genome. Because most angiosperms and the related genera Cucurbita and Citrullus show maternal transmission of the mtDNA, paternal transmission in Cucumis is likely the derived state. The Cucumis mitochondrial genomes are several-fold larger than those of other cucurbits. Based on 55 probe-enzyme combinations, mtDNA size differences could not be explained by duplication of the entire genome or partial duplication of regions hybridizing with the mitochondrial probes. Because the chloroplast, mitochondrial, and nuclear genomes of Cucumis are differentially transmitted, this genus is an excellent system to study the role of intergenomic transfer in the evolution of extremely large mitochondrial genomes. Received: 20 November 1997 / Accepted: 30 December 1997