The role of in vivo long terminal repeat (LTR) sequence variation of the lentivirus equine infectious anemia virus (EIAV) has not been explored. In this study, we investigated the heterogeneity found in the LTR sequences from seven EIAV-seropositive horses: three horses with clinical disease and four horses without any detectable signs of disease. LTR sequences were targeted in this study because the LTR U3 enhancer region of tissue culture-derived isolates has been identified as one of the few hypervariable regions of the EIAV genome. Furthermore, LTR variation may regulate EIAV expression in vivo. Both intra- and interanimal sequence variations were investigated. The intra-animal variation was low in seropositive, healthy horses (on average 0.44%). Intra-animal variation was consistently higher in clinically ill horses (0.99%), suggesting that greater numbers of quasispecies of EIAV are present when active virus replication is ongoing. Interanimal comparisons of consensus sequences generated from each horse demonstrated that the enhancer region is a hotspot of sequence variation in vivo. Thirty-seven of the 83 nucleotides that compose the U3 enhancer region were variable between the different in vivo-derived LTRs. The remainder of the LTR that was analyzed was more conserved, 8 of 195 nucleotide positions being variable. Results of electrophoretic mobility shift assays demonstrated that some nucleotide substitutions that occurred in the enhancer region eliminated or altered transcription factor binding motifs that are known to be important for EIAV LTR expression. These data suggested that the selective pressures exerted on the EIAV LTR enhancer sequences are different from those exerted on the remainder of the LTR. Our findings are consistent with the possibility that enhancer sequence hypervariability can alter expression of the virus in tissue macrophages and therefore contribute to clinical disease in infected horses.

The V3 region of the human immunodeficiency virus type 1 (HIV-1) envelope protein is known to have a major influence on macrophage tropism as well as the ability to cause syncytium formation or fusion in CD4-positive lymphocyte cultures. Using infectious molecular HIV-1 clones, a series of mutant clones was created which allowed detailed mapping of V3 amino acid positions involved in these properties. In these experiments the non-syncytium-inducing phenotype in T cells did not always correlate with macrophage tropism. Macrophage tropism appeared to depend on the presence of certain combinations of amino acids at five specific positions within and just outside of the V3 loop itself, whereas syncytium formation in lymphocytes was influenced by substitution of particular residues at two to four positions within V3. In most cases, different V3 amino acid positions were found to independently influence macrophage tropism and syncytium formation in T cells and position 13 was the only V3 location which appeared to simultaneously influence both macrophage tropism and syncytium formation in lymphocytes.

Aleutian mink disease parvovirus (ADV) DNA was identified by PCR in samples from mink and raccoons on commercial ranches during an outbreak of Aleutian disease (AD). Comparison of DNA sequences of the hypervariable portion of VP2, the major capsid protein of ADV, indicated that both mink and raccoons were infected by a new isolate of ADV, designated ADV-TR. Because the capsid proteins of other parvoviruses play a prominent role in the determination of viral pathogenicity and host range, we decided to examine the relationship between the capsid protein sequences and pathogenicity of ADV. Comparison of the ADV-TR hypervariable region sequence with sequences of other isolates of ADV revealed that ADV-TR was 94 to 100% related to the nonpathogenic type 1 ADV-G at both the DNA and amino acid levels but less than 90% related to other pathogenic ADVs like the type 2 ADV-Utah, the type 3 ADV-ZK8, or ADV-Pullman. This finding indicated that a virus with a type 1 hypervariable region could be pathogenic. To perform a more comprehensive analysis, the complete VP2 sequence of ADV-TR was obtained and compared with that of the 647-amino-acid VP2 of ADV-G and the corresponding VP2 sequences of the pathogenic ADV-Utah, ADV-Pullman, and ADV-ZK8. Although the hypervariable region amino acid sequence of ADV-TR was identical to that of ADV-G, there were 12 amino acid differences between ADV-G and ADV-TR. Each of these differences was at a position where other pathogenic isolates also differed from ADV-G. Thus, although ADV-TR had the hypervariable sequence of the nonpathogenic type 1 ADV-G, the remainder of the VP2 sequence resembled sequences of other pathogenic ADVs. Under experimental conditions, ADV-TR and ADV-Utah were highly pathogenic and induced typical AD in trios of both Aleutian and non-Aleutian mink, whereas ADV-Pullman was pathogenic only for Aleutian mink and ADV-G was noninfectious. Trios of raccoons experimentally inoculated with ADV-TR and ADV-Utah all became infected with ADV, but only a single ADV-Pullman-inoculated raccoon showed evidence of infection. Furthermore, none of the ADV isolates induced pathological findings of AD in raccoons. Finally, when a preparation of ADV-TR prepared from infected raccoon lymph nodes was inoculated into mink and raccoons, typical AD was induced in Aleutian and non-Aleutian mink, but raccoons failed to show serological or pathological evidence of infection. These results indicated that raccoons can become infected with ADV and may have a role in the transmission of virus to mink but that raccoon-to-raccoon transmission of ADV is unlikely.

In vitro cDNA libraries from scrapie-infected and non-infected murine neuroblastoma cell lines were screened with cDNA probes derived by subtractive hybridization from scrapie-infected and uninfected cells to identify genes with altered expression associated with scrapie infection. Eleven independent recombinant clones, whose expression was either increased or decreased in scrapie-infected cells, were identified. Expression of these genes was also analyzed in a panel of scrapie-infected and non-infected cell lines. Five genes had altered mRNA expression in most scrapie-infected neuroblastoma cell clones compared to non-infected clones. These genes were chromogranin B, intracisternal-A particle envelope, ornithine decarboxylase antizyme, heat shock protein 70 and one unidentified gene. None of these alterations in gene expression was uniquely scrapie-specific; however, the skewed association of increased expression with scrapie infection suggested that a causal relationship might exist between scrapie infection and altered expression of these genes in mouse neuroblastoma cells in vitro.

Human immunodeficiency virus (HIV) dementia is a common clinical syndrome of uncertain pathogenesis in patients with AIDS. In several animal models of retrovirus-induced brain disease, specific viral envelope sequences have been found to influence the occurrence of central nervous system disease. Therefore, to search for unique envelope sequences correlated with HIV dementia, we studied 22 HIV-infected patients who were neurologically assessed premortem and classified into demented (HIVD) (n = 14) and nondemented (ND) (n = 8) groups. Using DNA from autopsied brain and spleen, we amplified, cloned, and sequenced a 430-nucleotide region including the V3 loop and flanking regions. All brain-derived clones in both clinical groups showed marked homology to the macrophage-tropic consensus sequence within the V3 loop. Two amino acid positions within (position 305) and outside (position 329) the V3 region showed significant divergence between the two clinical groups. At position 305, a histidine was predominant in the HIVD group and was not observed in the ND group, but a proline was predominant in the ND group and was not observed in the HIVD group. Similarly, at position 329, a leucine was predominant in the HIVD group but rarely observed in the ND group, whereas an isoleucine was predominant in the ND group at this position. In addition, the HIVD group had 21 amino acid residues at specific positions that were unique relative to the ND group, whereas only 2 residues at specific positions were unique to the ND group. These data suggest that distinct HIV envelope sequences are associated with the clinical expression of HIV dementia.

The ADV-G strain of Aleutian mink disease parvovirus (ADV) is nonpathogenic for mink but replicates permissively in cell culture, whereas the ADV-Utah 1 strain is highly pathogenic for mink but replicates poorly in cell culture. In order to relate these phenotypic differences to primary genomic features, we constructed a series of chimeric plasmids between a full-length replication-competent molecular clone of ADV-G and subgenomic clones of ADV-Utah 1 representing map units (MU) 15 to 88. After transfection of the plasmids into cell culture and serial passage of cell lysates, we determined that substitution of several segments of the ADV-Utah 1 genome (MU 15 to 54 and 65 to 73) within an infectious ADV-G plasmid did not impair the ability of these constructs to yield infectious virus in vitro. Like ADV-G, the viruses derived from these replication-competent clones caused neither detectable viremia 10 days after inoculation nor any evidence of Aleutian disease in adult mink. On the other hand, other chimeric plasmids were incapable of yielding infectious virus and were therefore replication defective in vitro. The MU 54 to 65 EcoRI-EcoRV fragment of ADV-Utah 1 was the minimal segment capable of rendering ADV-G replication defective. Substitution of the ADV-G EcoRI-EcoRV fragment into a replication-defective clone restored replication competence, indicating that this 0.53-kb portion of the genome, wholly located within shared coding sequences for the capsid proteins VP1 and VP2, contained a determinant that governs replication in cell culture. When cultures of cells were studied 5 days after transfection with replication-defective clones, rescue of dimeric replicative form DNA and single-stranded progeny DNA could not be demonstrated. This defect could not be complemented by cotransfection with a replication-competent construction.

Previous experiments indicate that the V3 hypervariable region of the human immunodeficiency virus (HIV) envelope protein influences cell tropism of infection; however, so far no consistent V3 sequence can account for macrophage or T-cell tropism. In these experiments, we studied infectious recombinant HIV clones constructed by using V3 region sequences of HIV isolates from 16 patients to search for sequences associated with cell tropism. Remarkable homology was seen among V3 sequences from macrophage-tropic clones from different patients, and a consensus V3 region sequence for patient-derived macrophage-tropic viruses was identified. In contrast, V3 sequences of T-cell-tropic clones from different patients were highly heterogeneous, and the results suggested that sequence diversity leading to T-cell tropism might be generated independently in each patient. Site-specific mutations identified amino acids at several positions on each side of the GPGR motif at the tip of the V3 loop as important determinants of tropism for T cells and macrophages. However, a wide variety of mutant V3 sequences induced macrophage tropism, as detected in vitro. Therefore, the homogeneity of macrophage-tropic patient isolates appeared to be the result of selection based on a biological advantage in vivo.

An avirulent, field-derived isolate of equine infectious anemia virus (EIAV), designated MA-1, was molecularly cloned, and the complete nucleotide sequence was determined for the 3' half of the viral genome. Comparisons between MA-1 and the prototype Wyoming strain of EIAV identified a 66-nucleotide stretch between CAAT (-91) and TATAA (-25) in the U3 region of the long terminal repeat, where sequence divergence was as high as 39.3%. The polymerase chain reaction was used to amplify and clone long terminal repeat sequences from Th-1, the in vivo parental stock of MA-1. Results indicated that the nucleotide sequences of MA-1 and Th-1 clones were less variable than was observed between MA-1 and Wyoming. However, MA-1 and Th-1 markedly differed in the types of enhancer sequences located in the hypervariable region. These results suggest that variation in lentivirus regulatory sequences may be important in EIAV host cell tropism and pathogenesis.

The wild mouse ecotropic retrovirus CasBrE causes a spongiform neurodegenerative disease after neonatal inoculation, with an incubation period ranging from 2 to 12 months. We previously showed that introduction of long terminal repeat (LTR) and gag-pol sequences from a strain of Friend murine leukemia virus (FB29) resulted in a dramatic acceleration of the onset of the disease. The chimeric virus FrCasE, which consisted of the FB29 genome containing 3' pol and env sequences from the wild mouse virus, induced a highly predictable, lethal neurodegenerative disease with an incubation period of only 16 days. Here we report that the sequences which are primary determinants of the length of the incubation period are located in the 5' end of the viral genome between a KpnI site in the R region of the LTR and a PstI site immediately 5' of the start codon for pr65gag (R-U5-5' leader). This region contains the tRNA primer binding site, splice donor site for the subgenomic env mRNA, and the packaging sequence. Computer-assisted sequence analysis failed to find evidence of a consensus sequence for a DNA enhancer in this region. In addition, sequences within a region of the genome between a ClaI site at the 3' end of env to the KpnI site in the R region of the LTR (inclusive of U3) also influenced the incubation period of the disease, but the effect was distinctly weaker than that of the R-U5-5' leader sequence. This U3 effect, however, appeared to be independent of the number of direct repeats, since deletion of one of two duplicated 42-base repeats containing consensus sequences of nuclear-factor binding domains had no effect on the incubation period of the disease. On the basis of Southern blot analysis of total viral DNA in the tissues, the effect of these sequences on the incubation period appeared to be related to the level of virus replication in the central nervous system. All of the chimeric viruses analyzed, irrespective of neurovirulence, replicated to comparable levels in the spleen and induced comparable levels of viremia.

Infectious recombinant viruses were constructed from three molecularly cloned human immunodeficiency virus (HIV) strains varying in cell tropism. All recombinants showed a high infectivity titer on phytohemagglutinin-stimulated normal T lymphocytes. However, a 120-bp region of the envelope gene including the area of the V3 hypervariable loop was found to influence infectivity titer on both clone 1022 CD4-positive HeLa cells and CD4-positive CEM leukemia cells. Infectivity for macrophages was more complex. All viruses replicated in macrophages to a low level, but viral sequences both inside and outside the V3 loop region influenced the efficiency of replication. Two experiments showed that the mechanism of restriction of infection of 1022 cells by HIV strain JR-CSF was related to lack of virus entry. First, productive virus infection occurred after transfection of 1022 cells with viral plasmid DNA. Second, the nonpermissive HIV strain JR-CSF could infect 1022 cells when pseudotyped with the envelope of other retroviruses, including human T-cell leukemia virus type I (HTLV-I), HTLV-II, and amphotropic murine leukemia virus. These results demonstrate the possibility that unexpected cell types might be infected with HIV in human patients coinfected with HIV and HTLV-I or HTLV-II.

Friend murine leukemia virus is a replication-competent retrovirus that contains no oncogene and that exerts lytic and leukemogenic properties. Thus, newborn mice inoculated with Friend murine leukemia virus develop severe early hemolytic anemia before appearance of erythroleukemia. To identify the retroviral determinants regulating these effects, we used chimeric infectious constructions and site-directed point mutations between a virulent Friend murine leukemia virus strain and a naturally occurring variant attenuated in lytic and leukemogenic effects. We found that severe hemolytic anemia was always associated with higher numbers of blood reticulocytes with budding retroviral particles. Furthermore, a remarkably conservative leucine to isoleucine change in the extracellular SU component of the retroviral envelope was sufficient to attenuate this lytic effect. Also, this leucine at position 348 of the envelope precursor protein was located within the only stretch of five amino acids that is conserved in the extracellular SU component of all murine, feline, and primate type C and type D retroviral envelopes. This observation suggested an important structural function for this yet undescribed conserved sequence of the envelope. Lastly, we observed that lytic and leukemogenic effects were attenuated by a deletion of a second repeat in the transcriptional enhancer region of the viral long terminal repeats of the variant strain.

Complete nucleotide sequence of Friend murine leukemia virus, Strain FB29 Sylvia Peeryman, Sylvia Peeryman Laboratory of Persistent Viral Diseases, Rocky Mountain Laboratories, National Institute of Allergy and Infectious DiseasesHamilton, MT 59840, USA Search for other works by this author on: Oxford Academic PubMed Google Scholar Jane Nishio, Jane Nishio Laboratory of Persistent Viral Diseases, Rocky Mountain Laboratories, National Institute of Allergy and Infectious DiseasesHamilton, MT 59840, USA Search for other works by this author on: Oxford Academic PubMed Google Scholar Bruce Chesebro Bruce Chesebro Laboratory of Persistent Viral Diseases, Rocky Mountain Laboratories, National Institute of Allergy and Infectious DiseasesHamilton, MT 59840, USA Search for other works by this author on: Oxford Academic PubMed Google Scholar Nucleic Acids Research, Volume 19, Issue 24, 25 December 1991, Page 6950, https://doi.org/10.1093/nar/19.24.6950 Published: 25 December 1991 Article history Received: 12 November 1991 Published: 25 December 1991

The 5'-terminal palindrome of the ADV-G strain of Aleutian mink disease parvovirus (ADV) was molecularly cloned and sequenced. A full-length molecular clone of ADV-G, denoted pXVB, was then constructed. When this clone was transfected into cell cultures, infectious ADV could be rescued. Virus derived from pXVB was nonpathogenic for adult mink, as is the parent ADV-G strain.

We studied the transcription program of Aleutian mink disease parvovirus (ADV) by using a combination of cDNA cloning and sequencing, primer extension, and Northern (RNA) blot hybridization with splice-specific oligonucleotides. The 4.8-kilobase ADV genome was transcribed in the rightward direction, yielding plus-sense polyadenylated transcripts of 4.3 (R1 RNA), 2.8 (R2), 2.8 (R3), 1.1 (RX), and 0.85 (R2') kilobases. Each RNA transcript had potential translation initiation sites within open reading frames, suggesting protein translation, and a scheme encompassing ADV structural and nonstructural proteins is proposed. Each of the five RNA transcripts had a characteristic set of splices and originated from a promoter at nucleotide 152 (map unit 3 [R1, R2, R2', and RX]) or at nucleotide 1729 (map unit 36 [R3]). The transcripts terminated with a poly(A) tail at one of two positions: either at map unit 53 (R2' and RX) or at map unit 92 (R1, R2, and R3). Similarities with and differences from the transcription maps of other parvoviruses are discussed, and possible roles of the unique features found in ADV transcription are related to the special pathogenic features of this virus.

A DNA sequence of 4,592 nucleotides (nt) was derived for the nonpathogenic ADV-G strain of Aleutian mink disease parvovirus (ADV). The 3'(left) end of the virion strand contained a 117-nt palindrome that could assume a Y-shaped configuration similar to, but less stable than, that of other parvoviruses. The sequence obtained for the 5' end was incomplete and did not contain the 5' (right) hairpin structure but ended just after a 25-nt A + T-rich direct repeat. Features of ADV genomic organization are (i) major left (622 amino acids) and right (702 amino acids) open reading frames (ORFs) in different translational frames of the plus-sense strand, (ii) two short mid-ORFs, (iii) eight potential promoter motifs (TATA boxes), including ones at 3 and 36 map units, and (iv) six potential polyadenylation sites, including three clustered near the termination of the right ORF. Although the overall homology to other parvoviruses is less than 50%, there are short conserved amino acid regions in both major ORFs. However, two regions in the right ORF allegedly conserved among the parvoviruses were not present in ADV. At the DNA level, ADV-G is 97.5% related to the pathogenic ADV-Utah 1. A total of 22 amino acid changes were found in the right ORF; changes were found in both hydrophilic and hydrophobic regions and generally did not affect the theoretical hydropathy. However, there is a short heterogeneous region at 64 to 65 map units in which 8 out of 11 residues have diverged; this hypervariable segment may be analogous to short amino acid regions in other parvoviruses that determine host range and pathogenicity. These findings suggested that this region may harbor some of the determinants responsible for the differences in pathogenicity of ADV-G and ADV-Utah 1.