Widely variable endogenous retroviral methylation levels in human placenta

It is generally assumed that transposable elements, including endogenous retroviruses (ERVs), are silenced by DNA methylation/chromatin structure in mammalian cells. However, there have been very few experimental studies to examine the methylation status of human ERVs. In this study, we determined and compared the methylation status of the 5′ long terminal repeats (LTRs) of different copies of the human endogenous retrovirus (HERV) family HERV-E, which are inserted in various genomic contexts. We found that three HERV-E LTRs which function as alternative gene promoters in placenta are unmethylated in that tissue but heavily methylated in blood cells, where these LTRs are not active promoters. This difference is not solely due to global hypomethylation in placenta, since two general measures of methylation levels of HERV-E and HERV-K LTRs suggest only 10–15% lower overall HERV methylation in placenta compared to blood. Comparisons between methylation levels of the LTR-derived gene promoters and six random HERV-E LTRs in placenta showed that the former display significantly lower methylation levels than random LTRs. Moreover, the differences in methylation between LTRs cannot always be explained by their genomic environment, since methylation of flanking sequences can be very different from methylation of the LTR itself.     

Characterization of a cis-Acting Sequence in the pol Region Required To Transfer Human Foamy Virus Vectors

To identify cis-acting elements in the foamy virus (FV) RNA pregenome, we developed a transient-vector-production system based on cotransfection of indicator gene-bearing vector and gag-pol and env expression plasmids. Two elements which were critical for vector transfer were found and mapped approximately. The first element was located in the RU5 leader and the 5′ gag region (approximately up to position 650 of the viral RNA). The second element was located in an approximately 2-kb sequence in the 3′ pol region. Although small 5′ and 3′ deletions, as well as internal deletions of the latter element, were tolerated, both elements were found to be absolutely required for vector transfer. The functional characterization of the pol region-located cis-acting element revealed that it is essential for efficient incorporation or the stability of particle-associated virion RNA. Furthermore, virions derived from a vector lacking this sequence were found to be deficient in the cleavage of the Gag protein by the Pol precursor protease. Our results suggest that during the formation of infectious virions, complex interactions between FV Gag and Pol and the viral RNA take place.     

macroH2A1-dependent silencing of endogenous murine leukemia viruses

We show that macroH2A1 histone variants are important for repressing the expression of endogenous murine leukemia viruses (MLVs) in mouse liver. Intact MLV proviruses and proviruses with deletions in env were nearly silent in normal mouse liver and showed substantial derepression in macroH2A1 knockout liver. In contrast, MLV proviruses with a deletion in the 5′ end of pro-pol were expressed in normal liver and showed relatively low levels of derepression in knockout liver. macroH2A1 nucleosomes were enriched on endogenous MLVs, with the highest enrichment occurring on the 5′ end of pro-pol. The absence of macroH2A1 also led to a localized loss of DNA methylation on the 5′ ends of MLV proviruses. These results demonstrate that macroH2A1 histones have a significant role in silencing endogenous MLVs in vivo and suggest that specific internal MLV sequences are targeted by a macroH2A1-dependent silencing mechanism.     

Acquisition of HIV by African-Born Residents of Victoria,Australia: Insights from Molecular Epidemiology

African-born Australians are a recognised “priority population” in Australia''s Sixth National HIV/AIDS Strategy. We compared exposure location and route for African-born people living with HIV (PLHIV) in Victoria, Australia, with HIV-1 pol subtype from drug resistance assays and geographical origin suggested by phylogenetic analysis of env gene. Twenty adult HIV positive African-born Victorian residents were recruited via treating doctors. HIV exposure details were obtained from interviews and case notes. Viral RNA was extracted from participant stored plasma or whole blood. The env V3 region was sequenced and compared to globally representative reference HIV-1 sequences in the Los Alamos National Library HIV Database. Twelve participants reported exposure via heterosexual sex and two via iatrogenic blood exposures; four were men having sex with men (MSM); two were exposed via unknown routes. Eight participants reported exposure in their countries of birth, seven in Australia, three in other countries and two in unknown locations. Genotype results (pol) were available for ten participants. HIV env amplification was successful in eighteen cases. HIV-1 subtype was identified in all participants: eight both pol and env; ten env alone and two pol alone. Twelve were subtype C, four subtype B, three subtype A and one subtype CRF02_AG. Reported exposure location was consistent with the phylogenetic clustering of env sequences. African Australians are members of multiple transnational social and sexual networks influencing their exposure to HIV. Phylogenetic analysis may complement traditional surveillance to discern patterns of HIV exposure, providing focus for HIV prevention programs in mobile populations.     

Mouse Mammary Tumor Virus Superantigen Expression in B Cells Is Regulated by a Central Enhancer within the pol Gene

Expression of mouse mammary tumor virus (MMTV)-encoded superantigens in B lymphocytes is required for viral transmission and pathogenesis. The mechanism of superantigen expression from the viral sag gene in B cells is largely unknown, due to problems with detection and quantification of these low-abundance proteins. We have established a sensitive superantigen-luciferase reporter assay to study the expression and regulation of the MMTV sag gene in B-cell lymphomas. The regulatory elements for retroviral gene expression are generally located in the 5′ long terminal repeat (LTR) of the provirus. However, we found that neither promoters nor enhancers in the MMTV 5′ LTR play a significant role in superantigen expression in these cells. Instead, the essential regulatory regions are located in the pol and env genes of MMTV. We report here that maximal sag expression in B-cell lines depends on an enhancer within the viral pol gene which can be localized to a minimal 183-bp region. Regulation of sag gene expression differs between B-cell lymphomas and pro-B cells, where an enhancer within the viral LTRs is involved. Thus, MMTV sag expression during B-cell development is achieved through the use of two separate enhancer elements.     

Nucleotide sequence of rous sarcoma virus

We present the 9312 nucleotide sequence of the Prague C (Pr-C) strain of Rous sarcoma virus (RSV). A comparison of known protein sequences with the nucleotide sequence allows assignment of the coding regions for the gag, pol, env and src genes. The gag gene is terminated by an amber stop codon and is contained within a different reading frame than is the pol gene. The pol and env genes overlap. The sequences surrounding the src gene in the PrC and Schmidt-Ruppin (SR-A) strains of RSV have been compared, and they reveal that an element, E, of approximately 153 nucleotides is present on the 3′ side of the src gene in Pr-C, and on the 5′ side in SR-A. We hypothesize that E was part of a duplicated region of over 250 nucleotides flanking the src gene in an ancestral RSV, and that differential deletion of one copy of E led to its positional difference in Pr-C and SR-A.     

cis-Acting Sequences Required for Simian Foamy Virus Type 1 Vectors

We have constructed a series of vectors based on simian foamy virus type 1 (SFV-1) to define the minimum cis-acting elements required for gene transfer. To characterize these vectors, we inserted the coding sequence of the bacterial lacZ gene linked to the cytomegalovirus immediate-early gene promoter. Introduction of a deletion mutation in the leader region between the 5′ long terminal repeat and the start of the gag gene at position 1659 to 1694 completely abrogated gene transfer by the SFV-1 vector. Deletion of 39 nucleotides from position 1692 to 1731 in the leader region resulted in a significant reduction in the transducing-particle titer. Furthermore, we have identified a second cis-acting element located at the 3′ end of the pol gene between position 6486 and 6975 to be critical for SFV-1 vector transduction. These results identify the two important cis-acting elements required for SFV-1 vector construction, and the finding of a cis-acting element in the pol gene is unique among retroviruses.     

Presence ofenv-like sequences inQuercus suber retrotransposons

The main difference between LTR retrotransposons and retroviruses is the presence of theenvelope (env) gene in the latter, downstream of thepol gene. Theenv gene is involved in their infectious capacity. Here we report the presence ofenv-like sequences in the genome ofQuercus suber (cork oak), one of the most economically important Portuguese species. These gene sequences were isolated through DNA amplification betweenRNaseH conserved motifs and 3’ LTR, based on the structure ofcopia retrotransposons. Phylogenetic analysis revealed that almost all the clones isolated are clustered withCyclops-2, aTy3-gypsy element identified inPisum sativum, except one clustered withgypsy andcopia retroelements found in different species. This suggests the existence of a potential ancestral sequence of theenv gene, prior to the separation ofTy3-gypsy andTy1-copia retrotransposons. Additionally, the isolatedenv-like sequences showed 26–39% of homology withenv-like sequences characterized in viruses. The origin ofenv-like sequences in retrotransposons from host plant taxa is discussed.     

Dexamethasone Induction of the Intracellular RNAs of Mouse Mammary Tumor Virus

We have studied the kinetics of dexamethasone induction of mouse mammary tumor virus (MMTV) RNAs and proteins in virus-infected rat XC cells and GR mouse mammary tumor cells. A detectable increase in viral RNA in infected XC cells was present within 10 min after hormone addition, and half-maximal induction was achieved in less than 2 h. The increase in viral RNA concentration was apparent first in nuclear RNA and later in the cytoplasm. Within the first 15 min of induction, only genome-sized RNA (35S, 7.8 kilobases) was present in augmented amounts, whereas the major subgenomic RNA (24S, 3.8 kilobases) did not appear until at least 30 to 60 min postinduction. The sequential appearance of these RNAs, the probable mRNA's for the gag and env proteins, paralleled the order of appearance of the gag and env proteins, respectively, after hormone treatment. An additional species of viral RNA (20S, 2.5 kilobases) was detected during these induction experiments, but the role of this RNA is not known. Both subgenomic RNAs contain sequences derived from both the 5′ and 3′ termini of genomic RNA and are presumably spliced. After dexamethasone induction of infected XC cells, we detected two smaller env-related proteins which were not found in full hormone induction. The functional role of these smaller proteins is not known. A previously reported smaller species of RNA (13S, 1.0 kilobase) did not appear to be induced and was shown to be cellular rather than viral in origin. In the fully induced infected XC and GR mammary tumor cells, the only viral RNAs present were the 35S and 24S RNAs. In addition, mammary tumors contained only these two viral RNAs. Thus, tumor cells appear to contain only the viral RNAs which direct the synthesis of the gag, pol, and env proteins of the virion.     

The Pre-NH2-Terminal Domain of the Herpes Simplex Virus 1 DNA Polymerase Catalytic Subunit Is Required for Efficient Viral Replication

The catalytic subunit of herpes simplex virus 1 DNA polymerase (HSV-1 Pol) has been extensively studied; however, its full complement of functional domains has yet to be characterized. A crystal structure has revealed a previously uncharacterized pre-NH₂-terminal domain (residues 1 to 140) within HSV-1 Pol. Due to the conservation of the pre-NH₂-terminal domain within the herpesvirus Pol family and its location in the crystal structure, we hypothesized that this domain provides an important function during viral replication in the infected cell distinct from 5′-3′ polymerase activity. We identified three pre-NH₂-terminal Pol mutants that exhibited 5′-3′ polymerase activity indistinguishable from that of wild-type Pol in vitro: deletion mutants PolΔN43 and PolΔN52 that lack the extreme N-terminal 42 and 51 residues, respectively, and mutant PolA₆, in which a conserved motif at residues 44 to 49 was replaced with alanines. We constructed the corresponding pol mutant viruses and found that the polΔN43 mutant displayed replication kinetics similar to those of wild-type virus, while polΔN52 and polA₆ mutant virus infection resulted in an 8-fold defect in viral yield compared to that achieved with wild type and their respective rescued derivative viruses. Additionally, both polΔN52 and polA₆ viruses exhibited defects in viral DNA synthesis that correlated with the observed reduction in viral yield. These results strongly indicate that the conserved motif within the pre-NH₂-terminal domain is important for viral DNA synthesis and production of infectious virus and indicate a functional role for this domain.