Mojgan H. Naghavi

Frequent patient-to-patient transmission of hepatitis C virus in a haematology ward

Blood transfusion is a well-documented route of transmission of hepatitis C virus (HCV). However, a persisting high frequency of HCV infections was recorded in our haematology ward even after screening of blood donors had been introduced. We investigated the viral strains in 37 patients with haematological malignant diseases who had developed hepatitis C when treated in the ward during 1990-93. 17 of the patients acquired hepatitis C despite being transfused only with blood components screened by second-generation anti-HCV tests. The viral strains were characterised by PCR genotyping and nucleotide sequencing of the hypervariable region of the E2 gene. Five clusters of closely related or identical viruses were found involving 2, 3, 4, 6, and 15 patients, respectively. Blood components could be ruled out as the common source of infection because no donor had given blood to all patients sharing a specific strain, and even donors whose blood had been given to several patients were negative for HCV RNA. All patients in each cluster had been treated in the ward during overlapping periods. These findings suggest that despite strict hygienic control, HCV transmission occurred between patients treated in the same hospital setting, as has previously been reported in a smaller group of haemodialysis patients.

Long terminal repeat promoter/enhancer activity of different subtypes of HIV type 1

Transcription of the HIV-1 provirus genome is regulated by a complex interplay between viral regulatory proteins and cellular transcription factors that interact with the viral long terminal repeat (LTR) region of HIV-1. However, several cellular transcription factors have been identified that can interact with the HIV-1 LTR; the significance of all of these factors is not clearly understood. In this study we have characterized the LTR region of different subtypes of HIV-1 with regard to nucleotide sequence and promoter activity. The LTR regions of HIV-1 from peripheral blood mononuclear cells of 29 infected individuals originating from 10 different geographical regions were sequenced and further analyzed for promoter/enhancer activity in transient transfection of HeLa cells, in the context of a reporter gene and in the context of the complete virus genome. We found several subtype-specific LTR sequences of the various HIV-1 strains, such as an insertion that created a potential third NF-kappaB site in the LTR of the subtype C strains. The USF-binding site in the NRE also contained subtype-specific sequences. Interestingly, the promoter/enhancer activities of the subtype C LTRs were higher than the activities of the other subtypes analyzed here (subtypes A, B, D, E, and G), suggesting that the potential third NF-kappaB site may confer higher LTR activity or that the subtype C NRE may be less potent. Thus, our data suggest that genetic diversity of the LTR may result in HIV-1 subtypes with different replicative properties.

Moesin regulates stable microtubule formation and limits retroviral infection in cultured cells

In a functional screen of mammalian complementary DNA libraries, we identified moesin as a novel gene whose overexpression blocks infection by murine leukemia viruses and human immunodeficiency virus type 1 in human and rodent lines, before the initiation of reverse transcription. Knockdown of moesin by RNA interference resulted in enhanced infection, suggesting that even the endogenous basal levels of moesin in rat fibroblasts are sufficient to limit virus infection. Moesin acts as a crosslinker between plasma membrane and actin filaments, as well as a signal transducer in responses involving cytoskeletal remodeling. Moesin overexpression was found to downregulate the formation of stable microtubules, whereas knockdown of moesin increased stable microtubule formation. A virus‐resistant mutant cell line also displayed decreased stable microtubule levels, and virus‐sensitive revertants recovered from the mutant line showed restoration of the stable microtubules, suggesting that these cytoskeletal networks play an important role in early post‐entry events in the retroviral lifecycle. Together, these results suggest that moesin negatively regulates stable microtubule networks and is a natural determinant of cellular sensitivity to retroviral infection.

HIV-1 induces the formation of stable microtubules to enhance early infection.

Stable microtubule (MT) subsets form distinct networks from dynamic MTs and acquire distinguishing posttranslational modifications, notably detyrosination and acetylation. Acting as specialized tracks for vesicle and macromolecular transport, their formation is regulated by the end-binding protein EB1, which recruits proteins that stabilize MTs. We show that HIV-1 induces the formation of acetylated and detyrosinated stable MTs early in infection. Although the MT depolymerizing agent nocodazole affected dynamic MTs, HIV-1 particles localized to nocodazole-resistant stable MTs, and infection was minimally affected. EB1 depletion or expression of an EB1 carboxy-terminal fragment that acts as a dominant-negative inhibitor of MT stabilization prevented HIV-1-induced stable MT formation and suppressed early viral infection. Furthermore, we show that the HIV-1 matrix protein targets the EB1-binding protein Kif4 to induce MT stabilization. Our findings illustrate how specialized MT-binding proteins mediate MT stabilization by HIV-1 and the importance of stable MT subsets in viral infection.

MCEF, the Newest Member of the AF4 Family of Transcription Factors Involved in Leukemia, Is a Positive Transcription Elongation Factor-b-Associated Protein

Positive transcription elongation factor-b (P-TEFb) contains CDK9 and cyclin T1. P-TEFb was affinity purified from a stably transfected cell line that expresses epitope-tagged CDK9, and proteins that appeared to be specifically bound were sequenced. In addition to CDK9, previously identified isoforms of cyclin T (including T1, T2A and T2B), HSP90 and CDC37, this analysis identified a novel protein named MCEF. Cloning of its cognate cDNA revealed that MCEF is the newest member of the AF4 family of transcription factors involved in acute lymphoblastic leukemia. MCEF RNA was expressed in all human tissues examined, and antisera directed against recombinant MCEF specifically immunoprecipitated P-TEFb. Ectopic expression of MCEF did not activate HIV-1 replication, and tethering of MCEF to a promoter did not activate transcription.

The Ezrin-Radixin-Moesin Family Member Ezrin Regulates Stable Microtubule Formation and Retroviral Infection

ABSTRACT We recently identified the cytoskeletal regulatory protein moesin as a novel gene that inhibits retroviral replication prior to reverse transcription by downregulation of stable microtubule formation. Here, we provide evidence that overexpression of ezrin, another closely related ezrin-radixin-moesin (ERM) family member, also blocks replication of both murine leukemia viruses and human immunodeficiency virus type 1 (HIV-1) in Rat2 fibroblasts before reverse transcription, while knockdown of endogenous ezrin increases the susceptibility of human cells to HIV-1 infection. Together, these results suggest that ERM proteins may be important determinants of retrovirus susceptibility through negative regulation of stable microtubule networks.

HIV-1 capsids bind and exploit the kinesin-1 adaptor FEZ1 for inward movement to the nucleus.

Intracellular transport of cargos, including many viruses, involves directed movement on microtubules mediated by motor proteins. While a number of viruses bind motors of opposing directionality, how they associate with and control these motors to accomplish directed movement remains poorly understood. Here we show that human immunodeficiency virus type 1 (HIV-1) associates with the kinesin-1 adaptor protein, Fasiculation and Elongation Factor zeta 1 (FEZ1). RNAi-mediated FEZ1 depletion blocks early infection, with virus particles exhibiting bidirectional motility but no net movement to the nucleus. Furthermore, both dynein and kinesin-1 motors are required for HIV-1 trafficking to the nucleus. Finally, the ability of exogenously expressed FEZ1 to promote early HIV-1 infection requires binding to kinesin-1. Our findings demonstrate that opposing motors both contribute to early HIV-1 movement and identify the kinesin-1 adaptor, FEZ1 as a capsid-associated host regulator of this process usurped by HIV-1 to accomplish net inward movement toward the nucleus.

Intracellular high mobility group B1 protein (HMGB1) represses HIV-1 LTR-directed transcription in a promoter- and cell-specific manner

We investigated whether the high mobility group B 1 (HMGB1), an abundant nuclear protein in all mammalian cells, affects HIV-1 transcription. Intracellular expression of human HMGB1 repressed HIV-1 gene expression in epithelial cells. This inhibitory effect of HMGB1 was caused by repression of long terminal repeat (LTR)-mediated transcription. Other viral promoters/enhancers, including simian virus 40 or cytomegalovirus, were not inhibited by HMGB1. In addition, HMGB1 inhibition of HIV-1 subtype C expression was dependent on the number of NF kappa B sites in the LTR region. The inhibitory effect of HMGB1 on viral gene expression observed in HeLa cells was confirmed by an upregulation of viral replication in the presence of antisense HMGB1 in monocytic cells. In contrast to what was found in HeLa cells and monocytic cells, endogenous HMGB1 expression did not affect HIV-1 replication in unstimulated Jurkat cells. Thus, intracellular HMGB1 affects HIV-1 LTR-directed transcription in a promoter- and cell-specific manner.

Upstream stimulating factor affects human immunodeficiency virus type 1 (HIV-1) long terminal repeat-directed transcription in a cell-specific manner, independently of the HIV-1 subtype and the core-negative regulatory element.

Human immunodeficiency virus type 1 (HIV-1) is classified into subtypes on the basis of phylogenetic analysis of sequence differences. Inter- and intra-subtype polymorphism extends throughout the genome, including the long terminal repeat (LTR). In this study, the importance of the upstream stimulating factor (USF)-binding site (E-box) in the core-negative regulatory element (NRE) of the LTR of HIV-1 subtypes A, B, C, D, E and G was investigated. In vivo, USF was found to repress transcription directed from representative HIV-1 LTR sequences of all the subtypes tested in an epithelial cell line, yet activate the same transcription in a T-cell line. Mutation of the core-NRE USF site of the representative subtype B LTR did not affect the cell-specific, subtype-independent, dual role of USF. In vitro binding assays showed that recombinant USF(43) interacts with the core-NRE from subtypes B and C, but not A, D, E or G. Thus, USF affects LTR-directed transcription in a cell-specific manner, independently of both the HIV-1 subtype from which the LTR was derived and the core-NRE USF site sequences.

DNA Sequence of the Long Terminal Repeat of Human Immunodeficiency Virus Type 1 Subtype A through G

485 THE RAPID ACCUMULATION of mutations and recombination occurs by processes that actively contribute to the evolution of HIV-1 and its genetic diversity. At least 10 distinct genetic subtypes of HIV-1 have been identified on the basis of the phylogenetic analysis of nucleotide sequences in the envV3 and gagp17 regions.1,2 According to an analysis of the 1993 Los Alamos HIV Database, 10% of the reported sequences were V3/p17 recombinants. Intersubtype mosaic forms of HIV-1 have been reported in geographic areas where multiple HIV-1 subtypes cocirculate. While many studies have been focused on phylogenetic analysis of the structural genes,1,2,5 relatively little attention has been paid to the genetic variation in the HIV1 long terminal repeat (LTR) region. Here we have characterized a collection of the LTR sequences from seven different HIV-1 subtypes and intersubtype recombinants representing different geographic origins. HIV-1 DNA was extracted from peripheral blood mononuclear cells (PBMCs) of 28 randomly chosen HIV-1-infected patients (21 African and 7 Swedish patients), as previously described. HIV-1 was subtyped by direct sequencing of the V3 and p17 regions of all of the patients, followed by phylogenetic analyses, as previously described. Full-length HIV genomic sequences (subtypes A±H), as presented in the 1997 Los Alamos HIV Database, were used as reference sequences in the alignments for V3 and p17 regions. For some subtypes unpublished full-length sequences (subtype H: VI991-3, VI997-2; subtype F: BZ126, F9363, ITM850; subtype A: SE8538, SE8131-3, SE8891, SE7253, SE7535; and subtype J: SE91733, SE9280-9) were available. The LTR was amplified by a nested polymerase chain reaction (PCR), using the previously described primers BJLTR1, BJLTR3, and BJLTR4.8 After cloning of the PCR fragments into the pCR2.1 vector (Invitrogen, San Diego, CA) the double-stranded (ds) DNA was used as a template for sequencing. The sequencing reactions were primed with CY5-labeled universal (or reverse) primers, provided in the Sequenase version 2.0 kit (United States Biochemical, Cleveland, OH), according to the protocol of the manufacturer. Multiple LTR clones (3±10 clones per patient) for each of the 28 HIV-1 strains were sequenced from both directions. The LTR sequences (nucleotides 2 382 to 1 113 relative to transcription start) were then aligned using Omiga 1.01 (ClustalW Oxford Molecular, Ltd., Oxford, UK) and DNASIS sequence analysis software (Hitachi Software Engineering America, San Bruno, CA). The LTR sequences presented in this study were aligned with the various subtype LTR sequences from the Los Alamos HIV Database of 1997 (data not shown). The alignment was optimized manually. On the basis of the LTR, p17, and V3 phylogenetic analyses 1 of the samples was classified as subtype A, 10 as B, 6 as C, and 1 as G (Fig. 1A, B, and C, respectively2,9). A discrepant topology was observed in the V3, p17, or LTR phylogenetic tree in 5 of the 28 HIV-1 strains (marked with asterisks in Fig. 1). The HIV-1 strains that gave discrepant clustering in the phylogenetic trees were therefore putatively classified as recombinant strains (SO5549, A/C; GM6139, A/G; IC5381, A/G; GM6452, E/A; and TH6098, E/A). On the basis of the LTR analysis five of the strains were classified as subtype D (UG6476, UG6083, UG4696, UG6357, and UG5609); however, a clear subtype classification based on p17 and V3 (except for patient UG6476) could not be obtained for those strains (marked with double asterisks in Fig. 1B and C). A possible explanation may be that in those strains these regions contain a recombination point so that one part of the sequence belong to one subtype and one part to another, leading to unclear classification of those isolates. Interestingly, all of these strains are from Uganda, where a high frequency of HIV-1 hybrid genomes can be expected owing to the cocirculation of multiple divergent HIV-1 strains, mainly subtypes A and D.1 The LTR analysis revealed a distinction between the LTR region of subtype C and that of all the other subtypes studied (A, B, D, E, F, and G), in that subtype C isolates (except the sample SO5549) contained an additional potential NFk B-binding site (NFk B1) (GGGCGKTCY). Such a unique pattern for the LTR region of HIV-1 clade C has previously been reported from Ethiopia,