Gag-pol Region Research Articles

Hypervariable 3′ UTR region of plant LTR-retrotransposons as a source of novel satellite repeats

The repetitive sequence PisTR-A has an unusual organization in the pea ( Pisum sativum) genome, being present both as short dispersed repeats as well as long arrays of tandemly arranged satellite DNA. Cloning, sequencing and FISH analysis of both PisTR-A variants revealed that the former occurs in the genome embedded within the sequence of Ty3/gypsy-like Ogre elements, whereas the latter forms homogenized arrays of satellite repeats at several genomic loci. The Ogre elements carry the PisTR-A sequences in their 3′ untranslated region (UTR) separating the gag-pol region from the 3′ LTR. This region was found to be highly variable among pea Ogre elements, and includes a number of other tandem repeats along with or instead of PisTR-A. Bioinformatic analysis of LTR-retrotransposons mined from available plant genomic sequence data revealed that the frequent occurrence of variable tandem repeats within 3′ UTRs is a typical feature of the Tat lineage of plant retrotransposons. Comparison of these repeats to known plant satellite sequences uncovered two other instances of satellites with sequence similarity to a Tat-like retrotransposon 3′ UTR regions. These observations suggest that some retrotransposons may significantly contribute to satellite DNA evolution by generating a library of short repeat arrays that can subsequently be dispersed through the genome and eventually further amplified and homogenized into novel satellite repeats.

Experimental evidence for splicing of intron-containing transcripts of plant LTR retrotransposon Ogre.

Ogre elements are a distinct group of plant Ty3/gypsy-like retrotransposons characterized by several specific features, one of which is a separation of the gag-pol region into two non-overlapping open reading frames: ORF2 coding for Gag-Pro, and ORF3 coding for RT/RH-INT proteins. Previous characterization of Ogre elements from several plant species revealed that part of their transcripts lacks the region between ORF2 and ORF3, carrying one uninterrupted ORF instead. In this work, we investigated a hypothesis that this region represents an intron that is spliced out from part of the Ogre transcripts as a means for preferential production of ORF2-encoded proteins over those encoded by the complete ORF2–ORF3 region. The experiments involved analysis of transcription patterns of well-defined Ogre populations in a model plant Medicago truncatula and examination of transcripts carrying dissected pea Ogre intron expressed within a coding sequence of chimeric reporter gene. Both experimental approaches proved that the region between ORF2 and ORF3 is spliced from Ogre transcripts and showed that this process is only partial, probably due to weak splice signals. This is one of very few known cases of spliced LTR retrotransposons and the only one where splicing does not involve parts of the element’s coding sequences, thus resembling intron splicing found in most cellular genes.Electronic supplementary materialThe online version of this article (doi:10.1007/s00438-008-0376-8) contains supplementary material, which is available to authorized users.

A new CRF01_AE/B recombinant structure of HIV type 1 found in Heilongjiang province, China

The HIV-1 epidemic in China has demonstrated both a complicated diversity and geographic characteristics. The second National HIV Molecular Epidemiology Survey revealed that HIV-1 subtypes A B C CRF01_AE CRF-BC and other unusual subtypes have been found in China. HIV-1 CRF_BC is the main epidemic component (50.2%) and subtype B (31.66%) and CRF01_AE (15.54%) take second and third places respectively (http://shgy.jhgl.org/shownews.asp?newsid=893). Heilongjiang province is located in the northeast of China neighbored with the far eastern part of the Russian Federation. Since the first AIDS case was officially reported in 1993 the spread of HIV/AIDS in Heilongjiang province has been comparatively slow. By the end of 2006 385 HIV-1-infected individuals with 106 AIDS cases have been reported (www.hlj.gov.cn). To clarify the molecular epidemiology and the genetic diversity of HIV-1 in Heilongjiang province we analyzed the sequence features of the gag pol and env gene regions of HIV-1 isolatescirculating locally. (excerpt)

Continuous Crossover(s) Events of HIV-1 CRF01_AE and B Subtype Strains in Malaysia: Evidence of Rapid and Extensive HIV-1 Evolution in the Region

The Asian HIV epidemic appears to be complex, characterized by the prevalence of multiple subtypes and circulating recombinant forms with gradual replacement of pure HIV-1 subtypes in several geographical regions. The main objectives of the present study are to identify and analyse the full-length viral genomes of three unique recombinant forms (URFs); the HIV-1 isolates 07MYKLD47, 07MYKLD48 and 07MYKLD49 from Malaysia. Long-range polymerase chain reaction (PCR) amplification of seven overlapping reading frames was used to derive near full-length HIV-1 genomes. Detailed phylogenetic and bootscanning analyses were performed to determine phylogenetic associations and subtypic assignments. We further confirmed the mosaic composition of these CRF01_AE/B inter-subtype recombinant forms, which are composed of B-subtype fragment(s) in the backbone of CRF01_AE. Both 07MYKLD47 and 07MYKLD48 have an insertion of B subtype (880 bp and 532 bp) in the gag-pol and gp41-env gene regions, respectively. Whereas the isolate 07MYKLD49 has three B-subtype fragments inserted in different gene region along the genome; one each in the gag-pol (1862 bp) and pol-vif (1935 bp) regions, and a short B-subtype insertion (541 bp) in the 5' LTR-gag region. This highlights the public health relevance of newly emerging second generation HIV-1 recombinant forms and their dispersal, along with their rapid and continuous evolution in the region.

Study on gag-pol region characteristics of HIV-1 strains epidemic in Liaoning province

To investigate the genetic subtypes and sequence characteristics of gag-pol region of HIV-1 epidemic in Liaoning province. 99 whole blood samples of HIV-1 infected individuals of Liaoning province were collected. Provirus DNA was extracted from anti-coagulated peripheral whole blood, and then the sequences of gag-pol region were amplified through nested PCR and then sequenced. Genetic subtypes were identified by phylogenetic analysis, controlled with new version subtype reference sequences of HIV sequence database. The genetic distances of different coding regions were calculated by MEGA2.0 with Kimura 2-parameter model. The synonymous (Ks) and anonymous (Ka) as well as the ratio of Ks/Ka were analyzed by SNAP tool in HIV sequence database. There are many HIV-1subtypes epidemic in Liaoning province. We found five subtype: A, Thai B, B, C and G, five CRFs: CRF01, CRF03, CRF06, CRF07, CRF08, and one inter CRF recombinant ICR07/08 in 99 HIV-1 infected individuals of Liaoning province. Thai B subtype was the main subtype epidemic in Liaoning, next were CRF01_AE. In gag-pol region, the genetic distances of p17, p2-p6 were higher than p24, protease and RT coding region. The genetic distance of p24 region in B subtype strains were relative high compared with other subtypes. The genetic distance and the value of Ks and Ka were rather low in each region of CRF07 and CRF08. The ratio of Ks/Ka of p2-p6 region was the lowest in each subtype, the mean value was below 1. The ratio of Ks/Ka of RT region was as high as 10.45. The HIV-1 genetic subtypes in Liaoning province were rather complex. The p24, protease and RT coding region coding region were more conserved in gag-pol region. P24 region of B subtype strains received more positive selective pressure, while the RT region of CRF01_AE strains received more negative selective pressure. CRF07_BC and CRF08_BC were the youngest strains transmitted to Liaoning province and showed strong founder effect.

Significant Expansion ofVicia pannonicaGenome Size Mediated by Amplification of a Single Type of Giant Retroelement

Amplification and eventual elimination of dispersed repeats, especially those of the retroelement origin, account for most of the profound size variability observed among plant genomes. In most higher plants investigated so far, differential accumulation of various families of elements contributes to these differences. Here we report the identification of giant Ty3/gypsy-like retrotransposons from the legume plant Vicia pannonica, which alone make up approximately 38% of the genome of this species. These retrotransposons have structural features of the Ogre elements previously identified in the genomes of pea and Medicago. These features include extreme size (25 kb), the presence of an extra ORF upstream of the gag-pol region, and a putative intron dividing the prot and rt coding sequences. The Ogre elements are evenly dispersed on V. pannonica chromosomes except for terminal regions containing satellite repeats, their individual copies show extraordinary sequence similarity, and at least part of them are transcriptionally active, which suggests their recent amplification. Similar elements were also detected in several other Vicia species but in most cases in significantly lower numbers. However, there was no obvious correlation of the abundance of Ogre sequences with the genome size of these species.

Genetic Diversification and Recombination of HIV Type 1 Group M in Kinshasa, Democratic Republic of Congo

As the HIV-1 pandemic becomes increasingly complex, the genetic characterization of HIV strains bears important implications for vaccine research. To better understand the molecular evolution of HIV-1 viral diversity, we performed a comparative molecular analysis of HIV strains collected from high-risk persons in Kinshasa, Democratic Republic of Congo (DRC). Analysis of the gag-p24, env-C2V3 and -gp41 regions from 83 specimens collected in 1999-2000 revealed that 44 (53%) had concordant subtypes in the three regions (14 subsubtype A1, 10 subtype G, 8 subtype D, 5 subtype C, 2 each subsubtype F1 and CRF01_AE, and one each of subtypes H and J, and subsubtype A2, while the remaining 39 (47%) had mosaic genomes comprising multiple subtype combinations. Similar multisubtype patterns were also observed in 24 specimens collected in 1985. Sequence analysis of the gag-pol region (2.1 kb) from 21 discordant specimens in the gag-p24, env-C2V3 and -gp41 regions in 1985 and 1999-2000 further confirmed the complex recombinant patterns. Despite the remarkable similarity in overall subtype distribution, the intra- and intersubtype distances of major subtypes A1 and G increased significantly from 1985 to 1999-2000 (p=0.018 and p=0.0016, respectively). Given the complexity of HIV-1 viruses circulating in DRC, efforts should focus on the development of vaccines that result in cross-clade immunity.

A robust measure of HIV-1 population turnover within chronically infected individuals.

A simple nonparameteric test for population structure was applied to temporally spaced samples of HIV-1 sequences from the gag-pol region within two chronically infected individuals. The results show that temporal structure can be detected for samples separated by about 22 months or more. The performance of the method, which was originally proposed to detect geographic structure, was tested for temporally spaced samples using neutral coalescent simulations. Simulations showed that the method is robust to variation in samples sizes and mutation rates, to the presence/absence of recombination, and that the power to detect temporal structure is high. By comparing levels of temporal structure in simulations to the levels observed in real data, we estimate the effective intra-individual population size of HIV-1 to be between 10(3) and 10(4) viruses, which is in agreement with some previous estimates. Using this estimate and a simple measure of sequence diversity, we estimate an effective neutral mutation rate of about 5 x 10(-6) per site per generation in the gag-pol region. The definition and interpretation of estimates of such "effective" population parameters are discussed.

Genetic diversity and high proportion of intersubtype recombinants among HIV type 1-infected pregnant women in Kisumu, western Kenya.

The high genetic diversity of HIV-1 continues to complicate effective vaccine development. To better understand the extent of genetic diversity, intersubtype recombinants and their relative contribution to the HIV epidemic in Kenya, we undertook a detailed molecular epidemiological investigation on HIV-1-infected women attending an antenatal clinic in Kisumu, Kenya. Analysis of gag-p24 region from 460 specimens indicated that 310 (67.4%) were A, 94 (20.4%) were D, 28 (6.1%) were C, 9 (2.0%) were A2, 8 (1.7%) were G, and 11 (2.4%) were unclassifiable. Analysis of the env -gp41 region revealed that 326 (70.9%) were A, 85 (18.5%) D, 26 (5.7%) C, 9 (2.0%) each of A2 and G, 4(0.9%) unclassifiable, and 1 (0.2%) CRF02_AG. Parallel analyses of the gag-p24 and env-gp41 regions indicated that 344 (74.8%) were concordant subtypes, while the remaining 116 (25.2%) were discordant subtypes. The most common discordant subtypes were D/A (40, 8.7%), A/D (27, 5.9%), C/A (11, 2.4%), and A/C (8, 1.7%). Further analysis of a 2.1-kb fragment spanning the gag-pol region from 38 selected specimens revealed that 19 were intersubtype recombinants and majority of them were unique recombinant forms. Distribution of concordant and discordant subtypes remained fairly stable over the 4-year period (1996-2000) studied. Comparison of amino acid sequences of gag-p24 and env-gp41 regions with the subtype A consensus sequence or Kenyan candidate vaccine antigen (HIVA) revealed minor variations in the immunodominant epitopes. These data provide further evidence of high genetic diversity, with subtype A as the predominant subtype and a high proportion of intersubtype recombinants in Kenya.

Naturally occurring amino acid polymorphisms in human immunodeficiency virus type 1 (HIV-1) Gag p7(NC) and the C-cleavage site impact Gag-Pol processing by HIV-1 protease.

Human immunodeficiency virus type 1 (HIV-1) protease activity is targeted at nine cleavage sites comprising different amino acid sequences in the viral Gag-Pol polyprotein. Amino acid polymorphisms in protease and in regions of Gag, particularly p7(NC) and the C-cleavage site between p2 and p7(NC), occur in natural variants of HIV-1 within infected patients. Studies were designed to examine the role of natural polymorphisms in protease and to identify determinants in Gag that modulate protease processing activity. Closely related Gag-Pol regions from an HIV-1-infected mother and two children were evaluated for processing in an inducible expression system, for protease activity on cleavage-site analogues, and for impact on replication by recombinant viruses. Gag-Pol regions displayed one of three processing phenotypes based on the appearance of Gag intermediates and accumulation of mature p24(CA). Gag-Pol regions that were processed rapidly to produce p24(CA) resulted in high-level replication by recombinant viruses, while slow-processing Gag-Pol variants resulted in recombinant viruses that replicated with reduced kinetics in both T cell lines and peripheral blood mononuclear cells. Direct impact by Gag sequences on processing by protease was assessed by construction of chimeric Gag-Pol regions and by site-directed mutagenesis. Optimal protease activity occurred when Gag and Pol regions were derived from the same gag-pol allele. Heterologous Gag regions generally diminished rates and extent of protease processing. Natural polymorphisms in novel positions in p7(NC) and the C-cleavage site have a dominant effect on protease processing activity. Accumulation of Gag products after processing at the C site appears to delay subsequent cleavage and production of mature p24(CA).

The centromere composition of multiple repetitive sequences on rice chromosome 5.

The large-scale primary structure of the centromeric region of rice chromosome 5 was analyzed, the first example in a cereal species. The yeast artificial chromosome (YAC) and bacterial artificial chromosome (BAC) contigs aligned on the centromere of rice chromosome 5 (CEN5) covered a distance of more than 670 kb. Strong suppression of genetic recombination, one of the features of a functional centromere, occurred along the contig region. The most remarkable feature of CEN5 is the composition of the multiple repetitive elements. Oryza-specific RCS2 short tandem repeats were clustered along less than 100 kb at one end of the contig. At least 15 copies of the conserved domain of the 1.9 kb RCE1 centromeric repeats, which are similar to the long terminal repeats (LTRs) of gypsy-type retrotransposon RIRE7, were dispersed mainly in 320 kb stretches next to RCS2 tandem clusters. Many copies of the LTR-like sequences of RIRE3 and RIRE8, another gypsy-type retrotransposon, were also found throughout the contig. On the other hand, the gagpol region was less conserved in the contig. These results indicate that the rice centromere is composed of multiple repetitive sequences with the RCS2 tandem cluster probably being situated as the core of a functional centromere of some hundreds of kilobases to megabases in length.

Functional association between the nef gene product and gag-pol region of HIV-1

Nef gene function is diverse among virus isolates of primate immunodeficiency viruses. We found differential effects of nef mutation on the virus replication between two HIV-1 clones, NL432 and LAI. The nef mutation in NL432 affected the infectivity more severely compared with that in LAI, although the Nef functions of both clones were comparable. Analysis with a series of chimeric viruses between NL432 and LAI revealed that the gag-pol region was responsible for the differential effect of nef mutation. The functional association between Nef and gag-pol suggested that one of the potential targets of Nef was located within the gag-pol region.

RIRE2, a novel gypsy-type retrotransposon from rice.

The 441-bp DNA segment in a PCR-amplified fragment from Oryza sativa cv. IR36 was found to have a sequence with features characteristic of LTRs of retroelements, which was named RIRE2 (Rice retroelement #2) and further analyzed. Cloning and sequencing analyses of the DNA segments connected to LTR-like sequence showed that RIRE2 has a long internal region almost 10 kb long that is flanked by LTR-like sequences. This internal region carries a primer binding site (PBS) and polypurine tract (PPT) which are necessary for cDNA synthesis of retroelements. The PBS sequence is complementary to the 3' end region of tRNA(Arg). The internal region has an rt gene homologous to that of gypsy-type retrotransposons, evidence that RIRE2 is indeed a retrotransposon related to gypsy from Drosophila. RIRE2 has an extra sequence more than 4 kb long in the region downstream of gag-pol. Phylogenetic analysis of the putative amino-acid sequences of the rt gene as well as the int gene showed that RIRE2 is related to a group of gypsy-type retrotransposons of a large size that include Grande1-4 of teosinte, Tat4-1 and Athila1-1 of Arabidopsis thaliana, and Cyclops-2 of pea, but distantly related to any other group of gypsy-type retrotransposons, including RIRE3 and RIRE8 of rice. RIRE2 and Grande1-4 had the highest homology in the gag-pol region, but the nucleotide sequences of the LTR regions differed. Both elements had significant homology in the middle area of the extra regions downstream of gag-pol, in which they had an open reading frame encoding a protein with no known function on the opposite strand from that coding for gag-pol.

The non-env tropism of HIV/SIV (Review).

The tropism of human and simian immunodeficiency viruses (HIV and SIV) determined by sequences other than env has been studied. The restriction of HIV-1 replication in monkey cells was demonstrated to be regulated by viral non-env sequence. Likewise, the gag-pol region of SIVagm (virus of the African green monkey) genome was found to be responsible for growth restriction in human cells of the virus. No viral DNA synthesis was detected in cells nonpermissive for the viruses. In addition, a number of HIV-1 gag gene mutants, which have an early defect in viral replication cycle and direct no viral DNA synthesis in some cells, exhibited a phenotype of host range mutant. Taken together, it can be concluded that the viral tropism associated with the uncoating/ reverse transcription process does exist in HIV/SIV replication. Furthermore, many of the accessory gene mutants of HIV/SIV exhibit host cell-dependent replication property. In this review, we summarize these examples of non-env tropism of HIV/SIV.

Deleted HTLV-1 provirus in cord-blood samples of babies born to HTLV-1-carrier mothers.

We screened 596 cord-blood samples by nested short PCRs for the gag and pX regions of HTLV-1 which are capable of detecting a single copy. The samples were derived from 449 and 147 babies born to seropositive and seronegative mothers respectively. Of these, 20 samples were positive in at least one of the PCRs: 9 (45%) were positive in both PCRs, but 10 and 1 samples in either the pX or the gag PCR respectively. These samples were tested further in nested long PCRs directed for gag-pX, gag-pol and pol-pX regions capable of detecting 8, 1 and 2 copies respectively. Of 9 dually positive samples, 7 (77%) showed the predicted 6.2-kbp band in the gag-pX PCR; only 2 of them had the predicted band alone; 7 samples had discrete bands shorter than the predicted size. In the gag-pol PCR, all 9 samples showed the predicted 2.2-kbp band alone. In the pol-pX PCR, 819 samples showed the predicted 4.2-kbp band, including one with an additional 2.1-kbp band, and the last a 1.0-kbp band alone. Thus, all of the dually positive samples had proviruses harboring gag, pol and pX priming sites. In contrast, none of the 11 singly positive samples showed the predicted band in the gag-pX PCR: 5 had no visible band, and the other 6 had shorter bands only. None of these 11 samples showed any positive signal in either gag-pol or pol-pX PCR. Our results suggest that HTLV-1 proviruses in the cord blood are frequently defective.

Deleted HTLV‐1 provirus in cord‐blood samples of babies born to HTLV‐1‐carrier mothers

We screened 596 cord-blood samples by nested short PCRs for the gag and pX regions of HTLV-1 which are capable of detecting a single copy. The samples were derived from 449 and 147 babies born to seropositive and seronegative mothers respectively. Of these, 20 samples were positive in at least one of the PCRs: 9 (45%) were positive in both PCRs, but 10 and 1 samples in either the pX or the gag PCR respectively. These samples were tested further in nested long PCRs directed for gag-pX, gag-pol and pol-pX regions capable of detecting 8, 1 and 2 copies respectively. Of 9 dually positive samples, 7 (77%) showed the predicted 6.2-kbp band in the gag-pX PCR; only 2 of them had the predicted band alone; 7 samples had discrete bands shorter than the predicted size. In the gag-pol PCR, all 9 samples showed the predicted 2.2-kbp band alone. In the pol-pX PCR, 8/9 samples showed the predicted 4.2-kbp band, including one with an additional 2.1-kbp band, and the last a 1.0-kbp band alone. Thus, all of the dually positive samples had proviruses harboring gag, pol and pX priming sites. In contrast, none of the 11 singly positive samples showed the predicted band in the gag-pX PCR: 5 had no visible band, and the other 6 had shorter bands only. None of these 11 samples showed any positive signal in either gag-pol or pol-pX PCR. Our results suggest that HTLV-1 proviruses in the cord blood are frequently defective. Int. J. Cancer 77:701–704, 1998. © 1998 Wiley-Liss, Inc.

Gag-Pol region determines the tropism of SIVagm for human cells.

Simian immunodeficiency virus isolated from African green monkeys (SIVagm) does not grow in many of human cell lines such as CEM x 174, H9, and MT-4, but could replicate in some human cell lines. Sequence of SIVagm responsible for its narrow host range was determined by making and monitoring growth potential of chimeric clones between SIVagm and human immunodeficiency virus type 1 (HIV-1). The results obtained indicated that the gag-pol region determines the observed narrow host range. By monitoring virus DNA synthesis and progeny virion production, the defect(s) of SIVagm in the replication in the restricted cells was demonstrated to be located at early phase.

Association between the presence of sequences homologous to gag-pol region of the HIV-1 in DNA from sets of connective disease patients and the detection of autoantibodies against cell nucleus particles: Prokop J. Postepy Dermatol 1995;12:455–461. (In Polish)

Association between the presence of sequences homologous to gag-pol region of the HIV-1 in DNA from sets of connective disease patients and the detection of autoantibodies against cell nucleus particles: Prokop J. Postepy Dermatol 1995;12:455–461. (In Polish)

Integration Patterns of HTLV-I Provirus in Relation to the Clinical Course of ATL: Frequent Clonal Change at Crisis From Indolent Disease

AbstractWe examined human T-lymphotropic virus type I (HTLV-I) DNA integration in 68 patients with adult T-cell leukemia/lymphoma (ATL) by Southern blotting using EcoRI, which does not cut within the 9 kb of the genome and probes for pX and gag-pol region of HTLV-I. We detected defective proviral integration as a monoclonal band of various sizes with the pX but not with the gag-pol probe, or a monoclonal band of less than 9 kb with the pX probe, in 20 patients (29.4%). These were designated defective (D) type. With both probes, a single band greater than 9 kb was detected in 34 (50.0%), designated complete (C) type, and two or more bands greater than 9 kb, were designated multiple (M) type, in 14 (20.6%). Advanced age, a high LDH value, and hypercalcemia were more frequent in D type patients. The median survival time (MST) was 6.8, 24.4, and 33.3 months, for D, C, and M types, respectively (log rank P = .006). Among 52 sequentially examined patients, the HTLV-I integration patterns changed in 4 (7.5%). In three of these four, the rearrangements of the T-cell receptor (TCR)b gene concomitantly changed, suggesting the appearance of a new ATL clone. Another patient had the same rearrangement of the TCRb gene, indicating clonal evolution. The HTLV-I integration pattern changed at crisis from indolent to aggressive ATL in three patients. These findings suggested that the HTLV-I integration patterns have clinical implications in ATL pathophysiology. In contrast to the clonal evolution characteristic of the multistep carcinogenesis of most human malignancies, the frequent clonal change of ATL at crisis is a peculiar phenomenon, probably reflecting the emergence of multiple premalignant clones in viral leukemogenesis as suggested in Epstein-Barr virus associated lymphomagenesis in the immunocompromised host.

Characterization of antisense RNA-mediated inhibition of SIV replication

Antisense RNA-mediated inhibition of HIV showed mixed success in previous experiments. In order to elucidate the parameters influencing the efficacy of an antisense RNA approach, retroviral vectors encoding 4.5 kb, 3.5 kb, or 2.5 kb antisense RNA of the gag-pol region of SIV (simian immunodeficiency virus) were constructed and used to transduce a CD4-positive CEM174 cell line. The growth rate of transduced cells was measured, and results showed that antisense RNAs have no detrimental effect on cell growth. Similar levels of antisense RNA expression were observed in all transduced cells by Northern analysis. The transduced cells were challenged with uncloned SIVmac239 at a m.o.i. (multiplicity of infection) of 1.0, 0.1, or 0.01. At a m.o.i. of 1.0 or 0.1, no significant inhibition of viral replication was observed in any antisense construct transduced cells up to 9 days postinfection. At a lower m.o.i. (0.01), viral replication was effectively inhibited in 3.5 kb antisense transduced cells as compared to 4.5 kb and 2.5 kb antisense transduced cells at 15 days postinfection. These data suggest that the size of antisense RNA and the challenge dose play a significant role in achieving effective inhibition.