Viral DNA Copy Number Research Articles

Epstein-Barr virus DNA in body fluids.

Advances in polymerase chain reaction technology have greatly simplified the ability to detect and monitor Epstein-Barr virus DNA copy number in a variety of settings. An initial focus on cell-associated viruses by many investigators has shown some interesting results regarding the dynamics of Epstein-Barr virus infection. Several findings are unexpected. A relation between HIV load or CD4 T-cell counts and Epstein-Barr virus copy number is not seen. Furthermore, highly active antiretroviral treatment therapy in HIV patients that results in a rise of CD4 T cells may sometimes be associated with a rise in cell-associated Epstein-Barr virus load. Detection of Epstein-Barr virus in spinal fluid is useful in the diagnosis of primary central nervous system lymphoma, and monitoring of Epstein-Barr virus DNA copy number in spinal fluid may be useful in assessing response. Cell-free DNA in serum or plasma is emerging as a useful diagnostic tool in several settings. Fetal DNA can be detected in maternal serum or plasma. Tumor DNA can be detected in serum or plasma in association with a variety of cancers. Epstein-Barr virus DNA in serum or plasma has been found in infectious mononucleosis, nasopharyngeal carcinoma, posttransplant lymphoma, and nasal lymphoma. In each of these malignancies, its detection or quantification has been shown to be of prognostic significance. The utility of Epstein-Barr virus DNA detection and quantification in the serum or plasma of patients with HIV malignancies has yet to be determined but holds great promise.

The use of nested polymerase chain reaction and restriction fragment length polymorphism for the detection and typing of mucosal human papillomaviruses in samples containing low copy numbers of viral DNA

Mucosal human papillomaviruses (HPVs) that infect the genital area have also been shown to infect the oral cavity. In this study a restriction fragment length polymorphism (RFLP) method was developed on a nested polymerase chain reaction (PCR) product to identify ten high risk HPV types 16, 18, 31, 33, 35, 45, 51, 52, 58 and 59 as well as the low risk HPV 11. HPV DNA was detected in 23/31 (74%) of buccal specimens using a sensitive nested PCR employing degenerate consensus primers ( Williamson and Rybicki, 1991). Consensus PCR using the PGMy09/11 primers. was able to detect HPV in only 29% of the specimens that had tested positive using the nested HPV PCR primers. HPV 11 type specific primers detected HPV 11 DNA in only 66% of the specimens showing HPV 11 DNA by means of nested PCR and RFLP. A Genbank search revealed that the PCR primers could detect a wide range of mucosal HPV types including types HPV 70, 72 and 73 which have all been isolated from immunocompromised patients. Of the 23 buccal specimens that were positive for HPV DNA, 13 were single infections, five were dual infections and three were triple infections. The HPV types identified by RFLP were: HPV 11 (18/23), HPV 18 (8/23), HPV 16 (3/23), and HPV 33 (1/23). HPV 13 (2/23) was identified by direct sequencing of the inner amplicon of the PCR product.

Mathematic modeling of the risk of HBV, HCV, and HIV transmission by window‐phase donations not detected by NAT

Blood transfusion centers around the world have introduced minipool NAT to reduce the risk of HBV, HCV, and HIV transmission by blood donations drawn in the infectious window phase. What would be the reduction in the residual risk when minipool NAT would be replaced by single-donation NAT? A mathematic model was developed to estimate the probability of virus transmission by blood transfusion when NAT screening methods are used for virologic safety testing. The major assumptions used are threefold: 1) The viral nucleic acid concentrations in the early window phase of infection double in 2.8 (HBV), 0.74 (HCV), and 0.90 (HIV) days. 2) The detectability of low copy numbers of viral DNA or RNA by the screening assay can be described with a probit model. 3) The probability of infection depends linearly on the logarithm of the administered dose, with 50-percent infectivity rates at 10 (HBV and HCV) or 1000 (HIV) viral nucleic acid copies per transfusion unit (estimates based on NAT studies with samples of known infectivity in chimpanzees). A reasonably simple equation was obtained that allows studying the effect of the sensitivity of the NAT assay and of the pool size used for screening on the residual risk of transfusion-transmitted infection. The computations are illustrated by using observed sensitivity estimates of various NAT methods. By using epidemiologic data among European donors over 1997 as baseline, the calculations predict that the incidence of virus transmission per 10-million RBC transfusions reduces with the following numbers when lowering the test pool size from 96 to 1 (single-donation testing): HBV from 11 to 13 to 3.3 to 5.1, HCV from 1.7 to 2.0 to 0.5 to 0.8, and HIV from 0.47 to 0.62 to 0.010 to 0.045 (ranges for the different NAT screening methods). A proper mathematic model for the calculation of residual infection risk by blood transfusion helps understand the impact of introducing new NAT methods for blood safety testing.

Development of strain-specific real-time PCR and RT-PCR assays for quantitation of chicken anemia virus

Chicken anemia virus (CAV) is a ubiquitous pathogen of poultry. A CAV specific TaqMan™-based PCR and RT-PCR assay for real-time quantitation of viral load and relative quantitation of virus-specific transcript levels was developed. Detection of viral DNA copy number from infected MDCC-CU147 cells was determined by extrapolation from a CAV plasmid-based standard curve. Viral load increased proportionally with increasing cell number harvested, increasing from 4×10 2 copies in 250 cells with 38% virus positive cells in an indirect immunofluorescence assay to 8×10 5 copies in 250,000 cells with 64% infected cells. The estimated average viral copy number per infected cell ranged from 5 to 14. Strain-specific primers were developed to distinguish between the Cux-1 and CIA-1 strains of CAV. These primers exhibited a 3 to 4 log differential in amplification comparing homologous versus heterologous virus-primer combinations. The sensitivity of the real-time assay was found to be comparable to a nested PCR assay using DNA samples from a SPF poultry flock exposed to the SH-1 strain of CAV. The real-time PCR detected from 1.7 to 4.2 target molecules in three out of four samples that were positive by nested PCR using 50% of the DNA used in the nested PCR. Relative viral transcript levels for Cux-1 and CIA-1 infected cell cultures increased proportionally with increasing cell numbers harvested for RNA extraction. This assay will be important for both diagnosis and in understanding the complex pathogenesis of CAV infection.

Serum/Plasma Viral DNA

Following reports describing circulating tumor DNA, serum/plasma viral nucleic acid has shown its potential as a new diagnostic target in cancer. In the majority of examples of viral carcinogenesis, the viral genome is consistently present in certain tumors and serves as an effective marker. This article reviews recent findings, proposes possible mechanisms, and examines the potential clinical application of serum/plasma Epstein-Barr virus (EBV) DNA in nasopharyngeal cancer (NPC) and human papillomavirus (HPV) DNA in cervical carcinoma (CC). These tumors share a DNA viral etiology and present similar histopathological findings. However, plasma EBV and HPV DNA are distinct in several aspects, including incidence, mechanism of release from tumor, and clinical application. Both circulating cell-free EBV and HPV DNA reveal the same viral type as their matched tumors, indicating both are derived from the neoplastic tissue. Plasma viral DNA incidence and copy number are high in NPC, but low in HPV-associated cancers. Whereas much EBV DNA in NPC is episomal, the resistance to DNase treatment of serum EBV DNA and evidence confirming lytic EBV replication in NPC suggest that a reasonable proportion of plasma EBV DNA is virions. On the contrary, plasma HPV genomes, as in CC, integrate into host chromosome. Plasma EBV DNA copy number, by quantitative PCR, is related to tumor mass, predicts prognosis, measures immediate response to treatment, and is useful in early detection of recurrence. Plasma HPV DNA, on the other hand, is associated with and can be considered as an early tumor marker for distant metastasis.

Detection of SV40 DNA sequences in human tissue.

AbstractSeveral recent investigations using polymerase chain reaction (PCR) detection methods have identified SV40-like DNA sequences in human neoplasms, particularly choroid plexus tumors (1), ependymomas (1), mesotheliomas (2), and osteosarcomas (3,4). Difficulties have arisen because of the use of paraffin-fixed tissue, loss of possible viral episomes during DNA isolation, low copy numbers of viral DNA, PCR contamination, and confusion with BKV and JCV sequences. Nonetheless, the authentication of amplified products by DNA sequencing, the finding of a single 72-bp viral-enhancer repeat element, variability of large tumor antigen (T-ag) carboxy terminal DNA sequences in many specimens, and the isolation of SV40 virus from a choroid plexus tumor all support the validity of the basic observation that SV40 is present in humans, and may therefore contribute to oncogenesis in special situations.KeywordsPolymerase Chain ReactionPolymerase Chain Reaction ProductPolymerase Chain Reaction MachineChoroid Plexus TumorFreeze Tumor SampleThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Analysis of individual human trigeminal ganglia for latent herpes simplex virus type 1 and varicella-zoster virus nucleic acids using real-time PCR.

Herpes simplex virus type 1 (HSV-1) and varicella-zoster virus (VZV) establish latent infections in the peripheral nervous system following primary infection. During latency both virus genomes exhibit limited transcription, with the HSV-1 LATs and at least four VZV transcripts consistently detected in latently infected human ganglia. In this study we used real-time PCR quantitation to determine the viral DNA copy number in individual trigeminal ganglia (TG) from 17 subjects. The number of HSV-1 genomes was not significantly different between the left and right TG from the same individual and varied per subject from 42.9 to 677.9 copies per 100 ng of DNA. The number of VZV genomes was also not significantly different between left and right TG from the same individual and varied per subject from 37.0 to 3,560.5 copies per 100 ng of DNA. HSV-1 LAT transcripts were consistently detected in ganglia containing latent HSV-1 and varied in relative expression by >500-fold. Of the three VZV transcripts analyzed, only transcripts mapping to gene 63 were consistently detected in latently infected ganglia and varied in relative expression by >2,000-fold. Thus, it appears that, similar to LAT transcription in HSV-1 latently infected ganglia, VZV gene 63 transcription is a hallmark of VZV latency.

Impaired Angiogenic Balance and Suppression of Tumorigenicity in HeLa Cells Chronically Exposed to Interferon-α

We have previously reported that IFNα-chronic treatment for 41 days induced a partial phenotype reversion on HeLa cells along with a down-regulation of HPV18 mRNA levels. However, tumorigenicity of these cells in nude mice was unchanged. Interestingly, after 1 year of IFNα-chronic exposition, HeLa cells failed to induce s.c. tumors when injected into nude mice. In such experimental conditions both HPV18 DNA integration pattern and viral DNA copy number present in HeLa cells remained intact in the nontumorigenic phenotype cells. As result of the treatment with IFNα, HeLa cells rendered more resistant to lysis mediated by activated natural killer cells in vitro. Furthermore, IFNα-chronic treatment was able to induce VEGF and decrease bFGF mRNA expression, suggesting a potential effect on the angiogenic behavior of these tumoral cells. Thus, long-term treatment of HeLa cells with IFNα can accomplish a reversion of the malignant phenotype by a sequential multistep mechanism, in which the antiangiogenic effect of IFNα could be one of the contributing events.

Proteasome-mediated degradation of the papillomavirus E2-TA protein is regulated by phosphorylation and can modulate viral genome copy number.

The bovine papillomavirus E2 proteins regulate viral transcription, replication, and episomal genome maintenance. We have previously mapped the major phosphorylation sites of the E2 proteins to serine residues 298 and 301 and shown that mutation of serine residue 301 to alanine leads to a dramatic (10- to 20-fold) increase in viral DNA copy number. In this study we analyzed how phosphorylation regulates E2 protein function. S301 is located in a PEST sequence; these sequences are often found in proteins with a short half-life and can be regulated by phosphorylation. We show here that the E2 protein is ubiquitinated and degraded by the proteasome. Mutation of serine 301 to alanine increases the half-life of E2 from approximately 50 min to 160 min. Furthermore, the A301 E2 protein shows greatly reduced ubiquitination and degradation by the proteasome. These results suggest that the E2 protein level is regulated by phosphorylation, which in turn determines viral episomal copy number.

Inverse relationship between the expression of the human papillomavirus type 16 transcription factor E2 and virus DNA copy number during the progression of cervical intraepithelial neoplasia.

The human papillomavirus type 16 (HPV-16) status of 43 cervical biopsies, which had been characterized histologically as normal, various grades of cervical intraepithelial neoplasia (CIN) and invasive squamous cell carcinoma, was examined by using (i) a novel antibody against the HPV-16 E2 protein, (ii) sensitive HPV-16 DNA in situ hybridization and (iii) microdissection/PCR for the E2 ORF. The data indicate that E2 protein expression is highest in koilocytes in lower-grade CIN (I), but decreases with increasing grade, whereas the detection of HPV DNA is delayed until CIN I/II, rising to the highest levels in carcinoma cells. Co-localization of E2 with HPV-16 DNA-positive cells was most commonly observed in koilocytes in CIN II lesions. PCR analyses of microdissected epithelium from the same or serial sections indicated that E2 ORFs were retained in an intact form in a number of higher-grade CIN lesions and invasive carcinomas.

Consistent high viral load of human papillomavirus 16 and risk of cervical carcinoma in situ: a nested case-control study

Persistent infection with certain types of human papillomavirus (HPV) is believed to be a prerequisite for the development of cervical neoplasia. Persistence may depend on certain characteristics, such as viral load, which has so far been given little attention. We investigated the association between HPV 16 viral load and cervical carcinoma in situ. We did a nested case-control study of women participating in cytological screening in Sweden. We used a sensitive quantitative PCR assay to estimate HPV 16 load in multiple smears for each woman, taken during a period of up to 26 years before diagnosis. We calculated C, values, which decrease as the number of viral DNA copies increases. 2081 smears from 478 cases and 1754 smears from 608 controls were tested. Among cases, we found a consistently increased load of HPV 16 already 13 years or more before diagnosis, and when many smears were still cytologically normal. Women with high HPV 16 viral loads were at least 30 times the relative risk of HPV-16-negative women more than a decade before diagnosis. The increase in relative risk was constant over time. About 25% of women (95% CI 0.12-0.32) infected with a high viral load before age 25 years developed cervical carcinoma in situ within 15 years. Cervical carcinoma in situ associated with HPV 16 occurs mainly in HPV-16-positive women who have consistently high viral loads long term. Women at high risk could be identified by use of a quantitative HPV test in addition to cytological screening.

Effect of lamivudine on human T-cell leukemia virus type 1 (HTLV-1) DNA copy number, T-cell phenotype, and anti-tax cytotoxic T-cell frequency in patients with HTLV-1-associated myelopathy.

Patients with human T-cell leukemia virus type 1 (HTLV-1)-associated myelopathy/tropical spastic paraparesis (HAM/TSP) typically have a high HTLV-1 proviral load in peripheral blood mononuclear cells and abundant, activated HTLV-1-specific cytotoxic T lymphocytes (CTLs). No effective treatment for HAM/TSP has been described so far. We report a 10-fold reduction in viral DNA for five patients with HAM/TSP during treatment with the reverse transcriptase inhibitor lamivudine. In one patient with recent-onset HAM/TSP, the reduction in viral DNA was associated with a fall in the frequency of CTLs specific to two peptides in the immunodominant viral antigen Tax. The half-life of peripheral blood mononuclear cell populations was estimated from changes in viral DNA copy number, CTL frequency, reduction in CD25 expression, and the loss of dicentric chromosomes following radiation-induced damage. Each of these four different techniques indicated a cellular half-life of approximately 3 days consistent with continuous lymphocyte replication and destruction. These results indicate that viral replication through reverse transcription significantly contributes to the maintenance of HTLV-1 viral DNA load. The relative contribution of proliferation versus replication may vary between infected people.

Human papillomavirus (HPV) DNA copy number is dependent on grade of cervical disease and HPV type.

The association between human papillomavirus (HPV) DNA copy number and cervical disease was investigated. Viral DNA copy number for the most common high-risk HPV types in cervical cancer (types 16, 18, 31, and 45) was determined in cervical cytobrush specimens from 149 women with high-grade cervical intraepithelial neoplasias (CIN II-CIN III), 176 with low-grade CIN (CIN I), and 270 with normal cytology. Quantitative, PCR-based fluorescent assays for each of the HPV genotypes and for the beta-globin gene were used. The amount of cellular DNA increased significantly with increasing disease; thus, HPV was expressed as copies per microgram of cellular DNA. The assay had a dynamic range of >10(7), allowing documentation for the first time of the wide range of HPV copy numbers seen in clinical specimens. Median HPV DNA copy number varied by more than 10(4) among the viral types. HPV16 was present in the highest copy number; over 55% of HPV16-positive samples contained more than 10(8) copies/microgram. Median copy number for HPV16 showed dramatic increases with increasing epithelial abnormality, an effect not seen with the other HPV types. HPV16 increased from a median of 2.2 x 10(7) in patients with normal cytology, to 4.1 x 10(7) in CIN I patients, to 1.3 x 10(9) copies/microgram in CIN II-III patients. Even when stratified by cervical disease and viral type, the range of viral DNA copies per microgram of cellular DNA was quite large, precluding setting a clinically significant cutoff value for "high" copy numbers predictive of disease. This study suggests that the clinical usefulness of HPV quantitation requires reassessment and is assay dependent.

Expression of CD26/dipeptidyl peptidase IV in adult T cell leukemia/lymphoma (ATLL)

The association of CD26/dipeptidyl peptidase IV (DPPIV) and human T lymphotropic virus type I (HTLV-I) was studied by two approaches. First, we examined the expression of CD26 in peripheral blood mononuclear cells (PBMC) from the patients with adult T cell leukemia/lymphoma (ATLL), an HTLV-I-related malignancy. The expression of CD26 on the surface of PBMC was decreased in all 20 patients with ATLL compared with those from normal individuals ( P < 0.01) and the expression of the CD26 gene transcript was not detectable in seven out of eight patients with ATLL. Then we compared the quantity of viral DNA in CD26-negative (CD26−) and CD26-positive (CD26+) cells obtained from 17 HTLV-I healthy carriers by using a polymerase chain reaction method. The CD26− cells had a higher copy number of viral DNA than CD26+ cells. These findings indicate that HTLV-I has in vivo tropism to CD26− cells, suggesting that some phenotypes of ATLL cells reflect the in vivo cellular tropism of HTLV-I.

PCR and RT-PCR analysis of infection and transcriptional activity of walleye dermal sarcoma virus (WDSV) in organs of adult walleyes (Stizostedion vitreum).

The pathogenesis of walleye dermal sarcoma virus (WDSV) infection was investigated in adult walleyes (Stizostedion vitreum). Three tumor-bearing and three tumor-free walleyes were collected in the spring from Oneida Lake, New York, and analyzed for viral infection and transcriptional activity. Specifically, the target organs for viral infection and supporting viral transcriptional activity were determined by assessing for the presence of WDSV DNA and RNA in the brain, liver, kidney, skin, and spleen. For each organ, WDSV DNA and RNA were detected using the polymerase chain reaction (PCR) and reverse transcription PCR (RT-PCR) respectively. Quantitative estimates of the number of viral DNA and RNA copies were obtained in each case by comparing the signal intensity of the sample to that of external controls. WDSV RNA/DNA ratios, based on those quantitative estimates, were computed for each organ. An RNA/DNA ratio of 3 was arbitrarily chosen as the threshold above which there was viral transcriptional activity. Viral DNA was found in all the organs examined from the three tumor-free walleyes. In those three tumor-free walleyes, low levels of WDSV RNA were detected in only one kidney and two spleen samples. In the three tumor-bearing walleyes, viral DNA was found in one brain, one kidney, two liver, and two skin samples. In contrast to the few organs from tumor-free walleyes in which WDSV RNA was detected, in tumor-bearing walleyes WDSV RNA was present in the one brain examined and in 2/3 kidney, 2/3 liver, 3/3 skin, and 3/3 spleen samples. A WDSV RNA/DNA ratio above 3 was obtained in all three tumor-bearing walleyes but in only one tumor-free fish. These data indicated that 1) both tumor-bearing and tumor-free walleyes were infected by WDSV, 2) many cell types were targeted by WDSV and supported viral transcription, and 3) tumor-bearing walleyes harbored a transcriptionally active WDSV, whereas tumor-free walleyes contained mostly silent WDSV DNA.

Human Papillomavirus DNA in Genital Cancers, Metastases, and Lymph Nodes

To evaluate the utility of human papillomavirus (HPV) DNA as molecular marker for diagnostic screening of lymph nodes for cancer metastases, we examined 16 involved and 124 histologically cancer-free nodes of 34 patients with cervical, vulval, or vaginal carcinomas associated with HPV 6, 16, 18, 31, 33, 45, or X. All metastases of HPV-positive tumors contained viral DNA of the same type as the primary cancer but in one case the copy number of viral genomes was dramatically reduced. In a varying proportion of histologically normal nodes of 7 of 23 patients with HPV 16-positive cancers HPV 16 DNA was revealed by Southern blot hybridization and/or PCR. Histologic reevaluation showed a metastasis in one case. Three examples of tumor heterogeneity regarding physical state or copy number of viral DNA are presented. Tumor heterogeneity is discussed as possible explanation for apparently inconsistent results such as HPV-negative metastases of HPV-positive tumors.

Intravirion reverse transcripts in the peripheral blood plasma on human immunodeficiency virus type 1-infected individuals

Variable levels of viral DNA have been demonstrated within human immunodeficiency virus type 1 (HIV-1) virions purified from cell cultures. In the present studies, it is demonstrated that DNase-resistant viral DNA is associated with HIV-1 virions purified from the peripheral blood plasma of both symptomatic and asymptomatic HIV-1-infected individuals. The differences in viral DNA copy numbers, detected by quantitative PCR in various regions of the HIV-1 genome, indicated that the intravirion HIV-1 DNA is frequently, but perhaps not totally, the result of partial reverse transcription. These in vivo data suggest that it may be valuable to further investigate the impact of virion-associated viral DNA upon the efficiency of intra- and interhost HIV-1 transmission modes.

Epstein-Barr virus genomes in undifferentiated and squamous cell nasopharyngeal carcinomas in Italian patients.

Although undifferentiated carcinoma (UC) and squamous cell carcinoma (SCC) of the nasopharynx are regarded as two distinct histopathologic and clinical entities, it is unclear whether, like UC, SCC carries Epstein-Barr virus (EBV) genomes. We used the polymerase chain reaction (PCR) on paraffin-embedded biopsy specimens to test for the presence of EBV DNA in 20 cases of UC and 9 cases of SCC. Multiple copies of the viral genome were regularly detected in all UCs; however, of the nine cases of SCC, seven had no detectable EBV DNA and two contained viral genomes in a low copy number. In parallel, a marked difference in the serum levels of anti-EBV antibodies between patients with UC and SCC was found. Our findings provide evidence for the specific association of EBV with UC in Italian patients and prove by means of a highly sensitive molecular technique that SCC is occasionally related to EBV DNA. Because of the absence of EBV DNA in most cases of SCC and the minimal viral DNA copy number in the few EBV-associated cases of SCC, a different pathway of oncogenic transformation and growth of the nasopharyngeal epithelium is suggested for SCC and UC.

Antiviral therapy of chronic hepatitis B: past, present, and future

A wide variety of agents has been used to treat chronic hepatitis B, but none has proved effective with the exception of interferon. Toxicity has been a major problem with some drugs whereas in others a lack of antiviral potency has been demonstrated. Alpha-interferon represents a good compromise because it has both immunomodulatory and antiviral properties; moreover, it is generally well tolerated. Loss of HBeAg and hepatitis B virus DNA may be anticipated in 40-50% of patients who are treated with doses of 5 million units daily or 10 million units thrice weekly for 16 weeks. While drug-related adverse effects occur commonly, the majority of clinically stable patients are able to tolerate this regimen, and withdrawal from drug is necessary in approximately 5% of patients. Unlike the situation with chronic hepatitis C, loss of viral replication tends to be sustained years later. Disappearance of HBsAg only occurs in 10-15% of treated patients within the first year after therapy, but an increasing number of responders demonstrate HBsAg seroconversion upon prolonged follow-up. Hepatitis B virus DNA usually disappears from serum by polymerase chain reaction at the time of HBsAg loss. Low copy numbers of residual viral DNA are still detectable in liver tissue at this time, but this has uncertain significance. Marked improvement in histological features has been observed years after loss of HBsAg. Pre-therapy levels of circulating viral DNA and aminotransferase activity, degree of histologic activity, and HIV status appear to influence the response to therapy.(ABSTRACT TRUNCATED AT 250 WORDS)

A Tat antagonist inhibits HIV-1 induction in naturally infected and experimentally infected T cells

Ro 5-3335, a novel antagonist of human immunodeficiency virus type 1 (HIV-1) Tat activity, inhibits acute and chronic HIV-1 infection in T lymphocytes. Here we describe the effects of Ro 5-3335 on the accumulation of viral DNA during primary infection, the induction of virus from a latently infected cell line, and the expression of virus upon activation of naturally infected T cells. Ro 5-3335 permitted initial DNA synthesis during primary infection, but inhibited the subsequent increase in viral DNA copy number. The induction of HIV-1, as determined by the synthesis of p24 core antigen, was inhibited by 99% by Ro 5-3335 in both the model cell line and naturally infected T cells.