The simultaneous presence and expression of human hepatitis C virus (HCV), human herpesvirus-6 (HHV-6), and human immunodeficiency virus-1 (HIV-1) in a single human T-cell

1743-422X-4-106 1743-422X Research The simultaneous presence and expression of human hepatitis C virus (HCV), human herpesvirus-6 (HHV-6), and human immunodeficiency virus-1 (HIV-1) in a single human T-cell Salahuddin S Zaki zaki@cimm.net Snyder A Katherine ksnyder@clunet.edu Godwin Andre agodwin@clunet.edu Grewal Renu rtg602@yahoo.com Prichard G John johnprichard@mail.co.ventura.ca.us Kelley S Ann annzaki@aol.com Revie Dennis revie@clunet.edu

Department of Basic Research, California Institute of Molecular Medicine, Ventura, California, USA

Department of Biology, California Lutheran University, Thousand Oaks, California, USA

Departments of Medicine and Family PracticeVentura County Medical Center, Ventura, CA

Department of Hematology and Oncology, Ventura County Hematology-Oncology Specialists, Ventura, CA

Virology Journal 1743-422X 2007 4 1 106 http://www.virologyj.com/content/4/1/106 17958914 10.1186/1743-422X-4-106 04 10 2007 24 10 2007 24 10 2007 2007 Salahuddin et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

We have developed a system that isolates and replicates HCV in vitro. These isolates are called CIMM-HCV. This system has made it possible to analyze the biology, nature, and extent of HCV variability, among other things. Individuals that are infected with HIV-1 are often also infected with HCV and HHV-6. In addition to HCV, our lab has systems for replicating HIV-1 and HHV-6. We asked whether all these viruses could infect the same cells. We report here the successful infection of a T-cell (CEM) by CIMM-HCV, HHV-6, and HIV-1. PCR analyses demonstrated that the CEM cells were productively infected by HHV-6A. RT-PCR showed that the same cell culture was positive for HCV and HIV-1. Co-infection of a T-cell by all three viruses was confirmed by transmission electron microscopy (TEM). All these viruses are highly cytolytic; therefore, triply-infected cells were short lived. However, HIV-1 and HCV co-infected cells unexpectedly lasted for several weeks. Viral replication was unhindered and the phenomenon of 'dominance' was not observed in our experiments. In addition, CIMM-HCV was present in the perinuclear space, suggesting their possible synthesis in the nucleus. This report is based entirely on viruses produced in vitro in our laboratories. As part of the determinations of host ranges of these viruses, studies were designed to demonstrate the infection of a single cell by these viruses and to study the consequences of this phenomenon. All measurements were made on cultured cells and cell culture supernatants.

Background

Individuals harbouring more than one virus in acute or chronic diseases are frequently observed. A minority of patients that are infected with HIV-1 are at least doubly infected 1. Infection with HIV-1, HCV, and human hepatitis B virus (HBV) may result from a common route of infection. The majority of these individuals are also infected with HHV-6, and other DNA viruses such as Epstein-Barr virus (EBV) or Cytomegalovirus (CMV). Except for HIV-1, many of these viruses co-exist in healthy individuals without causing any pathological consequences. The host range of all these viruses is well known.

Although co-infection by HIV-1 and HCV has been extensively studied in AIDS patients, in vitro studies of co-infected cell cultures are few and limited in scope. AIDS patients are frequently infected with HHV-6 in addition to HIV-1 and HCV. In general, HIV-1 adversely affects HCV-infected patients while the effects of HCV on HIV-infected patients are less defined and controversial 23. Increased morbidity in co-infected individuals would not be surprising. HAART, however, may have changed the dynamics of AIDS by prolonging the lives of HIV-infected individuals irrespective of infections and other associated problems 4.

Our effort is to understand the impact of multiple infections at a cellular level, both on virus reproduction and its characteristics and on their effects on cell functionality. This may help explain some pathogenic consequences such as neuropathy or dementia.

Results

We previously reported an in vitro system that can replicate HCV for extended periods of time 5. Later reports from our laboratories included an analysis of the 5'UTR of CIMM-HCV 6 and the discovery of significant HCV variants 7. Rare insertions and deletions have also been seen by others 8. The analysis of the 5'UTR revealed that there were no significant differences between HCV-RNA found in patient's blood and the CIMM-HCV.

Host ranges of each virus

Since HIV-1, HCV, and HHV-6A are routinely isolated in our laboratories, it was decided that we should determine whether these agents can co-infect the same cells. In developing our co-infection system, we first needed to select a cell line that could be infected by all three viruses (Table 1). Macrophages and T-cells were the most suitable cell types for our co-infection experiments. B-cells do not lend themselves to studying this group of viruses, as HIV-1 and HHV-6 are T-cell tropic agents. Since T-cells are easier to continuously culture than macrophages, and also more productive, CEM were selected as target cells.

Table 1

Summary of viral transmission experiments with various hematopoetic and liver cells

HCV short term

HCV long term

HIV

HHV-6

A. T-cells ¹

B. B-cells ²

C. Monocytes/macrophages ³

D. Neuronal precursors ⁴

E. Liver cells ⁵

Kupffer's

+/-

Hepatocytes

+/-

¹T-cells isolated from human fetal chord blood.

²B-cells immortalized by infection with transforming EBV.

³Monocyte/Macrophages, adherent cells stimulated with PMA.

⁴Recently isolated neuronal cells from fetal human brain.

⁵Freshly isolated liver cells from liver biopsies. Kupffer's cells are liver macrophages and Hepatocytes are liver endothelial cells.

Infection of T-cells by all three viruses

CEM cells were infected individually by each of the three viruses (Fig. 1). To test the infectivity of HHV-6A, CEM cells were infected with the virus (Figure 1, K1) 910. Two methods were used to determine whether the cell culture was infected and actively producing HHV-6A particles. First, characteristic cytopathic effects (CPE) on the cells were observed. The cells have the appearance of a balloon, called "juicy cells" (Figure 2) 10. Second, the cell culture supernatants were tested by PCR using the appropriate primer set (Table 2). A band of about 400 bp was seen after PCR analysis, indicating HHV-6A was replicating in these cells (Figure 3A, Lane 3).

Table 2

Primers used to analyze HHV-6A, HCV, and HIV

Virus

Primer

Sequence (5' to 3')

Reference

HCV

HCV 9.1

gac act cca cca tag atc act c

[5]

HCV

HCV 9.2

cat gat gca cgc tct acg aga c

[5]

HCV

HCV 10.1

ctg tga gga act act gtc ttc acg cag

[5]

HCV

HCV 10.2

cac tcg caa cca ccc tat cag

[5]

HIV

Con-1f1

cca gcn cac aaa ggn ata gga gg

[33]

HIV

Con-1r1

acb acy gcn cct tch cct ttc

[33]

HIV

Con-1r2

ccc aat ccc ccc ttt tct tta aaa tt

[33]

HHV-6

U16-17F

cgt aga aca gaa gac cgg c

[34]

HHV-6

U16-17R

aga act gca aat cgt tcc g

[34]

Figure 1

Schematic showing the infection process

Schematic showing the infection process. The viral transmissions used cell-free supernatants. The designations K1 through K7 don't represent the order of the experiments. The respective viruses and cell types for each cell culture are indicated in the boxes.

Figure 2

Cytopathic effects in CEM cells

Cytopathic effects in CEM cells. This picture represents the commonly observed cytopathic effects in triply-infected CEM cells. Large cells are seen with increasing frequency in infected cultures.

Figure 3

Agarose gels of PCR products

Agarose gels of PCR products. The DNA standard is a 1 kb plus ladder (Invitrogen). Arrows indicate the expected band sizes. Positive bands for K7 are seen in gel B lane 6 (HHV-6), gel C lane 4 (HCV), and gel F lane 3 (HIV-1). A. HHV-6. Lane 1 is the ladder, Lane 2 is uninfected CEM cells, Lane 3 is K1, Lane 4 is K2, and Lane 5 is K3. The positive bands are 401 bp in size. B. HHV-6. Lane 6 is K7. This sample was run on a different gel than gel A. C. HCV. Lane 1 is the ladder. Lane 2 is K5, Lane 4 is K7. The positive bands are 269 base pairs in size. D. HCV. Lane 1 is the ladder. Lane 3 is K6. E. HIV-1. Lane 1 is the ladder. Lane 2 is uninfected CEM cell supernatant, Lane 3 is K1, Lane 4 is K2, Lane 5 is K3, and Lane 6 is K4. The positive bands are 650 base pairs in size. F. HIV-1. Lane 1 is a 100 bp ladder (Invitrogen). Lane 2 is K6 and Lane 3 is K7.

CEM cells were infected separately with HCV (K5). Evidence of HCV was seen after RT-PCR (Figure 3C, Lane 2). CEM cells were also separately infected with HIV-1 (data not shown). All three viruses could productively infect CEM cells.

The general procedure we used to produce triply-infected cells was to sequentially infect CEM cells with the viruses. For a successful co-infection with multiple viruses, the order of infection of a cell type may determine whether the experiment will succeed. In our case, to produce the triply-infected cells, infection proceeded with CIMM-HCV, followed by HHV-6A and HIV-1 (Figure 1). Cell cultures were incubated overnight post infection and allowed to incubate at 37°C for the remainder of the experiment.

HHV-6A infected CEM cells (K1) were infected with HCV using our standard infection process to produce K6. The presence of HCV was determined by RT-PCR (Figure 3D, lane 3). Our results demonstrate that the CEM cells were co-infected with both HHV-6A and HCV (Figure 1, K6).

HHV-6A infected CEM cells were also infected separately with HIV-1 (Figure 1, K2). The presence of HIV-1 was determined by RT-PCR (Figure 3E, Lane 4). We therefore demonstrated that CEM cultures could be co-infected by the combinations of two different viruses (cultures K2 and K6).

As a control, we infected macrophages obtained from cord blood cells with HHV-6A (K3) or both HHV-6A and HIV-1 (K4). We were able to show infection of these cells by each virus (Figure 3A, Lane 5; Figure 3E, Lanes 5 and 6).

Finally the HCV infected K5 cells were infected with HHV-6A and HIV-1 from culture K2. The presence of HCV (Figure 3C, lane 4) and HIV-1 (Figure 3F, Lane 3) were determined using RT-PCR, and HHV-6A by PCR (Figure 3B, Lane 6). In short, the CEM cells were found to be simultaneously infected with all three viruses (Figure 1, K7).

The triply infected CEM cells were observed by light microscopy for CPE. The cells looked polymorphic compared to uninfected cells. This has been reported previously for HHV-6A 10, and HIV-1 11, and HCV 5. All three viruses are cytolytic, so triply infected CEM cultures lasted for approximately three weeks. The HHV-6A infected cells were the first to die. They were followed by HIV-1 infected cells and finally the HCV infected ones died. At this point these experiments were terminated.

Infection of single cells

Since PCR analysis showed that all three viruses were present in the same cell culture, the next question was whether individual cells were similarly infected. To answer this question, we prepared samples for transmission electron microscopy (TEM).

Due to its large size and distinct morphology, TEM pictures of cells infected only with HHV-6A (K1) were easily identified (Figure 4A). The intracellular particles were around 150 to 200 nm in size. This allowed us to compare the HHV-6A infected cells with cells co-infected with HHV-6A and HIV-1 (K2). Distinct maturation of these viruses made it easy to determine their presence (Figure 4B).

Figure 4

Electron micrographs of infected cells

Electron micrographs of infected cells. A. K6 cell infected with HHV-6 (arrows) with margination of the chromatin in the nucleus and extensive vascularization. B. K2 cell showing HIV-1 particles (arrows). It is unclear whether the particles are outside the cell or in a vacuole inside the cell. C. K5 cell showing partial HCV particles inside a vacuole (arrow). D. K6 cell showing HCV particles (arrows). E. Inset showing HCV from Figure 4D.

TEM pictures of cells infected with HCV (K5) were examined next. Complete as well as incomplete HCV particles were observed in the cytoplasm of infected cells (Figure 4C and 4D). The complete HCV particles were 70 to 100 nm in size, while the incomplete particles were 50 to 70 nm in size. The HCV virions and the incomplete HCV particles resembled pictures published by other investigators 1213.

CEM cells that were co-infected with HHV-6A and HCV (K6) were also examined. The presence of both HHV-6A and HCV, including immature HCV particles inside the vesicles were noted.

TEM pictures show that HIV-1 and HCV were generally of similar size. In our analysis, HIV-1 usually appeared to be a little larger than HCV, which may be an artefact of fixation and processing.

As noted above, the K7 cell culture contained all three viruses. HIV-1 and HCV were seen outside the cells (Figure 5), and the cells contained both complete and incomplete HCV virions. One example of a triply infected cell is shown in Figure 6. Incomplete HHV-6A particles were seen budding from the nucleus (Figures 7A–7D). HIV-1 was seen budding from the plasma membrane of the cell (Figure 6). In addition, HCV virions were seen in the cytoplasm and in the vicinity of budding HHV-6A particles (Figure 7B), and other incomplete HCV particles were also seen in the perinuclear space (Figures 7A and 7D). These HCV particles were approximately 70 to 100 nm in size. HIV-1 was present outside but adjacent to or in the vicinity of the infected cell (Figure 7C).

Figure 5

Electron micrograph of HCV and HIV-1 extracellular particles in the vicinity of a K7 cell

Electron micrograph of HCV and HIV-1 extracellular particles in the vicinity of a K7 cell. Representative HIV-1 particles are indicated with black arrows, HCV with white arrows, and immature HIV-1 particles by an arrowhead. The HIV-1 particles are a little larger than HCV.

Figure 6

TEM of triply-infected K7 cell

TEM of triply-infected K7 cell. HIV-1 particle budding from the plasma membrane (arrow).

Figure 7

Enlargements of triply-infected cell from Figure 6

Enlargements of triply-infected cell from Figure 6. A. HHV-6 budding from the nuclear membrane (black arrows) and HCV in the perinuclear space (white arrow). B. HCV particles in the cytoplasm. C. HIV-1 particle outside the cell. D. HHV-6 (black arrows) and HCV (white arrow) in the perinuclear space.

Discussion

Studies that have compared HCV-infected patients with HCV-HIV-1 infected patients have observed that there is a longer half-life of HCV, and up to ten times higher HCV RNA levels in serum or liver 24. Co-infected patients are more likely to develop cirrhosis, which may rapidly progress to acute disease 14. The mechanisms for these effects are unclear, but loss of immune function by HIV-1 infection is thought to be a significant contributing factor. However, most studies of HAART suggest that it does not significantly affect the levels of HCV RNA 2, meaning that HIV-1 may have little effect on HCV RNA production. These studies are confusing, since evidence for HCV replication is absent, and HCV RNA levels do not correlate with the levels of infectious HCV in the blood. Unfortunately there is no commonly used system to determine these levels.

Other studies that have looked at the effects of HCV on HIV-1 infected individuals have also shown conflicting results. Early studies suggest that HCV increases the rate of progression to AIDS in co-infected individuals, but others have not seen this 23. A large Swiss study showed HCV serum-positivity was associated with more likely progression to AIDS and a less likely increase of CD4+ cell count after therapy 1516. Similar determinations can be made by comparing other viral infections such as HHV-6 and HIV. A strong case can be made for HHV-6 as a co-factor in the development of AIDS. Other short-term studies have not found a correlation between CD4+ cell number and negative consequences due to HCV infection 1718.

In vitro studies of HIV-HCV co-infected cells have been hampered by a lack of a system that can replicate both viruses. Laskus et al. 19 showed that HIV-1 stimulates HCV production in macrophages in vitro. The system, however, did not support HCV replication for extended periods of time. Macrophages, although they stay alive for extended periods of time, do not synthesize DNA and replicate.

Initial in vitro studies of HIV-HHV co-infected T-cells showed that HHV-6 increased HIV-1 production and cell death 20. Later studies showed that HHV-6 could decrease HIV-1 production in some cell types, such as dendritic cells 21 and macrophages 22. This difference in virus production may be accounted for by differences in activities of the LTR of HIV-1.

Patients and clinically normal individuals are frequently infected with multiple viruses. It is therefore important to understand the implications of simultaneous infection by multiple viruses. Since co-infections with HIV, HCV, and HHV-6 are frequently seen in the same individual, the development of a system to study the effects of the interactions among these viruses at the level of a single cell is important. Our experience in human viruses allowed us to develop this system. We realize that the occurrence of triple infections of single cells may be a rare event, yet it also establishes the fact that immunity and dominance are also limited statistical phenomena.

Our system for growing HCV starts with the infection of macrophages obtained from human cord blood. Macrophages are distributed all over the body, and they perform specialized functions, e.g., Kupffer's cells of liver, dendritic cells of skin, astrocytes and microglial cells of the nervous system. In addition, the infection of our human neuronal precursor cells with CIMM-HCV may be similar to the infection of macrophages with HIV-1. These macrophages may become multiply infected and function as a reservoir. Other investigators have shown the presence of HCV in human brains in the post-mortem analysis 2324. Similarly, HIV-1 has also been reported in the brain environment 252627. These observations may partly explain the diminished cognitive function, depression and fatigue in these individuals. The presence of HIV-1 and HCV in the same cell may greatly aggravate the malady.

Our TEM analysis showed HHV-6A and HCV in the same cells, with extracellular HIV-1 particles budding or adjacent to the infected cells. Since HIV-1 assembly is completed at the plasma membrane level, their presence is only seen outside the cells. The assembly and synthesis of HHV-6A begins in the nucleus, the virion acquires a membrane as it passes through the nuclear membrane, and it matures in the cytoplasm. The synthesis and maturation of HCV is presumed to occur in the cytoplasm. This, in our opinion, is an evolving concept. We have seen HCV particles in the nucleus, in the perinuclear space, in the cytoplasm, in vesicles, and outside cells. The presence of HCV particles in the perinuclear space may indicate that at least partial synthesis of this virus may occur in the nucleus. Recent suggestions that some nuclear proteins bind to HCV RNA support this possibility 28. The TEM results presented in Figures 6 and 7 suggest that HCV and HHV-6A may have similar or identical sites of replication. Others have proposed that HCV synthesis may occur in the perinuclear space 2930, which is unlikely.

Another question addressed by these experiments was to conclusively demonstrate that HCV can replicate well in T-cells. There was a difference in the level of virus replication when compared to B-cells but, as shown in the electron micrographs, it replicates well in T-cells. HCV has been shown to infect T-cells in addition to other cell types in infected individuals 31, so it is not surprising that significant production of HCV particles occurs in vitro. The claims of levels of virus titers for many human viruses are merely claims. There are no definitive and reproducible methods for titrating biologically active HIV-1, HTLV-I, HTLV-II, HHV-6, or HCV with accuracy. We generally use the RNA levels as a relative indicator of virus production.

This study, in addition to our previous reports, supports the notion that HCV infects multiple hematopoietic cell types, viz monocytes-macrophages, B-cells, and T-cells. We have also reported that neuronal cells, endothelial cells (hepatocytes), and Kupffer's cells of liver can also be infected. Replication of HCV in liver cells is generally very low, which may be clinically relevant.

Since HCV, HIV-1, and HHV-6A can coexist in culture, none of these viruses prevent the others from infection and replication. However, these effects may be variable due to a number of factors that affect replication. This has relevance to viral protein production that may induce or produce pathology. How the viruses interact will be the subject of later work.

Methods

Cell culture and viruses

HIV-1 911, HHV-6A 1032, and HCV 5 are routinely isolated in our laboratories at the California Institute of Molecular Medicine (CIMM), Ventura, CA. Freshly isolated viruses were used for this study. Stocks of these isolates were stored in aliquots of 1 ml at -70°C for experimental use. CEM cells were previously obtained from the American Type Culture Collection (ATCC), Bethesda, MD.

Transmission experiments were carried out using our standard protocol. Briefly, CEM cells were seeded at 10⁵cells/5 ml in complete medium supplemented with 10% fetal bovine serum containing 5 ng of polybrene, and incubated overnight. To infect these cells, they were centrifuged, the media was discarded, and the cells were re-suspended in 1 ml of stock virus, incubated at 37°C in a 5% CO₂atmosphere. Cell cultures were centrifuged, and the culture supernatants were filtered through 0.45 μ filter membranes for assays.

The CEM cells were infected sequentially using HCV followed by HHV-6A and HIV-1 (Figure 1). Cell cultures were incubated overnight each time post infection. Aliquots of all the infected cells and cell culture supernatants were saved in liquid nitrogen and at -70°C, respectively.

Detection of viruses using RT-PCR and PCR analyses

RNA was purified from the cell culture supernatants and nested RT-PCR was performed to amplify HCV RNA 5 or HIV-1 RNA 33. To detect HHV-6A, DNA was purified from the cell culture supernatants and PCR was performed using the U16-17F and U16-17R primers 34. The primers used for the PCR and RT-PCR experiments are listed in Table 2.

Transmission electron microscopy (TEM)

Cultured cells were placed in fixative provided by the electron microscopy group at the City of Hope, Duarte, CA. The fixed samples were shipped to them overnight for sectioning and analysis.

The cells analyzed by TEM were well fixed, although cut thick they were easy to interpret. Since HIV-1 particles are assembled at the plasma membrane level they are only found either budding from the cell membrane or outside the cells. Hence, they do not appear inside the triply infected cells. The intracellular presence of HHV-6A and HCV is impressive.

Competing interests

All intellectual rights are reserved by the California Institute of Molecular Medicine (CIMM), and all aspects of this work were performed by CIMM. There are no competing interests between California Lutheran University or any other body and CIMM.

Authors' contributions

K.A.S., A.G., and R.G. performed biological work. J.G.P. and A.S.K. performed the clinical work, recruitment of patients, and procurement of specimens. K.A.S. and A.G. performed molecular work. S.Z.S. and D.R. designed and conducted experiments, analyzed the data, and wrote the manuscript. All of the authors have read and approved the final manuscript.

Acknowledgements

We would like to thank John D. Hardy of the City of Hope EM Core lab for the electron micrography. Part of this study was supported by a Swenson Summer Research Fellowship awarded to Kathy Snyder. We would also like to thank the Community Memorial Hospital for their continued support with the clinical specimens.

Hepatitis C Virus prevalence among patients infected with Human Immunodeficiency Virus: a cross-sectional analysis of the US adult AIDS Clinical Trials Group Sherman KE Rouster SD Chung RT Rajicic N Clin Infect Dis 2002 34 6 831 837 10.1086/339042 11833007 Interaction between HIV-1 and HCV infections: towards a new entity? Winnock M Salmon-Ceron D Dabis F Chene G J Antimicrob Chemother 2004 53 6 936 946 10.1093/jac/dkh200 15117928 HIV and hepatitis C co-infection Jones R Dunning J Nelson M Int J Clin Pract 2005 59 9 1082 1087 10.1111/j.1742-1241.2005.00596.x 16115185 HIV and hepatitis C virus coinfection: bad bedfellows Schooley RT Top HIV Med 2005 13 4 112 116 16304454 Transmission of human hepatitis C virus from patients in secondary cells for long term culture Revie D Braich RS Bayles D Chelyapov N Khan R Geer C Reisman R Kelley AS Prichard JG Salahuddin SZ Virol J 2005 2 37 1090622 15840164 10.1186/1743-422X-2-37 Analysis of in vitro replicated human hepatitis C virus (HCV) for the determination of genotypes and quasispecies Revie D Alberti MO Braich RS Chelyapov N Bayles D Prichard JG Salahuddin SZ Virol J 2006 3 81 1609102 17010197 10.1186/1743-422X-3-81 Discovery of significant variants containing large deletions in the 5'UTR of human hepatitis C virus (HCV) Revie D Alberti MO Braich RS Bayles D Prichard JG Salahuddin SZ Virol J 2006 3 82 1599722 17010198 10.1186/1743-422X-3-82 Contribution of insertions and deletions to the variability of hepatitis C virus populations Torres-Puente M Cuevas JM Jimenez-Hernandez N Bracho MA Garcia-Robles I Carnicer F del Olmo J Ortega E Moya A Gonzalez-Candelas F J Gen Virol 2007 88 Pt 8 2198 2203 10.1099/vir.0.82855-0 17622623 Isolation of infectious human T-cell leukemia/lymphotropic virus type III (HTLV-III) from patients with acquired immunodeficiency syndrome (AIDS) or AIDS-related complex (ARC) and from healthy carriers: a study of risk groups and tissue sources Salahuddin SZ Markham PD Popovic M Sarngadharan MG Orndorff S Fladagar A Patel A Gold J Gallo RC Proc Natl Acad Sci U S A 1985 82 16 5530 5534 391156 2991931 10.1073/pnas.82.16.5530 Isolation of a new virus, HBLV, in patients with lymphoproliferative disorders Salahuddin SZ Ablashi DV Markham PD Josephs SF Sturzenegger S Kaplan M Halligan G Biberfeld P Wong-Staal F Kramarsky B Science 1986 234 4776 596 601 10.1126/science.2876520 2876520 Human T lymphotropic virus type III infection of human alveolar macrophages Salahuddin SZ Rose RM Groopman JE Markham PD Gallo RC Blood 1986 68 1 281 284 3013342 Hepatitis C virus: detection of intracellular virus particles by electron microscopy Shimizu YK Feinstone SM Kohara M Purcell RH Yoshikura H Hepatology 1996 23 2 205 209 10.1002/hep.510230202 8591842 Ultrastructural observations in hepatitis C virus-infected lymphoid cells Steffan A Marianneau P Caussin-Schwemling C Royer C Schmitt C Jaeck D Wolf P Gendrault J Stoll-Keller F Microbes Infect 2001 3 3 193 202 10.1016/S1286-4579(01)01369-7 11358713 Natural history and predictors of severity of chronic hepatitis C virus (HCV) and human immunodeficiency virus (HIV) co-infection Vallet-Pichard A Pol S J Hepatol 2006 44 1 Suppl S28 34 10.1016/j.jhep.2005.11.008 16343684 Clinical progression, survival, and immune recovery during antiretroviral therapy in patients with HIV-1 and hepatitis C virus coinfection: the Swiss HIV Cohort Study Greub G Ledergerber B Battegay M Grob P Perrin L Furrer H Burgisser P Erb P Boggian K Piffaretti JC Hirschel B Janin P Francioli P Flepp M Telenti A Lancet 2000 356 9244 1800 1805 10.1016/S0140-6736(00)03232-3 11117912 Coinfection with hepatitis viruses and outcome of initial antiretroviral regimens in previously naive HIV-infected subjects De Luca A Bugarini R Lepri AC Puoti M Girardi E Antinori A Poggio A Pagano G Tositti G Cadeo G Macor A Toti M D'Arminio Monforte A Arch Intern Med 2002 162 18 2125 2132 10.1001/archinte.162.18.2125 12374521 Impact of viral hepatitis co-infection on response to antiretroviral therapy and HIV disease progression in the HIV-NAT cohort Law WP Duncombe CJ Mahanontharit A Boyd MA Ruxrungtham K Lange JM Phanuphak P Cooper DA Dore GJ Aids 2004 18 8 1169 1177 10.1097/00002030-200405210-00010 15166532 Effect of hepatitis C infection on progression of HIV disease and early response to initial antiretroviral therapy Sullivan PS Hanson DL Teshale EH Wotring LL Brooks JT Aids 2006 20 8 1171 1179 10.1097/01.aids.0000226958.87471.48 16691069 Human immunodeficiency virus facilitates infection/replication of hepatitis C virus in native human macrophages Laskus T Radkowski M Jablonska J Kibler K Wilkinson J Adair D Rakela J Blood 2004 103 10 3854 3859 10.1182/blood-2003-08-2923 14739225 Productive dual infection of human CD4+ T lymphocytes by HIV-1 and HHV-6 Lusso P Ensoli B Markham PD Ablashi DV Salahuddin SZ Tschachler E Wong-Staal F Gallo RC Nature 1989 337 6205 370 373 10.1038/337370a0 2463490 Human herpesvirus 6 infects dendritic cells and suppresses human immunodeficiency virus type 1 replication in coinfected cultures Asada H Klaus-Kovtun V Golding H Katz SI Blauvelt A J Virol 1999 73 5 4019 4028 104181 10196298 Human herpesvirus 6A decreases the susceptibility of macrophages to R5 variants of human immunodeficiency virus 1: possible role of RANTES and IL-8 Csoma E Deli T Konya J Csernoch L Beck Z Gergely L Virus Res 2006 121 2 161 168 10.1016/j.virusres.2006.05.007 16815583 Search for hepatitis C virus negative-strand RNA sequences and analysis of viral sequences in the central nervous system: evidence of replication Radkowski M Wilkinson J Nowicki M Adair D Vargas H Ingui C Rakela J Laskus T J Virol 2002 76 2 600 608 136845 11752151 10.1128/JVI.76.2.600-608.2002 Pathogenesis of Hepatitis C Virus Coinfection in the Brains of Patients Infected with HIV Letendre S Paulino AD Rockenstein E Adame A Crews L Cherner M Heaton R Ellis R Everall IP Grant I Masliah E J Infect Dis 2007 196 3 361 370 10.1086/519285 17597450 HIV-1 DNA in brains in AIDS and pre-AIDS: correlation with the stage of disease An SF Giometto B Scaravilli F Ann Neurol 1996 40 4 611 617 10.1002/ana.410400411 8871581 Immunocytochemical quantitation of human immunodeficiency virus in the brain: correlations with dementia Glass JD Fedor H Wesselingh SL McArthur JC Ann Neurol 1995 38 5 755 762 10.1002/ana.410380510 7486867 Quantitation of human immunodeficiency virus in brains of demented and nondemented patients with acquired immunodeficiency syndrome Johnson RT Glass JD McArthur JC Chesebro BW Ann Neurol 1996 39 3 392 395 10.1002/ana.410390319 8602761 Nuclear factors are involved in hepatitis C virus RNA replication Isken O Baroth M Grassmann CW Weinlich S Ostareck DH Ostareck-Lederer A Behrens SE Rna 2007 17684232 Hepatitis C virus NS5B protein is a membrane-associated phosphoprotein with a predominantly perinuclear localization Hwang SB Park KJ Kim YS Sung YC Lai MM Virology 1997 227 2 439 446 10.1006/viro.1996.8357 9018143 Replication of hepatitis C virus RNA occurs in a membrane-bound replication complex containing nonstructural viral proteins and RNA El-Hage N Luo G J Gen Virol 2003 84 Pt 10 2761 2769 10.1099/vir.0.19305-0 13679611 Hepatitis C virus core protein up-regulates anergy-related genes and a new set of genes, which affects T cell homeostasis Dominguez-Villar M Munoz-Suano A Anaya-Baz B Aguilar S Novalbos JP Giron JA Rodriguez-Iglesias M Garcia-Cozar F J Leukoc Biol 2007 17711976 Human herpesvirus-6 strain groups: a nomenclature D.V. Ablashi HA Z. Berneman, G. Campadelli-Fiume, D.R. Carrigan, L. Ceccerini-Nelli, B. Chandran, S. Chou, H. Collandre, R. Cone, T.R. Dambaugh, S. Dewhurst, D. DiLuca, L. Foa′-Tomasi, B. Fleckenstein, N. Frenkel, R. Gallo, U.A. Gompels, C. Hall, M. Jones, G. Lawrence, M.E. Martin, L. Montagnier, F. Neipel, J. Nicholas, P.E. Pellett, A. Razzaque, G. Torrelli, B.J. Thomson, S.Z. Salahuddin, L. Wyatt and K. Yamanishi Arch Virol 1993 129 363 366 10.1007/BF01316913 8385923 Amplification of a complete simian immunodeficiency virus genome from fecal RNA of a wild chimpanzee Santiago ML Bibollet-Ruche F Bailes E Kamenya S Muller MN Lukasik M Pusey AE Collins DA Wrangham RW Goodall J Shaw GM Sharp PM Hahn BH J Virol 2003 77 3 2233 2242 140993 12525658 10.1128/JVI.77.3.2233-2242.2003 Temporal mapping of transcripts in herpesvirus 6 variants Mirandola P Menegazzi P Merighi S Ravaioli T Cassai E Di Luca D J Virol 1998 72 5 3837 3844 109607 9557667