TWiV 232: Gophers go viral

On episode #232 of the science show This Week in Virology, Vincent meets up with Roberto, Reuben, Lou, and Leslie at the University of Minnesota to talk about their work on HIV-1, APOBEC proteins, measles virus, and teaching virology to undergraduates.

You can find TWiV #232 at www.microbe.tv/twiv.

Antimicrobial peptides induced by herpesvirus enhance HIV-1 infection

Langerhans cellsThe risk of being infected with human immunodeficiency virus type 1 (HIV-1) is substantially enhanced in individuals with other sexually transmitted diseases. For example, infection with herpes simplex virus type 2 (HSV-2) increases the risk ratio of acquiring HIV from 2 to 4. Explanations for this increased risk include direct inoculation of HIV-1 into the blood through genital ulcers, and the induction of inflammatory cells by HSV-2 which act as sites of replication for HIV-1. The results of infections carried out in cell culture suggest a biological mechanism for the enhancement of HIV-1 infection by HSV-2.

Langerhans cells (LC) are believed to one of the first cells in which HIV-1 replicates after sexual exposure. LCs are dendritic cells which patrol the mucosal epithelium, taking up and processing antigens and presenting them to T cells in the lymph nodes. These cells express the HIV-1 receptors CD4 and CCR5, but not CXCR4, and can therefore be infected with CCR5-tropic* but not CXCR4-tropic HIV-1. Individuals who do not express CCR5 are resistant to HIV infection. For these and other reasons CCR5-tropic HIV-1 viruses are believed to be ones that transmit infection from one individual to another.

In human skin explant cultures, which contain LCs, co-infection with HSV-2 substantially increased the number of HIV-1 cells. This observation could not be explained by co-infection of individual cells because very few of these were observed in the cultures. When applied to fresh cells, the supernatant of cultures infected with HSV-2 also stimulated the number of HIV-1 infected LCs. These observations suggested that HSV-2 infection stimulates the production of one or more substances from infected cells which in turn improve HIV-1 infection.

Human epithelial and epidermal cells are known to produce antimicrobial peptides such as defensins and cathelicidin. These are short, evolutionarily conserved peptides that inhibit the growth of bacteria, viruses, and fungi. HSV-2 infected keratinocytes were found to produce a number of antimicrobial peptides, but the most important one is called LL-37. This peptide enhanced the expression of HIV-1 receptors CD4 and CCR5 on LCs, leading to increased susceptibility of the cells to HIV-1. Removing LL-37 from the supernatant of HSV-2 infected cells reduces the ability of the medium to stimulate susceptibility to HIV-1.

These findings provide a plausible mechanism by which HIV-1 infection is enhanced by HSV-2. When HSV-2 infects the genital mucosa, the epithelial cells produce LL-37. This antimicrobial peptide enhances the production of CD4 and CCR5 on LCs, allowing more efficient infection by HIV-1. This mechanism is supported by the observation that elevated levels of LL-37 correlate with HIV-1 infection in sex workers.

I wonder why antimicrobial peptides up-regulate CD4 and CCR5. In addition to their antimicrobial properties, the cathelicidins possess chemotactic, immunostimulatory, and immunomodulatory effects, and the upregulation of CD4 and CCR5 are likely part of these activities.

These are exciting findings, and if they are further correlated in humans, they might lead to novel ways of interfering with HIV-1 infection, such as by antagonizing LL-37.

*CCR5 and CXCR4-tropic refer to HIV-1 virions that bind to chemokine receptors CCR5 or CXCR4, respectively, in addition to CD4, to initiate infection.

TWiV 143: Live at ASV in Minneapolis

asv minneapolisHosts: Vincent Racaniello, Rich Condit, Julie Overbaugh, and Stacey Schultz-Cherry

Vincent, Rich, Julie and Stacey recorded TWiV at the 30th Annual Meeting of the American Society for Virology in Minneapolis, where they discussed the role of neutralizing antibodies in protection against HIV-1 infection, and astroviruses, agents of gastroenteritis.

Click the arrow above to play, or right-click to download TWiV 143 (48 MB .mp3, 66 minutes).

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Weekly Science Picks

Rich – Iter – building a fusion reactor
Vincent – American girls sweep at Google science fair (NY Times)

Listener Pick of the Week

JingBees by Rudolph Steiner

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Not-so-similar fate of identical twins infected with HIV-1

identical twinsFor extra credit in my recently concluded virology course, I asked students to summarize a virology finding in the style of this blog. I received many excellent submissions which I plan to post here in the coming months.

by Amanda Carpenter

In 1983, identical twin boys simultaneously received a contaminated blood transfusion immediately after birth, and were subsequently diagnosed with HIV-1. Years later, one of the twins is faring very well and has a near normal immune system, while the other is in poor health and has experienced many complications. How could the same virus, infecting two individuals at the same time, with the same genetic background, yield such different clinical courses? This unfortunate natural experiment has allowed researchers to study viral evolution while holding host genetic make-up constant. Brigham Young University Chairman of Biology Keith Crandall has studied the virus in this interesting case and recently published his findings in BMC Evolutionary Biology.

Crandall’s team examined blood samples obtained from the twins when they were 15 years old. They collected nucleotide sequence data from several genes essential to the success of HIV infection, which allowed them to extrapolate rates of viral growth, recombination, and genetic diversity. They reported that the virus of the healthier twin exhibited generally higher growth rates, higher genetic diversity, and higher recombination rates than the virus of the sicker twin (2).

How is this possible? Infected individuals produce an estimated 1010 new HIV virions every day, with errors occurring at a rate of about 1 per 104 nucleotides incorporated (1). These frequent point mutations are a simple starting point to explain the divergence of a once identical virus. In addition, HIV virions are capable of exchanging their genetic material with a different strain of virus via a process called recombination. Recombination is likely a random event, but has important implications for the host immune system.

Part of the immune system’s response to HIV is the utilization of cytotoxic T lymphocytes (CD8+ cells), which target and kill virus-infected cells. These cells are very specific, and if a recombination event occurs, these cytotoxic cells may not be able to recognize the new viral strain as readily as the original. The immune system may adapt to the new strain, but the virus may recombine again and again, and the immune system will not be able to keep up. These recombinant strains are likely to become more prevalent through natural selection. If recombined strains are better at evading the immune system, and are therefore more detrimental to the host, does this mean they are more successful? Why would the virus that has higher genetic diversity, a growth rate, and a higher recombination rate cause less disease? Perhaps the answer lies in the immune system.

Once out of the womb, these twins no longer exist in identical environments. They are exposed to different pathogens, bacteria, and microbes, all of which affect the make-up of the immune system. The healthier twin’s immune system may be better able to fight the virus, and so the virus must grow, diversify, and recombine in order to propagate the infection. In other words, because the sicker twin has a more depressed immune system, the virus is replicating with less resistance, and there is less incentive for the virus to evolve. Divergent viral evolution in the case of these monozygotic twins is likely due to random mutation and recombination events, combined with antiviral pressure from the hosts, whose immune systems are not identical at all.

(1) Yang, O., Church, J., Kitchen, C., Kilpatrick, R., Ali, A., Geng, Y., Killian, M., Sabado, R., Ng, H., Suen, J., Bryson, Y., Jamieson, B., & Krogstad, P. (2005). Genetic and Stochastic Influences on the Interaction of Human Immunodeficiency Virus Type 1 and Cytotoxic T Lymphocytes in Identical Twins Journal of Virology, 79 (24), 15368-15375 DOI: 10.1128/JVI.79.24.15368-15375.2005

(2) Tazi L, Imamichi H, Hirschfeld S, Metcalf JA, Orsega S, Pérez-Losada M, Posada D, Lane HC, & Crandall KA (2011). HIV-1 infected monozygotic twins: a tale of two outcomes. BMC evolutionary biology, 11 PMID: 21385447

TWiV 133: The HIV hideout

Dr. Kathleen CollinsHosts: Vincent Racaniello, Rich Condit, Dickson DespommierAlan Dove, and Kathleen Collins

Vincent, Rich, Alan, and Dickson discuss the cellular reservoir of HIV-1 with Kathleen Collins, MD, PhD.

Click the arrow above to play, or right-click to download TWiV #133 (42 MB .mp3, 87 minutes).

Subscribe to TWiV (free) in iTunes , at the Zune Marketplace, by the RSS feed, by email, or listen on your mobile device with the Microbeworld app.

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Kathleen – TRIM5 is an innate immune sensor (Nature)
Rich – A Biologist’s Mothers Day Song (YouTube)
Alan – World of Viruses
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Chris  – Periodic Tales by Hugh Aldersey-Williams

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XMRV not detected in seminal plasma

How XMRV, the new human retrovirus associated with prostate cancer and chronic fatigue syndrome, might be transmitted among humans is unknown. The finding that the virus can be detected in prostate cancer cells, and in prostatic secretions of men with prostate cancer suggests that it could be sexually transmitted. To address this question, the presence of XMRV in seminal plasma of men with HIV-1 was examined. Although the virus was not detected in 93 samples from 54 HIV-1 infected men, the study provides little information on possible transmission mechanisms of XMRV.

This study involved two groups of HIV-1 infected men from the Netherlands: 29 who have sex with men, and 25 heterosexual men. The rationale for examining HIV-1 infected men for XMRV was that “they have a higher chance of contracting sexually transmitted pathogens than non-HIV-1 infected men”. For 39 men a second sample was also available from another time point, bringing the total samples to 93.

To detect XMRV, semen samples were diluted 1:1 with buffer and centrifuged to remove cells, yielding seminal plasma. Total nucleic acid was then extracted and subjected to reverse-transcription and then polymerase chain reaction. This procedure assays for the presence of XMRV viral RNA. At the same time, the samples were also tested for the presence of HIV-1 RNA. The positive control for XMRV was total nucleic acid extracted from a prostate cancer cell line known to produce viral RNA.

The results show that HIV-1 was detected in 25% of the seminal plasma samples, while none contained XMRV nucleic acid. The authors conclude:

Although HIV-1 was amplified from 25% of the seminal plasma samples, no XMRV was detected, suggesting that either the prevalence of XMRV is very low in The Netherlands, or that XMRV is not naturally present in the seminal plasma.

In my opinion, these conclusions are not supported by the data obtained in this study. Here are my reasons:

  • The semen samples were subjected to centrifugation, which removes all cells, including spermatozoa, epithelial cells, and lymphocytes. Such cells could harbor virions.
  • The study was designed only to search for XMRV virions or viral RNA, not proviral DNA, which is integrated into cellular DNA.
  • No attempt was made to determine if the 54 men were infected with XMRV. This could have been done by taking blood samples and co-culturing them with LNCaP cells, then performing PCR. If none of the men were infected with the virus, then absence of the virus in their semen is meaningless.

To determine if XMRV could be transmitted in semen, I would obtain semen samples from patients known to be infected with the virus. Then I would co-culture total semen and seminal plasma with LNCaP cells to amplify any virus present, followed by PCR to detect either virions or proviral DNA. I realize it may be difficult to conduct the study in this way, but I don’t see the value of doing it any other way.

Marion Cornelissen, Fokla Zorgdrager, Petra Blom, Suzanne Jurriaans, Sjoerd Repping, Elisabeth van Leeuwen, Margreet Bakker, Ben Berkhout, & Antoinette C. van der Kuyl (2010). Lack of Detection of XMRV in Seminal Plasma from HIV-1 Infected Men in The Netherlands PLoS One : 10.1371/journal.pone.0012040

XMRV in human respiratory tract

An important question about the retrovirus XMRV, which has been implicated in prostate cancer and chronic fatigue syndrome, is where the virus replicates in humans. Such information would provide clues about how infection might be transmitted. To date the virus has been detected in malignant prostate cells and in the peripheral blood mononuclear cells and plasma of patients with CFS. A new study reveals that XMRV is present in respiratory secretions.

Polymerase chain reaction was used to detect XMRV in 267 respiratory samples taken from German patients. One group comprised sputum and nasal swab specimens from 75 travelers from Asia who had respiratory tract infections. The second group consisted of 31 bronchoalveolar lavage samples from patients with chronic obstructive pulmonary disease, while samples from the third group were from 161 immunosuppressed patients with severe respiratory tract infections. The study included 62 healthy controls. It should be noted that none of the patients had been diagnosed with CFS.

XMRV sequences were detected in 3 of 75 samples (2.3%) in group 1, 1 of 31 samples (3.2%) in group 2, and 16/161 (9.9%) in group 3. Six of the XMRV-positive samples in the second group also contained rhinovirus, adenovirus, or pathogenic fungi. The higher rate of detection of XMRV and other microbes in immunosuppressed individuals is not unexpected. The control group contained 2 of 62 samples (3.2%) positive for XMRV.

The presence of XMRV in PBMCs and plasma suggests a blood-borne route of transmission of the virus: transfusions, health care associated needle sticks, and intravenous drug use. Does finding XMRV in the respiratory tract prove that the virus can be transmitted by the respiratory route? No, not until we have other information, including the level of virus in respiratory secretions, and the infectivity of XMRV. In this context it is interesting to note that it was not possible to isolate infectious XMRV from the respiratory tract of the German patients.

Reviewing the transmission of another human retrovirus, HIV-1, is instructive in understanding the pathogenesis of XMRV infection. The main modes of transmission of HIV-1 are sexual, parenteral, and from mother to infant. These routes of transmission are consistent with levels of infectious virus in body fluids (shown in this table). Viral RNA can be detected at several levels of the respiratory tract, but respiratory secretions rarely transmit HIV.

FIsher, N., Schulz, C., Stieler, K., Hohn, O., Lange, C., Drosten, C., & Aepfelbacher, M. (2010). Xenotropic murine leukemia virus-related gammaretrovirus in respiratory tract Emerg. Inf. Dis. : 10.3201/eid1606.100066

TWiV 82: Immunology in silico

Hosts: Vincent Racaniello and Rich Condit

On episode #82 of the podcast This Week in Virology (TWiV), Vincent and Rich talk about how thymic selection of T cells might lead to better control of HIV-1 infection, and a mouse model for severe antibody-induced dengue virus disease.

This episode is sponsored by Data Robotics Inc. Use the promotion code TWIVPOD to receive $75-$500 off a Drobo.

Win a free Drobo S! Contest rules here.

Click the arrow above to play, or right-click to download TWiV #82 (59 MB .mp3, 82 minutes)

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Rich The Mold in Dr. Florey’s Coat: The Story of the Penicillin Miracle by Eric Lax
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Virology lecture #18: HIV pathogenesis


Download: .wmv (330 MB) | .mp4 (72 MB)

Visit the virology W3310 home page for a complete list of course resources.

Inhibitors of XMRV

Raltegravir inhibition XMRVXenotropic murine leukemia virus related virus (XMRV) has been implicated in prostate cancer and chronic fatigue syndrome (CFS). Because XMRV is a retrovirus, it might be susceptible to antiviral drugs that are licensed for the treatment of AIDS. AZT (azidothymidine) was previously found to block XMRV replication. A screen of forty-five compounds reveals that XMRV replication is inhibited by raltegravir and three other drugs.

The authors studied the effect of 45 compounds on the replication of XMRV in cell lines derived from human breast (MCF-7) and prostate (LNCaP) cancers. Twenty-eight of the drugs have been approved for use in humans, including treatment of HIV-1 infection. The drugs tested include nucleoside and non-nucleoside reverse-transcriptase inhibitors, and integrase and protease inhibitors. Other inhibitors used included aspirin, acylclovir (the anti-herpesvirus drug), and chloroquine (complete list here).

To test the ability of the drugs to inhibit XMRV replication, cells were infected with the virus, and increasing amounts of the compound were added to the cell culture medium. After incubation for six days, the virus in the culture medium was assayed by measuring reverse transcriptase activity. This enzyme, which converts RNA to DNA, is packaged within the viral particle. Its presence in the cell culture medium is therefore a measure of viral production. The cells were also monitored for cytotoxicity to ensure that a reduction in viral release was a consequence of specific antiretroviral activity, not toxicity of the compounds.

The most potent inhibitor of XMRV was raltegravir, the compound that blocks the viral integrase protein. This viral protein is essential for insertion of viral DNA into the host chromosome. The EC50 (the concentration of drug that inhibited virus production by 50%) was 0.005 µM in MCF-7 cells and 0.03 µM in LNCaP cells. Another integrase inhibitor, called L-000870812, also blocked XMRV replication, but at higher concentrations (EC50 of 0.16 µM in MCF-7 cells, and 0.7 µM in LNCaP cells).

Other drugs were found to inhibit XMRV replication at much higher concentrations. Among the most effective were zidovudine (ZDV; EC50 0.11 µM in MCF-7 cells and 0.14 µM in LNCaP cells) and tenofovir disoproxil fumarate (TDF). Protease inhibitors were less effective at blocking XMRV replication. Other non-HIV inhibitors were not effective at concentrations which were not cytotoxic.

The problem of drug resistance during treatment of AIDS was made manageable by using combinations of three antiviral compounds. Pairwise combinations of raltegravir, L-000870812, TDF and ZDV were tested for activity against XMRV in LNCaP cells.The effects were either additive or synergistic. All combinations that included raltegravir showed synergy without cytotoxicity. This observation is good news for treating XMRV infection. Based on the response of other retroviruses to combination therapy, the use of two antiviral drugs might suppress XMRV replication, reduce disease, and reduce emergence of resistant viral mutants.

How well do these drugs inhibit XMRV compared with HIV-1? The authors write that “Relative to HIV-1, the compounds were generally less potent against XMRV than HIV-1, especially at the EC90 level” – the concentration of drug needed to inhibit virus production by 90%. It’s difficult to predict from these data whether the drugs would be effective at effectively reducing XMRV levels in an infected individual. Chemical modification of the compounds could yield drugs that are more active against XMRV.

No matter what antiviral drugs are used, resistant viral variants inevitably emerge. For XMRV the process may be less problematic than for HIV-1. Compared with HIV-1, isolates of XMRV isolates have limited sequence diversity. The genomes of all the XMRV isolates obtained to date differ from each other at 27 out of 8,100 nucleotides. If this lack of diversity is a consequence of limited replication in the host, then the emergence of drug resistant variants could be significantly lower than HIV-1. A combination of two drugs might therefore be effective in treating XMRV infection. The authors note that after several months of propagating XMRV in the presence of raltegravir, drug resistant viruses have not emerged. But a human is very different from a dish of cultured cells.

Whether or not combinations of these drugs can be used to treat XMRV infection awaits the results of additional epidemiological studies, as well as clinical trials to determine efficacy. As the authors conclude:

If XMRV proves to be a causal factor in prostate cancer or CFS, these discoveries may allow for rational design of clinical trials.

Raltegravir has been previously shown to inhibit murine leukemia virus, which is highly related to XMRV. But the drug exacerbates autoimmune disease in mice which might rule out its use in treating CFS.

Ila R. Singh1, John E. Gorzynski, Daria Drobysheva, Leda Bassit, & Raymond F. Schinazi (2010). Raltegravir Is a Potent Inhibitor of XMRV, a Virus Implicated in Prostate Cancer and Chronic Fatigue Syndrome PLoS One : 10.1371/journal.pone.0009948