HIV Molecular Virology

David Harrich Staff
Funding
Collaborators
Key Publications
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Lab Head: Dr David Harrich

HIV Research

The HIV Pandemic
Globally HIV/AIDS now ranks as one of the largest killer of any infectious disease. More than 25 million people have now died from AIDS and there are 39.5 million (34.1 - 47.1 million) infected. While a large portion of infections have occurred in Sub-Saharan Africa, an estimated 8.5 million people are now living with HIV in Asia and the Pacific, including 1 million new infections in 2002. India, with approximately 5.7 million cases by year-end 2005, and China, with official estimates at 650 000 (390 000-1.1 million) were living with HIV at the end of 2005, are regions deserving great concern. Other countries with suspected high prevalence are Cambodia, Myanmar, and Thailand. By 2020, it is projected that another 45 million people will become infected without new intervention, such as increased access to antiretroviral therapy or an effective vaccine, which could greatly slow transmission rates.[Source: UNAIDS Fact Sheet 2006.]

Antiretroviral Drugs
TAT Current antiretroviral drugs that inhibit HIV growth act upon essential viral enzymes to inhibit its normal function. For example, nucleoside reverse transcriptase inhibitors (NRTI) such as zidovudine (AZT), lamivudine (3TC), or stavudine (d4T) are used by the viral polymerase, reverse transcriptase (RT), in place of the normal nucleotide and prevent further synthesis. HIV protease inhibitors (PI) prevent processing of the viral capsid proteins resulting in non-infectious virions. Highly aggressive antiretroviral therapy using three or more drugs has resulted in dramatic reductions of patient viral loads. The hypothesis behind combinatorial therapy is that resistance to drugs which act through distinct mechanisms requires multiple mutations that are not easily achieved by the virus. However, there are now reports of documented transmission of HIV strains with drug resistance to multiple NRTI and PI drugs. It is unclear whether this is due to an adapting virus or patients not adhering to strict treatment regimens. In either case, it is likely that new therapeutic options to drug resistant HIV infection will be required.

New Drug Target
The HIV Molecular Virology lab focus is the discovery of key viral or cellular molecules required for HIV to grow, and to target their action so that HIV growth can be effectively blocked.

Is Tat a good drug target?
Reverse transcription is the process by which the viral RNA genome is converted into a double strand of DNA. HIV-1 reverse transcription is affected by a number of viral proteins including one called Tat. Genetic analysis showed that Tat's function in reverse transcription was distinct from its role in HIV-1 gene expression where it can activate HIV as much as a thousand-fold. The mechanism by which Tat enhances reverse transcription remains unknown, but the observation that Tat-deficient virus can be trans-complemented for reverse transcription in a virus producer cell but not in the target cells indicates that Tat has an early effect on the virion or is itself a virion protein. Our experiments have shown that key mutations in Tat can effectively block virus replication by inhibiting reverse transcription.

Tat is a virion protein but exactly how this predominantly nuclear protein traffics to the plasma membrane is unknown. A current grant awarded by the National Health and Medical Research Council aims to investigate Tat-enhanced reverse transcription, mechanisms of Tat trafficing, and methods to block Tat function.

TAR

Viral RNA regulates reverse transcription
We have investigated a RNA stem-loop structure called the transactivation response element (TAR), which is best known for its role in supporting high levels of HIV-1 gene expression. Viruses with mutations in TAR show a greatly reduced ability to support reverse transcription. How TAR functions in reverse transcription is unknown, but at least two reasonable mechanisms can be proposed. TAR RNA may directly interact with viral protein(s) or with other RNA sequences in order to stabilise a reverse transcription complex. Alternatively, TAR may recruit to the virion a factor(s) which can enhance the reverse transcription reaction.

Cellular factors and HIV reverse transcription
The studies described above have primarily focused on the initiation of reverse transcription. Our data indicate that a post-entry event or cell factor may be involved in efficient HIV-1 reverse transcription in vivo. We seek to determine factors which enable late HIV-1 DNA synthesis with the long term aim to define new drug targets. We have demonstrated an in vitro system that can be used to identify cellular factors required by HIV-1 for efficient reverse transcription. A current grant awarded by the National Health and Medical Research Council aims to identify the key factors required for virus replication after entry into a cell.

Protein Arginine Methylation
Protein methylation is recognized as a major protein modification pathway regulating diverse cellular events such as protein trafficking, transcription, and signal transduction. More recently, protein arginine methyltransferase activity has been shown to regulate HIV-1 transcription via Tat. Recently, it has been shown that inhibiting protein methylation can modulate infectivity in some viruses. Our data have shown that protein methylation is required for optimal HIV-1 infectivity. When protein methylation was inhibited in cells producing HIV-1 using adenosine periodate, the resultant virus was affected in terms of virion size, virion composition and infectivity. We are investigating which viral proteins are methylated and to identify the relevant protein methyltransferase activity.

Staff

Labhead:	Dr David Harrich
Research Officers:	Dr Ann Apolloni Dr David Warrilow
PhD Students:	Luke Meredith Haran Sivakumaran Kylie Warren
Masters Student:	Min-Hwuan Lin

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Funding

National Health & Medical Research Council
Australian Centre for HIV and Hepatitis (ACH2)

Collaborators

Nitin Saksena, Westmead Millennium Institute, Sydney
Andreas Suhrbier, QIMR, Brisbane
Alex Khromykh, Sir Albert Sakzewski Virus Research Centre, Brisbane
Richard Gaynor, Eli Lilly, Indianapolis
Professor Harry Smith, Royal Brisbane and Women's Hospital, Brisbane
Sabine Piller, Westmead Millennium Institute, Sydney
David Jans, Monash University
Li Peng, IMVS Adelaide
Chris Burrell, Univ. of Adelaide
Rosemary Kiernan, Institut de Genetique Humaine
Monsef Benkirane, Institut de Genetique Humaine

Key Publications

Sivakumaran H, Harrich D. (2008) SOCS1: a host factor required for HIV-1 Gag trafficking. Future HIV Therapy, 3: 247-251, 2008

Warrilow D, Meredith L, Davis A, Burrell C, Li P, Harrich D. Cell factors stimulate human immunodeficiency virus type 1 reverse transcription in vitro. J. Virol, 82:1425-1437, 2008.

Sivakumaran H, Wang B, Gill JM, Beckholdt B, Saksena NK, Harrich D. Functional relevance of nonsynonymous mutations in the HIV-1 tat gene within an epidemiologically-linked transmission cohort. Virology J, 4:107, 2007

Warrilow D, Stenzel D, Harrich D. HIV-1 Core is Active for Reverse Transcription. Retrovirology, 4:77, 2007

Apolloni A, Meredith LW, Suhrbier A, Sivakumaran H, Kiernan R, and Harrich D. The HIV-1 Tat Protein Stimulates Reverse Transcription in vitro. Current HIV Research, 5:473-483, 2007.

Warrilow D and Harrich D. HIV-1 replication from after cell entry to the nuclear periphery. Current HIV Research, 5:293-9, 2007.

Willemsen NM, Hitchen EM, Bodetti TJ, Apolloni A, Warrilow D, Piller SC, Harrich D. Protein methylation is required to maintain optimal HIV-1 infectivity. Retrovirology 3:92, 2006.

Harrich D, McMillan N, Munoz N, Apolloni A, Meredith.L. Will diverse Tat interactions lead to novel antiretroviral drug targets? Current Drug Targets 7:1595-606, 2006

Warrilow D, Gardner J, Suhrbier A, Harrich D.? A protein kinase C activator reveals an unusual mechanism of HIV-1 inhibition. AIDS Research and Human Retroviruses 22:854-64, 2006

Darnell GA, Schroder WA, Gardner J, Harrich D, Yu H, Medcalf RL, Warrilow D, Antalis TM, Sonza S, Suhrbier A. Serpinb2 is an inducible host factor involved in enhancing HIV-1 transcription and replication. J Biol Chem 281:31348-58, 2006

Endo-Munoz L, Harrich D, and McMillan N. Phosphorylation of HIV Tat by PKR increases interaction with TAR RNA and enhances transcription. Virology J. 2005,2:17

Darnell GA, Antalis TM, Johnstone RW, Stringer BW, Ogbourne SM, Harrich D, and Suhrbier A. Inhibition of retinoblastoma protein degradation by interaction with the serpin PAI-2 via a novel consensus motif. Mol Cell Bio 23: 6520-6532, 2003.

Anraku I, Harvey TJ, Linedale R, Gardner J, Harrich D, Suhrbier A, and Khromykh AA. Kunjin replicon vectors for human immunodeficiency Virus Vaccine development. J Virol 77:7796-7803, 2003.

Apolloni A, Hooker CW, Mak J, and Harrich D. HIV-1 protease regulation of Tat activity is essential for efficient reverse transcription and replication. J Virol 77: 9912-9921, 2003

Hooker CW and Harrich D. The first strand transfer reaction of HIV-1 reverse transcription is more efficient in infected cells than in cell-free natural endogenous reverse transcription reactions. J Clin Virol 26: 229-238, 2003

Hooker CW, Scott J, Apolloni A, Parry E, and Harrich D. Human immunodeficiency virus type 1 reverse transcription is stimulated by Tat from other lentiviruses. Virology 300:226-35, 2002

Hooker CW, Lott WB, Harrich D. Inhibitors of human immunodeficiency virus type 1 reverse transcriptase target distinct phases of early reverse transcription. J Virol 75:3095-3104, 2001

Harrich D, Hooker CW, Parry E. The human immunodeficiency virus type 1 TAR RNA upper stem loop plays distinct roles in reverse transcription and packaging. J Virol 74: 5639-5646, 2000

Ulich, C, Dunne A, Parry E, Hooker CW, Gaynor RB, and Harrich D. Functional domains of tat required for efficient human immunodeficiency virus type 1 reverse transcription. J Virol73:2499-2508, 1999